Luận án Tiến sĩ Nghiên cứu tạo kháng nguyên Hemagglutinin (HA) tái tổ hợp của virus cúm AH5N1 bằng phương pháp biểu hiện tạm thời trên cây thuốc lá và đánh giá khả năng sinh miễn dịch

BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Phạm Thị Vân NGHIÊN CỨU TẠO KHÁNG NGUYÊN HEMAGGLUTININ (HA) TÁI TỔ HỢP CỦA VIRUS CÚM A/H5N1 BẰNG PHƯƠNG PHÁP BIỂU HIỆN TẠM THỜI TRÊN CÂY THUỐC LÁ VÀ ĐÁNH GIÁ KHẢ NĂNG SINH MIỄN DỊCH LUẬN ÁN TIẾN SĨ SINH HỌC Hà Nội – 2021 BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Phạm Thị Vân NGHIÊN CỨU TẠO KHÁNG NGUYÊN HEMAGGLUTININ (HA) TÁI TỔ HỢP CỦA VIRUS CÚM A/H5N1 BẰNG PHƯƠNG PHÁP BIỂU HIỆN TẠM THỜI TRÊN CÂY THUỐC LÁ VÀ ĐÁNH GIÁ KHẢ NĂNG SINH MIỄN DỊCH Chuyên ngành: Công nghệ sinh học Mã số: 42 02 01 LUẬN ÁN TIẾN SĨ SINH HỌC NGƯỜI HƯỚNG DẪN KHOA HỌC: TS Vũ Huyền Trang GS TS Lê Thanh Hoà Hà Nội – 2021 i LỜI CẢM ƠN Tôi xin bày tỏ lòng biết ơn sâu sắc tới thầy cô giáo, TS Vũ Huyền Trang và GS.TS Lê Thanh Hoà đã tận tình hướng dẫn, chỉ bảo, dìu dắt, giúp đỡ tôi thực và hoàn thành luận án Tôi xin bày tỏ lòng biết ơn sâu sắc tới PGS.TS Chu Hoàng Hà, PGS TS Phạm Bích Ngọc, Ths Hồ Thị Thương, ThS Nguyễn Thu Giang, KTV Trần Thị Loan cùng tập thể cán bộ Phòng Công nghệ Tế bào thực vật, phòng công nghệ ADN ứng dụng, Viện Công nghệ sinh học đã tạo điều kiện làm việc, hỗ trợ kinh phí, nhiệt tình giúp đỡ và truyền đạt nhiều kinh nghiệm quý báu cho tôi suốt thời gian thực và hoàn thành luận án Đặc biệt tôi xin bày tỏ lòng biết ơn sâu sắc tới GS TS Udo Conrad, TS Phan Trọng Hoàng, Viện nghiên cứu di truyền và cây trồng thực vật (IPK) CHLB Đức đã truyền đạt kiến thức và kinh nghiệm quý báu, giúp đỡ tôi suốt thời gian thực tập tại Viện IPK như thời gian tôi học tập tại Việt Nam Tôi xin cảm ơn sâu sắc tới TS Trần Xuân Hạnh Công ty CP thuốc thú y Ttrung ương NAVETCO đã giúp đỡ tôi thực thành công các thí nghiệm công cường độc gà Tôi xin chân thành cảm ơn Bộ Khoa học và Công nghệ Việt Nam với đề tài Nghị định thư mã số NĐT.07.GER.15 và Bộ giáo dục và nghiên cứu CHLB Đức với đề tài “Plantfluvac” mã số 031A283 đã hỗ trợ kinh phí cho tôi thực nghiên cứu luận án này Tôi xin bày tỏ lòng biết ơn sâu sắc tới Bộ phận Đào tạo, các phòng chức năng và Ban lãnh đạo Viện Công nghệ sinh học, Học viện Khoa học và Công nghệ, Viện Hàn lâm Khoa học và Công nghệ Việt Nam đã tạo điều kiện thuận lợi cho tôi quá trình học tập và bảo vệ luận án Cuối cùng, tôi xin dành lòng biết ơn sâu sắc tới người thân gia đình, bạn bè đã luôn bên tôi, động viên và góp ý cho tôi suốt quá trình thực luận án Tôi xin chân thành cảm ơn tất cả sự giúp đỡ quý báu đó! Hà Nội, ngày tháng Nghiên cứu sinh Phạm Thị Vân năm 2021 ii LỜI CAM ĐOAN Tôi xin cam đoan: Đây là công trình nghiên cứu của tôi, các số liệu và kết quả trình bày luận án là trung thực, đã được công bố các tạp chí khoa học chuyên ngành với sự đồng ý và cho phép của các đồng tác giả Hà Nội, ngày tháng Nghiên cứu sinh Phạm Thị Vân năm 2021 iii MỤC LỤC LỜI CẢM ƠN i LỜI CAM ĐOAN ii MỤC LỤC iii DANH MỤC CÁC TỪ VIẾT TẮT vi DANH MỤC CÁC BẢNG vii DANH MỤC CÁC HÌNH viii MỞ ĐẦU 1 Tính cấp thiết luận án Mục tiêu nghiên cứu luận án Các nội dung nghiên cứu luận án Chương TỔNG QUAN TÀI LIỆU 1.1 Virus cúm A/H5N1 1.1.1 Phân loại đặc tính của virus cúm A/H5N1 1.1.2 Cấu trúc, chức năng hệ gen của virus cúm A 1.1.3 Cấu trúc, chức năng của protein hemagglutinin 1.1.4 Biến đổi thành phần hemagglutinin tạo nên các clade của virus A/H5N1 13 1.2 Bệnh cúm gia cầm 16 1.2.1 Lịch sử và tình hình dịch cúm A/H5N1 thế giới 16 1.2.2 Tình hình bệnh cúm A/H5N1 Việt Nam 17 1.3 Vaccine phòng bệnh cúm cho gia cầm 22 1.3.1 Vaccine phòng bệnh cúm gia cầm thế giới 23 1.3.2 Vaccine phòng bệnh cúm gia cầm tại Việt Nam 24 1.3.3 Nghiên cứu vaccine cúm phổ rộng 27 1.3.4 Nghiên cứu vaccine từ thực vật 29 1.3.5 Nghiên cứu vaccine dựa vào HA ELP hoá 33 1.3.6 Nghiên cứu vaccine dựa vào HA oligomer hoá 34 1.4 Hệ thống biểu hiện protein tái tổ hợp tạm thời thực vật thông qua Agrobacterium (agroinfiltration) 36 1.4.1 Nguyên lý 36 1.4.2 Ứng dụng agroinfiltration nghiên cứu và sản xuất protein có hoạt tính sinh học thế giới 37 1.4.3 Tình hình nghiên cứu và sản xuất protein có hoạt tính sinh học thông qua agroinfiltration Việt Nam 40 Chương VẬT LIỆU VÀ PHƯƠNG PHÁP NGHIÊN CỨU 44 2.1 Vật liệu 44 2.1.1 Nguồn vật liệu thực vật 44 iv 2.1.2 Chủng vi sinh vật, plasmid, trình tự gen 44 2.1.3 Các cặp mồi sử dụng 45 2.1.4 Nguồn vật liệu động vật 45 2.1.5 Hóa chất 45 2.1.6 Thiết bị 45 2.1.7 Môi trường và đệm 46 2.2 Phương pháp 46 2.2.1 Phương pháp lựa chọn gen mã hoá kháng nguyên HA của các chủng virus cúm A/H5N1 46 2.2.2 Phương pháp sử dụng thiết kế cấu trúc vector biểu thực vật mang gen mã hoá kháng nguyên HA và đánh giá sự biểu tạm thời kháng nguyên HA của virus cúm A/H5N1 cây thuốc lá 48 2.2.3 Phương pháp tinh sạch, đánh giá trạng thái oligomer hoá và hoạt tính ngưng kết hồng cầu của các kháng nguyên HA tái tổ hợp 55 2.2.4 Phương pháp đánh giá khả năng gây đáp ứng miễn dịch của kháng nguyên HA tái tổ hợp động vật thí nghiệm 57 Chương KẾT QUẢ NGHIÊN CỨU 62 3.1 Lựa chọn gen mã hoá kháng nguyên HA chủng virus cúm A/H5N1… 62 3.1.1 Thu thập và phân loại trình tự HA theo năm 62 3.1.2 Phân tích đởi mã gen và tổng hợp nhân tạo chuỗi gen mã hóa kháng nguyên HA của chủng virus cúm A/H5N1 cơ sở trình tự đã biến đổi 63 3.1.3 Thiết kế trình tự HA COBRA đại diện các chủng virus cúm A/H5N1 quan tâm 67 3.1.4 Chuyển đổi các trình tự amino acid của kháng nguyên HACOBRA1 HACOBRA2 thành các trình tự nucleotide 71 3.1.5 Phân tích so sánh trình tự của các protein HA lựa chọn nghiên cứu… 72 3.2 Thiết kế cấu trúc vector biểu hiện thực vật mang gen mã hoá kháng nguyên HA đánh giá biểu hiện tạm thời kháng nguyên HA virus cúm A/H5N1 trên thuốc 74 3.2.1 Thiết kế vector biểu kháng nguyên HA trimer 76 3.2.2 Thiết kế vector biểu kháng nguyên HA oligomer ELP 79 3.2.3 Thiết kế vector biểu kháng nguyên HA oligomer TP 82 3.2.4 Lựa chọn điều kiện thích hợp cho sự biểu kháng nguyên HA cây thuốc lá 84 3.2.5 Đánh giá sự biểu các kháng nguyên HA tái tổ hợp dịch chiết thô thuốc lá 91 3.2.6 Đánh giá hoạt tính ngưng kết hồng cầu của kháng nguyên HA dịch chiết thô lá thuốc 92 3.3 Tinh sạch, đánh giá trạng thái oligomer hố hoạt tính ngưng kết hồng cầu kháng nguyên HA tái tổ hợp 94 v 3.3.1 Tinh sạch kháng nguyên HA oligomer ELP từ cây thuốc lá mITC 94 3.3.2 Tinh sạch kháng nguyên HA trimer và oligomer TP IMAC 98 3.3.3 Trạng thái oligomer hoá và hoạt tính ngưng kết hồng cầu của các kháng nguyên HA sau tinh sạch 100 3.4 Đánh giá khả gây đáp ứng miễn dịch kháng nguyên HA tái tổ hợp trên động vật thí nghiệm 103 3.4.1 Miễn dịch chuột thí nghiệm 103 3.4.2 Miễn dịch gà thí nghiệm 110 Chương THẢO LUẬN 122 4.1 Lựa chọn gen HA yếu tố quan trọng nghiên cứu phát triển vaccine phòng bệnh cúm gia cầm A/H5N1 Việt Nam 122 4.2 HA trimer dung hợp ELP làm tăng cường biểu hiện protein thực vật, đơn giản hoá tinh khơng làm tăng cường hoạt tính sinh học HA trimer 125 4.3 HA COBRA kích thích tạo kháng thể trung hồ phổ rộng trên động vật thí nghiệm 127 4.4 HA oligomer có hoạt tính sinh miễn dịch cao trên động vật 128 4.5 Dịch chiết thơ thuốc chứa kháng nguyên HA oligomer TP sử dụng làm ứng viên vaccine phòng bệnh cúm A trên động vật 129 KẾT LUẬN VÀ KIẾN NGHỊ 131 NHỮNG ĐÓNG GÓP MỚI CỦA LUẬN ÁN 132 DANH MỤC CƠNG TRÌNH CỦA TÁC GIẢ 133 TÀI LIỆU THAM KHẢO 134 PHỤ LỤC P1 vi DANH MỤC CÁC TỪ VIẾT TẮT bp base pair BS3 bis [sulfosuccinimidyl] suberate DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate E coli Escherichia coli EDTA ethylene diamine tetra-acetate acid ELISA enzyme-linked immunosorbent assay ELP elastin-like polypeptides EtBr ethidium bromide FAO The Food and Agriculture Organization HA Hemagglutinin HI hemagglutinin inhibition IMAC Immobilized metal ion chromatography kb kilobase kDa kilodalton LB Luria and Bertani mITC membrane-based inverse transition cycling OD optical density OIE The World Organisation for Animal Health PCR polymerase chain reaction SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC size exclusion chromatography TAE tris acetate EDTA Taq DNA polymerase Thermus aquaticus DNA polymerase v/p vòng/phút WHO World Health Organization vii DANH MỤC CÁC BẢNG Bảng 1.1 Các gốc amino acid các vị trí điểm cắt protease, vị trí bám dính thụ thể, vị trí liên kết thụ thể, vị trí epitope và một vị trí glycosyl hoá bị biến đổi của các H5 từ một số chủng virus A/H5N1 khác 11 Bảng 1.2 Danh sách các chủng virus cúm A/H5N1 phát triển vaccine 23 Bảng 1.3 Các protein có hoạt tính sinh học, kháng thể và vaccine được sản xuất từ thực vật thử nghiệm lâm sàng có mặt thị trường 30 Bảng 2.1 Danh sách mồi sử dụng nghiên cứu 45 Bảng 3.1 Số lượng trình tự gen HA của các chủng virus cúm A/H5N1 gây bệnh gia cầm Việt Nam 63 Bảng 3.2 Các vị trí sai khác so sánh các trình tự protein HACOBRA clade 1.1; 1.1.1 1.1.2 68 Bảng 3.3 Các vị trí sai khác so sánh các trình tự protein HACOBRA clade 2.3.2.1, 2.3.2.1a, b c 69 Bảng 3.4 Danh sách các cấu trúc vector thiết kế chứa HA tự nhiên và nhân tạo 75 Bảng 3.5 Hiệu suất thu hồi các kháng nguyên HA oligomer ELP 98 Bảng 3.6 Bảng tổng hợp kết quả thiết kế, biểu và đánh giá hoạt tính của các kháng nguyên HA tái tổ hợp 120 Bảng 4.1 Trình tự amino acid tại các vị trí phân cắt protein, liên kết thụ thể epitope kháng nguyên và vị trí glycosyl của các protein HA 124 viii DANH MỤC CÁC HÌNH Hình 1.1 Sơ đồ cấu trúc của virus cúm A [8] Hình 1.2 Mô hình cấu trúc hemagglutinin xuyên lớp màng bao lipid kép 10 Hình 1.3 Dòng thời gian xuất các clade của A/H5N1 dựa vào trình tự của gen HA Việt Nam [1] 18 Hình 1.4 Bản đồ các ổ dịch cúm A/H5N1 xảy các năm 2006 đến 2014 dựa vào số liệu được báo cáo OIE 19 Hình 1.5 Bản đồ các ổ dịch cúm A/H5N1 xảy các năm 2015 đến 2017 dựa vào số liệu được báo cáo OIE 20 Hình 1.6 Bản đồ các ổ dịch cúm gia cầm xảy các năm 2018 đến 11/09/2020 dựa vào số liệu được báo cáo OIE 21 Hình 1.7 Sơ đồ dạng lục thể IgM (A) và dạng đơn tiểu đơn vị (B) 35 Hình 1.8 Quy mô thương mại N benthamiana tăng trưởng (A) và khử trùng (B) 39 Hình 1.9 Quy trình sản xuất virus cúm mùa tại công ty Medicago, Canada 40 Hình 2.1 Sơ đồ mô tả các bước thí nghiệm cơ bản nghiên cứu 46 Hình 2.2 Hình ảnh cây thuốc lá thuỷ canh tuần tuổi (A) được loại bỏ bớt lá già, lá non và lá bánh tẻ trước biến nạp (B) 52 Hình 2.3 Biến nạp dịch khuẩn vào lá thuốc lá máy hút chân không 52 Hình 3.1 Hình ảnh so sánh trình tự amino acid của protein H5TG chủng A/duck/Vietnam/TG24-O1/05 đã được tối ưu biểu thuốc lá so với trình tự gốc 66 Hình 3.2 Hình ảnh so sánh trình tự amino acid của protein H5HT chủng A/duck/Vietnam/HT-O2/2014 đã được tối ưu biểu thuốc lá so với trình tự gốc 67 Hình 3.3 Mô hình thiết kế các trình tự HA COBRA (A) và hình ảnh xây dựng cây phân loại dựa vào các trình tự của kháng nguyên HA tự nhiên và nhân tạo 71 Hình 3.4 Hình ảnh biểu diễn và so sánh trình tự amino acid của kháng nguyên HA tự nhiên và HA nhân tạo 73 Hình 3.5 Các loại cassette biểu kháng nguyên HA thực vật 75 Hình 3.6 Mô hình thiết kế vector biểu protein H5TG trimer thực vật 76 Hình 3.7 Hình ảnh điện di sản phẩm PCR khuyếch đại đoạn gen H5TG (A) và sản phẩm cắt vector tách dòng pRTRA_H5TG trimer enzyme BamHI PspOMI (B) 77 Hình 3.8 Hình ảnh điện di sản phẩm của quá trình thiết kế cấu trúc vector biểu kháng nguyên HA trimer 78 viii P6 A/duck/Khanhhoa/CVVI04/2013_KM821609 62 A/duck/Khanhhoa/CVVI05/2013_KM821610 A/duck/Khanhhoa/CVVI01/2013_KM821606 A/duck/Vietnam/QB1207/2012_KF182741 A/chicken/Vietnam/NCVD-1927/2012_EPI_ISL_136138 85 A/duck/Vietnam/NCVD-1933/2012_EPI_ISL_136189 A/duck/Vietnam/NCVDKA300/2012_KP097906 A/duck/Vietnam/NCVD-1943/2012_EPI_ISL_136185 A/chicken/Vietnam/NCVD-1942/2012_EPI_ISL_136193 A/duck/Khanhhoa/CVVI14/2013_KM821619 A/duck/Vietnam/NCVD-1930/2012_EPI_ISL_136249 A/chicken/Vietnam/NCVDKA275/2012_KP097903 A/duck/Vietnam/NCVD-1593/2012_EPI_ISL_136256 A/duck/Vietnam/NCVD-1937/2012_EPI_ISL_136191 A/duck/Vietnam/NCVD-1936/2012_EPI_ISL_136252 A/duck/Khanhhoa/CVVI21/2013_KM821626 64 42 A/duck/Khanhhoa/CVVI15/2013_KM821620 14 A/duck/Khanhhoa/CVVI33/2014_KM821638 A/duck/Khanhhoa/CVVI23/2013_KM821628 A/muscovy-duck/Vietnam/LBM344/2013_AB828360 15 A/chicken/Khanhhoa/CVVI13/2013_KM821618 A/duck/Khanhhoa/CVVI07/2013_KM821612 31 79 64 A/duck/Khanhhoa/CVVI16/2013_KM821621 A/duck/Khanhhoa/CVVI17/2013_KM821622 A/duck/Khanhhoa/CVVI03/2013_KM821608 31 A/duck/Khanhhoa/CVVI08/2013_KM821613 A/chicken/Khanhhoa/CVVI02/2013_KM821607 A/duck/Khanhhoa/CVVI19/2013_KM821624 94 A-Dk-VN-KH24-2013(H5N1)H5l(1704bp) 22 A/duck/Khanhhoa/CVVI10/2013_KM821615 A/chicken/Khanhhoa/CVVI12/2013_KM821617 A/duck/Khanhhoa/CVVI22/2013_KM821627 80 A/chicken/Khanhhoa/CVVI11/2013_KM821616 A/duck/Khanhhoa/CVVI20/2013_KM821625 A/chicken/Khanhhoa/CVVI09/2013_KM821614 A/duck/Vietnam/NCVDKA345/2012_KP097911 629 A/chicken/Vietnam/NCVDKA432/2013_KP097918 A/duck/Vietnam/NCVD-1905/2012_EPI_ISL_136255 A/duck/Vietnam/NCVD-1904/2012_EPI_ISL_136210 A/duck/Vietnam/NCVD-1878/2012_EPI_ISL_136173 A/duck/Vietnam/NCVD-1901/2012_EPI_ISL_136205 A/duck/Vietnam/NCVD-1897/2012_EPI_ISL_136136 A/duck/Vietnam/NCVDKA324/2012_KP097910 A/duck/Vietnam/NCVD-1902/2012_EPI_ISL_136209 A/duck/Vietnam/NCVD-1875/2012_EPI_ISL_136318 18 A/duck/Vietnam/NCVD-1872/2012_EPI_ISL_136187 20 A/duck/Vietnam/NCVD-1869/2012_EPI_ISL_136134 A/duck/Vietnam/NCVDKA262/2012_KP097902 A/duck/Vietnam/NCVD-1884/2012_EPI_ISL_136174 A/duck/Vietnam/NCVDKA311/2012_KP097909 A/duck/Vietnam/NCVD-1896/2012_EPI_ISL_136135 A/duck/Vietnam/NCVD-1898/2012_EPI_ISL_136137 A/duck/Vietnam/NCVD-1899/2012_EPI_ISL_136188 A/duck/Vietnam/NCVD-1968/2012_EPI_ISL_136253 A/muscovy-duck/Vietnam/LBM295/2012_AB807880 A/duck/Vietnam/LBM578/2014_AB972731 A/duck/Quang Ninh/53/2013_AB828589 19 67 70 77 A/muscovy-duck/Quang Ninh/49/2013_AB828693 A/muscovy-duck/Quang Ninh/48c141/2013_LC010712 A/duck/Quanh Ninh/21/2013_AB824279 99 A/muscovy-duck/Vietnam/LBM669/2014_LC010728 100 71 A/muscovy-duck/Vietnam/LBM668/2014_LC010720 A/muscovy-duck/Vietnam/LBM670/2014_LC010736 A/duck/Vietnam/LBM570/2014_AB972707 93 29 A/muscovy-duck/Vietnam/LBM573/2014_AB972723 A/duck/Vietnam/LBM568/2014_AB972691 86 A/duck/Vietnam/LBM569/2014_AB972699 63 A/muscovy-duck/Vietnam/LBM571/2014_AB972715 A/muscovy-duck/Vietnam/LBM567/2014_AB972683 43 46 A/ muscovy-duck/Vietnam/LBM566/2014_AB972675 100 A/duck/Vietnam/HU3-562/2015_LC115019 A/muscovy-duck/Vietnam/HU3-355/2015_LC115018 99 94 69 A/chicken/Vietnam/HU3-1055/2015_LC115021 A/muscovy-duck/Vietnam/HU3-21/2015_LC115016 66 A/muscovy-duck/Vietnam/HU3-159/2015_LC115017 A/chicken/Vietnam/HU3-881/2015_LC115020 A/muscovy-duck/LongAn/90/2014_LC010776 77 A/muscovy-duck/LongAn/89/2014_LC010768 A/muscovy-duck/LongAn/88/2014_LC010760 100 A/muscovy-duck/LongAn/87/2014_LC010752 A/muscovy-duck/LongAn/86/2014_LC010744 A/chicken/VietNam/TMU009/2008_CY095689 A/muscovy-duck/Vietnam/LBM438/2013_AB847565 0.005 Hình P Cây phả hệ của các chủng virus A/H5N1 thuộc clade 2.3.2.1c Việt Nam dựa vào trình tự amino acid của HA P7 (A) 10 20 30 40 50 60 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | MEKIVLLFATISLVKSDHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDL 60 60 60 60 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | NGVKPLILKDCSVAGWLLGNPLCDEFTNVPEWSYIVEKANPANDLCYPGNFNDYEELKHL I P I I 120 120 120 120 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | LSRINHFEKIQIIPKDSWSDHEASLGVSAACSYQGNSSFFRNVVWLIKKDNAYPTIKKGY N N N V 180 180 180 180 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | NNTNREDLLILWGIHHPNDEAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKINGQSG P 240 240 240 240 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | RIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTIMRSEVEYGNCNTRCQTPIGA W 300 300 300 300 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | INSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQ 360 360 360 360 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | GMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLER 420 420 420 420 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | RIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLKDNAKELGN 480 480 480 480 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | | | | | | | | GCFEFYHKCNNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVAS 540 540 540 540 A/duck/Vietnam/NCVD-1584/2012_ A-Dk-VN-HT2-2014 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 | | | | | SLVLAIMMAGLSLWMCSNGSLQCRICI A A AP 567 567 567 567 (B) Percent Identity 1 99.4 99.6 99.1 A-Dk-VN-NCVD-1584-2012 99.8 99.3 A-Dk-VN-HT2-2014 99.4 A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 0.6 0.4 0.2 0.9 0.7 0.6 Hình P So sánh trình tự amino acid gen HA của chủng ứng viên vaccine A/duck/Vietnam/NCVD-1584/2012 với các chủng phân lập Hà Tĩnh Việt Nam A-Dk-VN-HT2-2014, A-Dk-VN-HT4-2014 A-Dk-VN-HT5-2014 GS.TS Lê Thanh Hoà cung cấp P8 Ngoài clade 1.1 và 2.3.2.1c, có một số clade mới hình xuất tại Việt Nam Đó là, clade 1.1.1 và clade 1.1.2 và clade 2.3.2.1a, 2.3.2.1b Dựa vào các trình tự đã công bố, luận án đã thu thập được 38, 34, 143 và 28 lần lượt là trình tự gen HA của các chủng virus A/H5N1 clade 1.1.1, 1.1.2, 2.3.2.1a và 2.3.2.1b Các trình tự này đã được so sánh và phân tích để tìm trình tự đại diện cho nhóm Trong đó có một số trình tự đại diện có thể được sử dụng cho nghiên cứu sản suất vaccine cho clade (Bảng P 1) Bảng P Một số chủng đại diện cho nhóm 1.1.1, 1.1.2, 2.3.2.1a 2.3.2.1b Clade đại diện Chủng virus 1.1.1 A/chicken/Vietnam/NCVD117/2008 1.1.2 A/muscovy duck/Vietnam/OIE3312/2011 2.3.2.1a A/duck/Vietnam/QT801/2011 10 20 30 40 50 60 70 | | | | | | | | | | | | | | ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACC 70 H5TG_Origin H5TG_Optimization C A C C 22 80 90 100 110 120 130 140 | | | | | | | | | | | | | | ATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGA 140 H5TG_Origin H5TG_Optimization T T AGT A A C G A G A 92 150 160 170 180 190 200 210 | | | | | | | | | | | | | | CATACTGGAAAAGACACACAACGGAAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTGAGAGAT 210 H5TG_Origin H5TG_Optimization T C T G T T A T C T T.G CC.T 162 220 230 240 250 260 270 280 | | | | | | | | | | | | | | TGTAGTGTAGCTGGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTT 280 H5TG_Origin H5TG_Optimization TCC T .T.GT.A T T T G T C T A AG 232 290 300 310 320 330 340 350 | | | | | | | | | | | | | | ACATAGTGGAGAAGGCCAATCCAGTCAATGATCTCTGTTACCCAGGGGATTTCAATGACTATGAAGAATT 350 H5TG_Origin H5TG_Optimization C T A A C T C T.G T A C T C GC 302 360 370 380 390 400 410 420 | | | | | | | | | | | | | | H5TG_Origin GAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAGATCATCCCCAAAAGTTCTTGGCCCAGT 420 H5TG_Optimization T G T GC.ATCT T C C G A G GTCC T 372 430 440 450 460 470 480 490 | | | | | | | | | | | | | | CATGAAGCCTCATTAGGGGTGAGCTCAGCATGTCCATACCAGGGAAAGTCCTCCTTTTTCAGAAATGTGG 490 H5TG_Origin H5TG_Optimization A TC.T A T T T T A A T A C C T 442 500 510 520 530 540 550 560 | | | | | | | | | | | | | | TATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAGGAGCTACAATAATACCAACCAAGAAGA 560 H5TG_Origin H5TG_Optimization G T.G A G TCC C T C C G G 512 570 580 590 600 610 620 630 | | | | | | | | | | | | | | TCTTTTGGTACTGTGGGGGATTCACCATCCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAACCCA 630 H5TG_Origin H5TG_Optimization GC.T T A A T A C A A A 582 640 650 660 670 680 690 700 | | | | | | | | | | | | | | ACCACCTATATTTCCGTTGGGACATCAACACTAAACCAGAGATTGGTACCAAGAATAGCTACTAGATCCA 700 H5TG_Origin H5TG_Optimization T A C A A C T C A C.T T T G AGT 652 710 720 730 740 750 760 770 | | | | | | | | | | | | | | AAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCGAATGATGCAATCAATTT 770 H5TG_Origin H5TG_Optimization G T T T C A A T C AC.C T C T 722 780 790 800 810 820 830 840 P9 | | | | | | | | | | | | | | H5TG_Origin CGAGAGTAATGGAAATTTCATTGCCCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATT 840 H5TG_Optimization T TCC G T T G C C G A C C 792 850 860 870 880 890 900 910 | | | | | | | | | | | | | | ATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACACCAATGGGGGCGATAAATTCTA 910 H5TG_Origin H5TG_Optimization C GC G C T T T C C T A T T A 862 920 930 940 950 960 970 980 | | | | | | | | | | | | | | H5TG_Origin GTATGCCATTCCACAATATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATT 980 H5TG_Optimization C T T T T C G T A T G G G T TC.T 932 990 1000 1010 1020 1030 1040 1050 | | | | | | | | | | | | | | AGTCCTTGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGGAAAAAGAGAGGATTATTTGGAGCT 1050 H5TG_Origin H5TG_Optimization G T C T TT.AC G G G AC.TC. - T G T 993 1060 1070 1080 1090 1100 1110 1120 | | | | | | | | | | | | | | ATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATG 1120 H5TG_Origin H5TG_Optimization T T C A T T T T T T T CTCA 1063 1130 1140 1150 1160 1170 1180 1190 | | | | | | | | | | | | | | AGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGT 1190 H5TG_Origin H5TG_Optimization A TTCA T T T T A G A T T G T C A 1133 1200 1210 1220 1230 1240 1250 1260 | | | | | | | | | | | | | | CAACTCGATCATTGACAAAATGAACACTCAGTTTGAAGCCGTTGGAAGGGAATTTAACAACTTAGAAAGG 1260 H5TG_Origin H5TG_Optimization G T T T T T A C C C T TC.T C.T 1203 1270 1280 1290 1300 1310 1320 1330 | | | | | | | | | | | | | | AGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTC 1330 H5TG_Origin H5TG_Optimization C T A C.C T A G T C G A T.GT 1273 1340 1350 1360 1370 1380 1390 1400 | | | | | | | | | | | | | | TGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGT 1400 H5TG_Origin H5TG_Optimization G T C.T T.G C TAGC G A T A 1343 1410 1420 1430 1440 1450 1460 1470 | | | | | | | | | | | | | | CCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGAAATGGTTGTTTCGAGTTCTATCATAAATGTGAT 1470 H5TG_Origin H5TG_Optimization A.G T .C A T G C T T C C 1413 1480 1490 1500 1510 1520 1530 1540 | | | | | | | | | | | | | | AATGAATGTATGGAAAGTGTAAGAAACGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCAAAACTAA 1540 H5TG_Origin H5TG_Optimization .G C G C G G T C A T T C T G C T.G 1483 1550 1560 1570 1580 1590 1600 1610 | | | | | | | | | | | | | | AAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGACAATTTATTCCAC 1610 H5TG_Origin H5TG_Optimization C.G A TTCA G G G 1515 1620 1630 1640 1650 1660 1670 1680 | | | | | | | | | | | | | | AGTAGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCG 1680 H5TG_Origin H5TG_Optimization 1515 1690 1700 | | | | TTACAATGCAGAATTTGCATTTAG 1704 H5TG_Origin H5TG_Optimization 1515 Hình P Hình ảnh so sánh trình tự nucleotide gen H5TG đã được tối ưu biểu thuốc lá so với trình tự gốc 10 20 30 40 50 60 70 | | | | | | | | | | | | | | ATGGAGAAAATAGTTCTTCTCTTTGCAACAATCAGCCTTGTCAAAAGCGATCATATTTGCATTGGTTATC 70 H5HT_Origin H5HT_Optimization C C 22 80 90 100 110 120 130 140 | | | | | | | | | | | | | | H5HT_Origin ATGCAAATAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTCACACATGCCCAAGA 140 H5HT_Optimization T C AGT A A T G C A A T C A 92 150 160 170 180 190 200 210 | | | | | | | | | | | | | | H5HT_Origin CATACTGGAAAAGACACACAATGGGAAGCTCTGCGATCTAAATGGAGTGAAGCCTCTGATTTTAAAAGAT 210 H5HT_Optimization T C A G T C A T.G CT C T T CC G 162 220 230 240 250 260 270 280 | | | | | | | | | | | | | | TGTAGTGTAGCAGGATGGCTCCTCGGAAATCCATTGTGTGACGAATTCATCAATGTGCCAGAATGGTCTT 280 H5HT_Origin H5HT_Optimization TCC T T T.G A T A T A C T G AG 232 290 300 310 320 330 340 350 | | | | | | | | | | | | | | ACATAGTAGAGAAGCCCAATCCAGCCAATGACCTCTGTTACCCAGGAAATTTCAACGATTATGAAGAATT 350 H5HT_Origin H5HT_Optimization T C T A C T T TT.G T .C G G 302 360 370 380 390 400 410 420 | | | | | | | | | | | | | | P10 H5HT_Origin GAAACACCTATTGAGCAGGATAAACCATTTTGAGAAAATACAGATCATCCCCAAAGATTCTTGGTCAAAC 420 H5HT_Optimization A G T G ATC A C C G C A T T G G C C 372 430 440 450 460 470 480 490 | | | | | | | | | | | | | | H5HT_Origin CATGAAGCCTCATTGGGGGTGAGCGCAGCATGTTCATACCAGGGAAATTCCTCCTTCTTCAGAAATGTGG 490 H5HT_Optimization .G A T A A T T T T AGT T A T C T A TC C T 442 500 510 520 530 540 550 560 | | | | | | | | | | | | | | TGTGGCTTATCAAAAAGGACAATGCATACCCAACAATAAAGAAAGGCTACAATAATACCAACCGAGAAGA 560 H5HT_Origin H5HT_Optimization T T.G T G C T G T C C T G G 512 570 580 590 600 610 620 630 | | | | | | | | | | | | | | H5HT_Origin TCTATTGATACTGTGGGGAATCCACCATCCTAATGATGAGGCGGAACAGACAAGGCTCTACCAAAACCCA 630 H5HT_Optimization TC TT.A T A C A A T A T T T 582 640 650 660 670 680 690 700 | | | | | | | | | | | | | | H5HT_Origin ACTACCTATATTTCCATTGGGACTTCAACACTAAACCAGAGATTGGTACCAAAAATAGCCACTAGATCCA 700 H5HT_Optimization A A C AG C C G T T T T C T G A G T 652 710 720 730 740 750 760 770 | | | | | | | | | | | | | | H5HT_Origin AAATAAACGGGCAAAGTGGCAGGATAGATTTCTTCTGGACAATTTTAAAACCGAATGACGCAATCCACTT 770 H5HT_Optimization G T TCC A A C T C G T A 722 780 790 800 810 820 830 840 | | | | | | | | | | | | | | CGAGAGTAATGGGAATTTCATTGCTCCAGAATATGCGTACAAAATTGTCAAGAAGGGAGACTCCACAATC 840 H5HT_Origin H5HT_Optimization T C T T C G G C A G A G TAG C A 792 850 860 870 880 890 900 910 | | | | | | | | | | | | | | ATGAGAAGTGAAGTGGAATATGGTAACTGCAACACCAGGTGTCAGACTCCAATAGGGGCGATAAACTCTA 910 H5HT_Origin H5HT_Optimization C G T C A T T T A C A A T TAGC 862 920 930 940 950 960 970 980 | | | | | | | | | | | | | | H5HT_Origin GTATGCCATTCCACAACATACACCCTCTCACCATCGGAGAATGTCCCAAATATGTGAAATCAAACAAATT 980 H5HT_Optimization C T T T T T T T G C T C 932 990 1000 1010 1020 1030 1040 1050 | | | | | | | | | | | | | | AGTCCTTGCAACTGGGCTCAGAAATAGTCCTCAAAGAGAGAGAAGAAGAAAAAGAGGACTGTTTGGAGCT 1050 H5HT_Origin H5HT_Optimization G T G T AT.GC TC C C A G - T T A 993 1060 1070 1080 1090 1100 1110 1120 | | | | | | | | | | | | | | H5HT_Origin ATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCACAGCAATG 1120 H5HT_Optimization T T C T A T .T T T TTCT 1063 1130 1140 1150 1160 1170 1180 1190 | | | | | | | | | | | | | | H5HT_Origin AACAGGGGAGTGGTTACGCTGCAGACAAAGAATCTACTCAAAAGGCGATAGACGGGGTCACCAATAAGGT 1190 H5HT_Optimization .T T T T G A T T T C G T C A 1133 1200 1210 1220 1230 1240 1250 1260 | | | | | | | | | | | | | | H5HT_Origin CAATTCGATCATTGACAAAATGAACACTCAGTTTGAGGCTGTAGGAAGGGAATTTAATAATTTAGAGAGG 1260 H5HT_Optimization AGT T T T .C GC.T C C.T AC.T 1203 1270 1280 1290 1300 1310 1320 1330 | | | | | | | | | | | | | | AGAATAGAAAATTTAAACAAGAAGATGGAAGACGGATTCCTAGATGTCTGGACTTATAATGCTGAACTTC 1330 H5HT_Origin H5HT_Optimization C.T T G G C T T G .G T 1273 1340 1350 1360 1370 1380 1390 1400 | | | | | | | | | | | | | | TGGTTCTCATGGAGAATGAGAGAACTCTAGACTTCCATGACTCAAATGTCAAGAACCTTTACGATAAGGT 1400 H5HT_Origin H5HT_Optimization C T A C.T T T T C T T G A T C T C A 1343 1410 1420 1430 1440 1450 1460 1470 | | | | | | | | | | | | | | CCGACTACAGCTTAAGGATAATGCAAAAGAGCTGGGAAACGGTTGTTTCGAGTTCTATCACAAATGTAAT 1470 H5HT_Origin H5HT_Optimization GA.G C C A C A C G T G T A T C 1413 1480 1490 1500 1510 1520 1530 1540 | | | | | | | | | | | | | | AATGAATGTATGGAAAGTGTAAGAAACGGGACGTATGACTACCCGCAGTATTCAGAAGAAGCAAGATTAA 1540 H5HT_Origin H5HT_Optimization C TCA G G T T T T T C GC.C 1483 1550 1560 1570 1580 1590 1600 1610 | | | | | | | | | | | | | | AAAGAGAGGAAATAAGTGGAGTAAAACTGGAATCAATAGGAATCTACCAAATACTGTCAATTTATTCAAC 1610 H5HT_Origin H5HT_Optimization G A TTC C G T 1515 1620 1630 1640 1650 1660 1670 1680 | | | | | | | | | | | | | | H5HT_Origin AGTGGCGAGTTCCCTAGCGCTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTGTTCCAACGGATCG 1680 H5HT_Optimization 1515 1690 1700 | | | | H5HT_Origin TTACAGTGCAGGATCTGCATCTAA 1704 H5HT_Optimization 1515 Hình P Hình ảnh so sánh trình tự nucleotide gen HA của H5HT đã được tối ưu biểu thuốc lá so với trình tự gốc P11 Phụ lục Phân tích trình tự đa chuỗi để chọn lọc trình tự protein HA đại diện cho chủng thuộc subclade 1.1 (ký hiệu: HACOBRA clade 1.1) HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 10 20 30 40 50 60 70 80 90 100 | | | | | | | | | | | | | | | | | | | | MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVRPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN .K K K K N K K D .K K .K .L K .K .K .K .K .K .K .K .K .K .K .K .K .K .K K K K K K K K K K K K .K T K V K K K .V .K - .T E I T D N N T E E R .T - R .T .I .K A I .I .K A I .I .K R .A I 100 100 100 100 100 100 100 100 100 100 1 84 84 100 84 84 84 84 84 84 84 84 84 84 84 84 84 84 84 84 84 84 100 100 100 100 100 100 100 100 100 100 100 84 100 100 100 100 84 100 100 100 100 84 84 1 1 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 110 120 130 140 150 160 170 180 190 200 | | | | | | | | | | | | | | | | | | | | PVNDLCYPGVFNDYEELKHLLSRINHFEKIQIIPKSSWPSHEASLGVSAACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDA D S .P I I I .D 200 200 200 200 200 200 200 200 200 200 P12 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 .D I Q D D R D D - .N .D - I H.N D D D D R N I I G .D D .R .D .G S I - .N D D D N D S D .N D .V D .V D .V D D .V D D D D D D .R D D D D D S S S - D D D .V 1 184 184 200 184 184 184 184 184 184 184 184 184 184 184 184 183 184 184 179 184 184 200 200 200 200 200 200 200 200 200 200 200 184 200 200 200 200 184 200 200 200 200 184 184 1 1 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY 210 220 230 240 250 260 270 280 290 300 | | | | | | | | | | | | | | | | | | | | AEQTKLYQNPTTYISVGTSTLNQRLTPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA I PI I.H R .S- T .- Y .S - .- .T- - - V L 300 300 300 300 300 300 300 300 300 300 1 284 284 300 283 284 284 284 284 284 284 283 282 284 284 284 283 283 284 275 P13 A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 V T R S .R LR .Q T K V K I - A A I - I .S S - 284 284 300 300 300 300 300 300 300 300 300 300 300 284 300 300 300 300 284 300 300 300 300 284 284 1 1 300 300 300 300 300 300 300 299 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ 310 320 330 340 350 360 370 380 390 400 | | | | | | | | | | | | | | | | | | | | INSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQREGRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNS N - - -L - - R - PRE. R.KR G M - 400 400 400 400 400 400 400 400 400 400 1 381 384 400 383 384 384 384 384 384 384 383 382 384 384 384 383 383 384 367 384 384 400 400 400 400 400 400 400 400 400 400 400 384 400 400 400 400 384 400 400 P14 A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 - - - - - T T T T T T T T D T T T - T T T I T 400 400 384 384 1 1 400 400 400 400 400 400 400 399 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 400 HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 410 420 430 440 450 460 470 480 490 500 | | | | | | | | | | | | | | | | | | | | IIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRN S - S S S S -T .- - - - S S S S S - HW.I .D K .S S 500 500 500 500 500 500 500 500 500 500 101 100 481 483 500 483 484 484 484 484 484 484 483 482 484 484 484 483 483 484 462 484 484 500 500 500 500 500 500 500 500 500 500 500 484 500 500 500 500 484 500 500 500 499 484 484 101 101 101 101 101 500 500 500 500 500 500 500 499 500 500 500 500 P15 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 V N .S HACOBRA-clade1.1 A/duck/Vietnam/An_Giang_680B/2 A/Duck/Vietnam/Tien_Giang_409/ A/Muscovy_Duck/Vietnam/Ben_Tre A/Duck/Vietnam/Ca_Mau498A/2006 A/Duck/Vietnam/Ca_Mau498/2006_ A/Muscovy_duck/Ca_Mau/1181/200 A/chicken/Ca_Mau/1180/2006_JX4 A/Muscovy_duck/Ca_Mau/1185/200 A/Muscovy_duck/Ca_Mau/1159/200 A/duck/Viet_Nam/CM-1/2006_FJ81 A/duck/Viet_Nam/CM-2/2006_FJ81 A/duck/Vietnam/NCVD-2/2007_CY0 A/Muscovy_duck/Vietnam/NCVD-4/ A/Muscovy_duck/Vietnam/4/2007_ A/chicken/Vietnam/NCVD-3/2007_ A/duck/Vietnam/NCVD6/2007_CY03 A/duck/Vietnam/NCVD-8/2007_CY0 A/duck/Vietnam/NCVD9/2007_CY03 A/chicken/Vietnam/NCVD-10/2007 A/duck/Vietnam/NCVD-12/2007_CY A/duck/Vietnam/NCVD-13/2007_CY A/duck/Vietnam/NCVD-18/2007_CY A/chicken/Vietnam/NCVD-15/2007 A/duck/Vietnam/NCVD16/2007_CY0 A/duck/Vietnam/NCVD-17/2007_CY A/duck/Vietnam/NCVD19/2007_CY0 A/chicken/Vietnam/NCVD-21/2007 A/Muscovy_duck/Vietnam/NCVD-23 A/duck/Vietnam/NCVD-25/2007_CY A/duck/Vietnam/NCVD-26/2007_CY A/duck/Vietnam/NCVD-30/2007_CY A/Muscovy_duck/Vietnam/NCVD29/ A/chicken/Vietnam/29/2007_CY02 A/duck/Hau_Giang/07-12/2007_GU A/duck/Bac_Lieu/07-09/2007_GU0 A/chicken/Bac_Lieu/07-10/2007_ A/duck/Vietnam/6/2007_CY029591 A/duck/Vietnam/5/2007_CY029583 A/duck/Vietnam/8/2007_CY029607 A/duck/Soc_Trang/07-25/2007_GU A/duck/Bac_Lieu/07-06/2007_GU1 A/duck/Vietnam/7/2007_CY029599 A/duck/Vietnam/2/2007_CY029567 A/Muscovy_duck/Vietnam/NCVD-5/ A/duck/Vietnam/18/2007_CY02962 A/Muscovy_duck/Vietnam/33/2007 A/duck/Vietnam/34/2007_CY02964 A/Muscovy_duck/Ca_Mau/07-04/20 A/chicken/Vietnam/NCVD-24/2007 A/duck/Vietnam_Hau_Giang/NCVDA/duck/Vietnam_BacLiu/NCVD-10/ A/chicken/Bac_Lieu/1214/2007_J A/duck/Bac_Lieu/1213/2007_JX42 A/Muscovy_duck/Vietnam/NCVD-11 A/duck/Vietnam/NCVD-16/2007_CY A/duck/Viet_Nam/12A-LA/2008_FJ A/duck/Viet_Nam/15A-TV/2008_FJ A/duck/Viet_Nam/17A-TV/2008_FJ A/duck/Viet_Nam/2B-TV/2008_FJ8 A/duck/Viet_Nam/38ST/2008_FJ81 A/duck/Vietnam/NCVD-118/2008_E A/duck/Vietnam/NCVD-103/2008_E A/duck/Vietnam/NCVD-010/2008_E A/duck/Vietnam/NCVD-101/2008_E A/chicken/Vietnam/NCVD-117/200 A/duck/Vietnam/NCVD-011/2008_E A/duck/Vietnam/NCVD-365/2009_E A/duck/Vietnam/NCVD-364/2009_E A/duck/Vietnam/NCVD-370/2009_E A/chicken/Vietnam/NCVD-380/200 A/chicken/Vietnam/NCVD-369/200 A/chicken/Vietnam/NCVD-359/200 A/duck/Vietnam/NCVD-371/2009_E A/duck/Vietnam/NCVD-366/2009_E A/duck/Vietnam/NCVD-368/2009_E A/duck/Vietnam/NCVD-362/2009_E A/duck/Vietnam/NCVD-361/2009_E A/ostrich/Vietnam/NCVD-373/200 A/duck/Vietnam/NCVD-372/2009_E A/duck/Vietnam/NCVD-363/2009_E A/chicken/Vietnam/NCVD-387/200 A/duck/Vietnam/NCVD-367/2009_E A/duck/Vietnam/NCVD-360/2009_E A/Vietnam/26-1/2009_JF710592 510 520 530 540 550 560 | | | | | | | | | | | | | GTYDYPQYSEEARLKREEISGVKLESIGVYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIX I V I I I I I I I G I K .Q I - I - - I I I - I I I I I I I I I I I I I I I I I I I I I I G I - I I I I I I I S I G I G I - I I I I I I R I I - - - - - F F K I F - 569 568 568 568 568 568 568 568 568 568 160 159 548 551 559 551 552 552 552 552 552 552 551 550 552 552 552 551 551 552 530 552 552 568 568 568 568 565 566 566 568 568 566 566 552 568 568 563 568 552 568 568 568 567 552 552 160 160 160 160 160 568 568 568 568 568 568 568 567 568 568 568 568 568 568 568 568 568 568 568 568 568 568 568 168 500 500 500 500 500 500 500 500 500 500 500 100 500 500 500 500 500 500 P16 A/chicken/Vietnam/NCVD-424/200 A/chicken/Vietnam/NCVD-425/200 A/muscovy_duck/Vietnam/NCVD-42 A/chicken/Vietnam/NCVD-775/201 A/chicken/Vietnam/NCVD-780/201 A/chicken/Vietnam/NCVD-883/201 - 568 568 568 568 568 568 Hình P Hình ảnh so sánh trình tự amino acid của gen HA của các chủng virus A/H5N1 thuộc clade 1.1 gia cầm Việt Nam để thiết kế trình tự gen HACOBRA clade 1.1 HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized NcoI 10 20 30 40 50 60 70 | | | | | | | | | | | | | | ccatggctGAAAAAATCGTGTTATTGTTCGCAATTGTTTCATTAGTTAAGAGTGACCAAATCTGCATTGG 70 M A E K I V L L F A I V S L V K S D Q I C I G 80 90 100 110 120 130 140 | | | | | | | | | | | | | | TTATCACGCGAATAATAGCACTGAGCAAGTAGATACCATTATGGAAAAGAATGTGACTGTCACACATGCG 140 Y H A N N S T E Q V D T I M E K N V T V T H A 150 160 170 180 190 200 210 | | | | | | | | | | | | | | CAAGATATCCTTGAGAAGACCCATAATGGAAAACTTTGCGATCTTGATGGAGTGCGTCCTCTGATTTTAA 210 Q D I L E K T H N G K L C D L D G V R P L I L 220 230 240 250 260 270 280 | | | | | | | | | | | | | | GAGACTGCTCAGTGGCAGGCTGGCTCCTGGGAAACCCGATGTGTGACGAGTTTATAAATGTTCCTGAATG 280 R D C S V A G W L L G N P M C D E F I N V P E W 290 300 310 320 330 340 350 | | | | | | | | | | | | | | GTCCTATATAGTTGAAAAGGCAAATCCAGTCAATGACTTGTGTTATCCAGGTGTCTTTAATGACTACGAA 350 S Y I V E K A N P V N D L C Y P G V F N D Y E 360 370 380 390 400 410 420 | | | | | | | | | | | | | | GAGTTAAAACACCTTCTATCGAGAATTAATCACTTTGAAAAGATCCAGATAATACCTAAGAGCAGTTGGC 420 E L K H L L S R I N H F E K I Q I I P K S S W 430 440 450 460 470 480 490 | | | | | | | | | | | | | | CATCACATGAGGCCTCACTAGGGGTGTCTGCTGCTTGTCCTTACCAAGGACAGTCTTCTTTCTTTAGGAA 490 P S H E A S L G V S A A C P Y Q G Q S S F F R N 500 510 520 530 540 550 560 | | | | | | | | | | | | | | CGTAGTATGGCTTATCAAGAAGAACTCTACCTATCCAACAATTAAAAGGTCGTATAACAATACAAATCAG 560 V V W L I K K N S T Y P T I K R S Y N N T N Q 570 580 590 600 610 620 630 | | | | | | | | | | | | | | GAAGATCTACTCGTAATGTGGGGAATCCATCATCCGAACGATGCAGCCGAACAAACAAAGTTGTATCAAA 630 E D L L V M W G I H H P N D A A E Q T K L Y Q 640 650 660 670 680 690 700 | | | | | | | | | | | | | | ATCCAACCACATACATAAGCGTGGGAACTTCTACACTGAATCAACGCTTGACACCTCGAATTGCTACTCG 700 N P T T Y I S V G T S T L N Q R L T P R I A T R 710 720 730 740 750 760 770 | | | | | | | | | | | | | | TAGTAAAGTTAATGGGCAGTCCGGAAGAATGGAATTCTTCTGGACCATTCTTAAACCAAACGACGCTATT 770 S K V N G Q S G R M E F F W T I L K P N D A I 780 790 800 810 820 830 840 | | | | | | | | | | | | | | AATTTTGAGTCCAACGGAAATTTCATTGCTCCCGAATATGCCTATAAGATTGTAAAAAAAGGTGACTCCA 840 N F E S N G N F I A P E Y A Y K I V K K G D S 850 860 870 880 890 900 910 | | | | | | | | | | | | | | CGATTATGAAATCGGAACTCGAATACGGGAACTGTAACACGAAGTGCCAGACACCGATGGGCGCCATTAA 910 T I M K S E L E Y G N C N T K C Q T P M G A I N 920 930 940 950 960 970 980 | | | | | | | | | | | | | | TTCAAGTATGCCTTTTCACAACATTCATCCACTAACTATAGGTGAGTGCCCTAAGTACGTTAAGAGCAAT 980 S S M P F H N I H P L T I G E C P K Y V K S N 990 1000 1010 1020 1030 1040 1050 | | | | | | | | | | | | | | CGACTCGTGCTTGCGACTGGCCTTAGAAACTCTCCTCAGCGTGAGGGTCGCGGACTGTTCGGAGCAATAG 1050 R L V L A T G L R N S P Q R E G R G L F G A I 1060 1070 1080 1090 1100 1110 1120 | | | | | | | | | | | | | | CTGGGTTTATCGAGGGTGGCTGGCAGGGTATGGTTGATGGTTGGTACGGATATCACCATAGTAATGAGCA 1120 A G F I E G G W Q G M V D G W Y G Y H H S N E Q 1130 1140 1150 1160 1170 1180 1190 | | | | | | | | | | | | | | GGGATCTGGGTATGCTGCCGACAAAGAATCCACACAAAAAGCTATCGACGGTGTGACTAATAAGGTTAAT 1190 G S G Y A A D K E S T Q K A I D G V T N K V N 1200 1210 1220 1230 1240 1250 1260 | | | | | | | | | | | | | | TCAATCATTGATAAAATGAACACACAGTTTGAAGCTGTTGGGCGTGAATTTAACAACTTGGAGAGGCGGA 1260 S I I D K M N T Q F E A V G R E F N N L E R R 1270 1280 1290 1300 1310 1320 1330 | | | | | | | | | | | | | | TTGAAAATTTAAATAAAAAGATGGAAGATGGTTTCTTGGATGTCTGGACGTACAATGCAGAACTCTTAGT 1330 I E N L N K K M E D G F L D V W T Y N A E L L V 1340 1350 1360 1370 1380 1390 1400 | | | | | | | | | | | | | | TCTTATGGAGAATGAACGAACCCTGGATTTCCATGATTCAAATGTGAAGAACTTGTACGATAAGGTCCGG 1400 L M E N E R T L D F H D S N V K N L Y D K V R 1410 1420 1430 1440 1450 1460 1470 | | | | | | | | | | | | | | CTTCAGCTTAGAGATAACGCTAAAGAACTTGGAAACGGCTGCTTTGAGTTTTATCATAAGTGTGATAATG 1470 P17 L HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized HACOBRA1 codon optimized Q L R D N A K E L G N G C F E F Y H K C D N 1480 1490 1500 1510 1520 1530 1540 | | | | | | | | | | | | | | AGTGTATGGAGTCGGTTAGGAATGGTACTTATGATTATCCCCAATACTCTGAGGAGGCTAGACTAAAAAG 1540 E C M E S V R N G T Y D Y P Q Y S E E A R L K R 1550 1560 1570 1580 1590 1600 1610 | | | | | | | | | | | | | | GGAGGAAATATCAGGTGTTAAGTTGGAATCTATAGGCGTCTACCAAATTTTGAGCATTTATTCTACGGTT 1610 E E I S G V K L E S I G V Y Q I L S I Y S T V 1620 1630 1640 1650 1660 1670 1680 | | | | | | | | | | | | | | GCCTCTAGCTTGGCTTTGGCAATTATGGTAGCAGGGTTAAGTCTCTGGATGTGTAGTAATGGGTCACTCC 1680 A S S L A L A I M V A G L S L W M C S N G S L 1690 1700 NotI | | | | | AATGTCGGATCTGCATCTAGgcggccgc 1708 Q C R I C I * A A Hình P Trình tự đởi gen HACOBRA1 đã tối ưu mã biểu thuốc lá N benthamiana 10 HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized 20 30 40 50 60 70 | | | | | | | | | | | | | | ccatggctGAGAAGATAGTTCTCCTCTTCGCTACCATTAGCCTGGTTAAGTCTGACCACATCTGCATAGG 70 M A E K I V L L F A T I S L V K S D H I C I G 80 90 100 110 120 130 140 | | | | | | | | | | | | | | ATATCACGCTAATAATAGCACTGAGCAAGTAGATACAATAATGGAAAAAAATGTAACAGTAACACATGCA 140 Y H A N N S T E Q V D T I M E K N V T V T H A 150 160 170 180 190 200 210 | | | | | | | | | | | | | | CAGGATATTCTTGAGAAAACTCATAATGGGAAGTTATGCGACCTTAATGGTGTTAAACCTCTGATCTTAA 210 Q D I L E K T H N G K L C D L N G V K P L I L 220 230 240 250 260 270 280 | | | | | | | | | | | | | | AAGACTGCTCAGTGGCAGGGTGGCTATTAGGTAATCCATTGTGTGATGAATTTACTAACGTGCCCGAATG 280 K D C S V A G W L L G N P L C D E F T N V P E W 290 300 310 320 330 340 350 | | | | | | | | | | | | | | GTCATATATTGTTGAGAAGGCCAATCCAGCAAACGATCTTTGCTATCCCGGAAATTTTAACGATTATGAG 350 S Y I V E K A N P A N D L C Y P G N F N D Y E 360 370 380 390 400 410 420 | | | | | | | | | | | | | | GAATTGAAACACTTGCTTTCAAGAATTAATCACTTTGAAAAAATTCAGATCATTCCTAAGGATTCTTGGT 420 E L K H L L S R I N H F E K I Q I I P K D S W 430 440 450 460 470 480 490 | | | | | | | | | | | | | | CTGACCATGAGGCTAGTTTGGGTGTGTCCGCCGCTTGCTCTTACCAGGGTAATAGCTCATTCTTCAGGAA 490 S D H E A S L G V S A A C S Y Q G N S S F F R N 500 510 520 530 540 550 560 | | | | | | | | | | | | | | TGTTGTTTGGTTGATTAAGAAGGACAATGCGTACCCTACCATTAAAAAAGGCTACAATAACACTAATAGG 560 V V W L I K K D N A Y P T I K K G Y N N T N R 570 580 590 600 610 620 630 | | | | | | | | | | | | | | GAGGATCTGTTGATACTATGGGGTATTCATCACCCAAATGATGAGGCTGAACAAACAAGGCTATACCAAA 630 E D L L I L W G I H H P N D E A E Q T R L Y Q 640 650 660 670 680 690 700 | | | | | | | | | | | | | | ACCCAACTACTTATATCTCCATAGGCACCAGTACGCTTAATCAACGCCTCGTGCCTAAAATAGCAACCAG 700 N P T T Y I S I G T S T L N Q R L V P K I A T R 710 720 730 740 750 760 770 | | | | | | | | | | | | | | ATCAAAGATTAATGGCCAGTCAGGACGGATCGACTTCTTTTGGACAATTCTAAAGCCTAACGACGCAATC 770 S K I N G Q S G R I D F F W T I L K P N D A I 780 790 800 810 820 830 840 | | | | | | | | | | | | | | CACTTTGAGAGTAATGGCAATTTCATCGCTCCCGAATACGCGTATAAGATTGTAAAGAAAGGTGATTCCA 840 H F E S N G N F I A P E Y A Y K I V K K G D S 850 860 870 880 890 900 910 | | | | | | | | | | | | | | CTATCATGAGATCAGAGGTTGAGTATGGAAACTGTAACACGCGCTGTCAGACACCAATAGGCGCCATAAA 910 T I M R S E V E Y G N C N T R C Q T P I G A I N 920 930 940 950 960 970 980 | | | | | | | | | | | | | | TAGTTCTATGCCGTTCCATAATATCCATCCTCTTACCATAGGGGAGTGTCCGAAGTACGTGAAGTCTAAT 980 S S M P F H N I H P L T I G E C P K Y V K S N 990 1000 1010 1020 1030 1040 1050 | | | | | | | | | | | | | | AAGTTGGTCCTGGCTACGGGACTTCGAAACTCCCCTCAAAGAGAGCGTAGGGGATTATTTGGCGCAATCG 1050 K L V L A T G L R N S P Q R E R R G L F G A I 1060 1070 1080 1090 1100 1110 1120 | | | | | | | | | | | | | | CTGGTTTCATCGAAGGTGGTTGGCAAGGAATGGTCGATGGATGGTATGGGTACCATCATAGTAATGAACA 1120 A G F I E G G W Q G M V D G W Y G Y H H S N E Q 1130 1140 1150 1160 1170 1180 1190 | | | | | | | | | | | | | | GGGTTCGGGATACGCTGCCGACAAAGAAAGCACTCAAAAAGCTATTGATGGAGTAACAAACAAAGTTAAC 1190 G S G Y A A D K E S T Q K A I D G V T N K V N 1200 1210 1220 1230 1240 1250 1260 | | | | | | | | | | | | | | AGTATAATTGATAAAATGAACACACAATTTGAAGCTGTTGGGCGTGAATTTAATAACCTTGAGCGAAGAA 1260 S I I D K M N T Q F E A V G R E F N N L E R R 1270 1280 1290 1300 1310 1320 1330 P18 HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized HACOBRA2 codon optimized | | | | | | | | | | | | | | TTGAGAACCTGAATAAGAAGATGGAGGATGGATTTCTCGATGTTTGGACTTACAATGCCGAGCTGTTGGT 1330 I E N L N K K M E D G F L D V W T Y N A E L L V 1340 1350 1360 1370 1380 1390 1400 | | | | | | | | | | | | | | CCTGATGGAAAACGAAAGGACATTGGATTTCCATGATTCTAATGTCAAGAATTTATATGATAAAGTTAGA 1400 L M E N E R T L D F H D S N V K N L Y D K V R 1410 1420 1430 1440 1450 1460 1470 | | | | | | | | | | | | | | CTTCAGCTCAAGGATAATGCAAAGGAGTTGGGGAACGGCTGCTTTGAATTTTATCACAAGTGTAATAACG 1470 L Q L K D N A K E L G N G C F E F Y H K C N N 1480 1490 1500 1510 1520 1530 1540 | | | | | | | | | | | | | | AATGTATGGAGTCTGTGCGTAATGGTACCTATGATTACCCACAGTATTCGGAAGAAGCACGGCTAAAAAG 1540 E C M E S V R N G T Y D Y P Q Y S E E A R L K R 1550 1560 1570 1580 1590 1600 1610 | | | | | | | | | | | | | | GGAAGAGATTTCTGGTGTCAAGCTCGAAAGCATTGGTATCTACCAGATACTTTCTATTTATTCAACGGTT 1610 E E I S G V K L E S I G I Y Q I L S I Y S T V 1620 1630 1640 1650 1660 1670 1680 | | | | | | | | | | | | | | GCTTCGTCCCTCGTGCTTGCTATTATGATGGCAGGATTGTCATTGTGGATGTGTTCCAATGGTAGTCTTC 1680 A S S L V L A I M M A G L S L W M C S N G S L 1690 1700 | | | | | AATGTAGAATTTGTATTTAGgcggccgc 1708 Q C R I C I * A A Hình P Trình tự đổi gen HACOBRA2 đã tối ưu mã biểu thuốc lá N benthamiana HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 HACOBRA1 HACOBRA_clade1.1 HACOBRA_clade1.1.1 HACOBRA_clade1.1.2 10 20 30 40 50 60 70 | | | | | | | | | | | | | | MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVRPLILRD 70 70 V N I 70 70 80 90 100 110 120 130 140 | | | | | | | | | | | | | | CSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGVFNDYEELKHLLSRINHFEKIQIIPKSSWPS 140 140 140 140 150 160 170 180 190 200 210 | | | | | | | | | | | | | | HEASLGVSAACPYQGQSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDAAEQTKLYQNP 210 .K 210 .K G 210 210 220 230 240 250 260 270 280 | | | | | | | | | | | | | | TTYISVGTSTLNQRLTPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI 280 280 280 280 290 300 310 320 330 340 350 | | | | | | | | | | | | | | MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQREGRRKKRGLFG 350 350 350 R T .E 350 360 370 380 390 400 410 420 | | | | | | | | | | | | | | AIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLE 420 420 420 420 430 440 450 460 470 480 490 | | | | | | | | | | | | | | RRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKC 490 490 490 490 500 510 520 530 540 550 560 | | | | | | | | | | | | | | DNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGVYQILSIYSTVASSLALAIMVAGLSLWMCSNG 560 560 560 560 | SLQCRICI- 568 X 569 X 569 - 568 Hình P Hình ảnh so sánh để thiết kế nên trình tự protein HACOBRA1 đại diện cho các chủng virus thuộc subclade 1.1, 1.1.1 và 1.1.2 P19 Bảng P Hệ số tương đồng các trình tự protein HA tự nhiên và nhân tạo P20 Phụ lục Phát hiện IgY đặc hiệu H5HT dịch chiết thô Western blot Các phản ứng miễn dịch đặc hiệu được tạo H5HT trimer oligomer TP chiết xuất thô thực vật gà Liên kết đặc hiệu H5 của các kháng thể từ hỗn hợp của 12 huyết gà chống lại các chất chiết xuất từ thực vật tương ứng được phát Western blot Để phát kháng thể IgY đặc hiệu với các dạng chiết xuất thực vật khác và vaccine NAVET-VIFLUVAC thương mại (http://www.navetco.com.vn/vi/product/navet-vifluvac), 700 ng H5TG tinh khiết từ SEC protein được tách làn của gel SDS-PAGE biến tính (10% polyacrylamide) và chuyển sang màng nitrocellulose Màng được chặn sữa bột không béo 5% (w/v) được hòa tan dung dịch đệm PBS giờ Sau đó, làn của màng được phân lập cách cắt và ủ nhiệt độ phòng 90 phút với sự pha loãng 1:20 của hỗn hợp huyết từ mười hai gà của nhóm sau tiêm lần thứ hai Các màng này được ủ với độ pha loãng 1: 5000 của kháng thể thứ hai Anti-chicken IgY (whole molecule) Alkaline phosphatase giờ nhiệt độ phòng Các tín hiệu cụ thể được phát cách ủ màng với 3,3– diaminobenzidine (DAB, Thermo Scientific Pierce) hòa tan 0,05 M Tris – HCl và 0,04% hydrogen peroxide 10 phút bóng tối