Nghiên cứu biểu hiện kháng nguyên (m, GP5, GP5ectoM) của virus gây hội chứng rối loạn sinh sản và hô hấp ở lợn trong cây thuốc lá bằng phương pháp thẩm lọc nhờ agrobacterium

i VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM VIỆN CÔNG NGHỆ SINH HỌC NGUYỄN THỊ MINH HẰNG NGHIÊN CỨU BIỂU HIỆN KHÁNG NGUYÊN (M, GP5, GP5ectoM) CỦA VIRUS GÂY HỘI CHỨNG RỐI LOẠN SINH SẢN VÀ HÔ HẤP Ở LỢN TRONG CÂY THUỐC LÁ BẰNG PHƯƠNG PHÁP THẨM LỌC NHỜ AGROBACTERIUM LUẬN ÁN TIẾN SĨ SINH HỌC HÀ NỘI – 2018 i ii VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM VIỆN CÔNG NGHỆ SINH HỌC NGUYỄN THỊ MINH HẰNG NGHIÊN CỨU BIỂU HIỆN KHÁNG NGUYÊN (M, GP5, GP5ectoM) CỦA VIRUS GÂY HỘI CHỨNG RỐI LOẠN SINH SẢN VÀ HÔ HẤP Ở LỢN TRONG CÂY THUỐC LÁ BẰNG PHƯƠNG PHÁP THẨM LỌC NHỜ AGROBACTERIUM Chuyên ngành: Hoá sinh học Mã số: 94 20 116 LUẬN ÁN TIẾN SĨ SINH HỌC Người hướng dẫn khoa học: TS Nguyễn Trung Nam Viện Công nghệ sinh học PGS TS Chu Hồng Hà Viện Cơng nghệ sinh học HÀ NỘI – 2018 ii iii LỜI CẢM ƠN Luận án thực Phòng thí nghiệm Trọng điểm Cơng nghệ gen, Phòng Cơng nghệ tế bào thực vật, Phòng thử nghiệm sinh học, Viện Cơng nghệ sinh học, Viện Hàn lâm Khoa học & Công nghệ Việt Nam Trung tâm Chẩn đoán Thú y Trung ương Với hỗ trợ kinh phí từ đề tài thuộc nhiệm vụ nghiên cứu thường xun Phòng thí nghiệm Trọng điểm Công nghệ gen, Viện Công nghệ sinh học Trong suốt q trình học tập, nghiên cứu hồn thành luận án, nhận giúp đỡ tập thể cán hướng dẫn, thầy cô giáo, nhà khoa học, anh chị em đồng nghiệp q quan, phòng ban Tơi xin chân thành cảm ơn Ban Lãnh đạo, Bộ phận đào tạo sau đại học, phòng chức Viện Cơng nghệ sinh học tạo điều kiện cho học tập hồn thành luận án Tơi xin bày tỏ lòng biết ơn sâu sắc tới hai thầy, PGS.TS Chu Hoàng Hà TS Nguyễn Trung Nam định hướng nghiên cứu, tận tình hướng dẫn, tạo điều kiện thuận lợi, giúp đỡ suốt thời gian thực hồn thành luận án Tơi xin chân thành cảm ơn GS.TS Lê Trần Bình, PGS.TS Phạm Bích Ngọc, PGS.TS Đinh Duy Kháng, PGS.TS Tô Long Thành, TS Nguyễn Tường Vân, ThS Hồ Thị Thương tập thể cán Phòng Cơng nghệ tế bào thực vật, Phòng Thí nghiệm trọng điểm Công nghệ gen giúp đỡ, chia sẻ kiến thức quý báu tạo điều kiện tốt cho tơi suốt q trình thực luận án Tôi xin chân thành cảm ơn Ban Giám hiệu Trường Đại học Lâm nghiệp, Ban Lãnh đạo, tồn thể cán Viện Cơng nghệ sinh học Lâm nghiệp tạo điều kiện tốt yên tâm học tập thực luận án Cuối cùng, xin cảm ơn gia đình, bạn bè đồng nghiệp ln động viên, giúp đỡ tơi suốt q trình học tập thực luận án Hà Nội, ngày tháng năm 2018 Nghiên cứu sinh Nguyễn Thị Minh Hằng iii i LỜI CAM ĐOAN Tôi xin cam đoan: Đây cơng trình nghiên cứu tơi hướng dẫn khoa học PGS.TS Chu Hoàng Hà, TS Nguyễn Trung Nam số kết cộng tác với cộng khác Các số liệu kết trình bày luận án trung thực, phần cơng bố tạp chí khoa học chuyên ngành với đồng ý cho phép đồng tác giả Phần kết lại luận án chưa công bố cơng trình khác Hà Nội, ngày tháng năm 2018 Tác giả Nguyễn Thị Minh Hằng i ii MỤC LỤC LỜI CẢM ƠN LỜI CAM ĐOAN DANH MỤC CÁC CHỮ VIẾT TẮT DANH MỤC BẢNG DANH MỤC HÌNH MỞ ĐẦU Chương TỔNG QUAN TÀI LIỆU Chương 43 VẬT LIỆU VÀ PHƯƠNG PHÁP NGHIÊN CỨU Chương 61 KẾT QUẢ NGHIÊN CỨU Chương 108 BÀN LUẬN KẾT QUẢ NGHIÊN CỨU 108 KẾT LUẬN VÀ KIẾN NGHỊ 130 NHỮNG CƠNG TRÌNH CƠNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN 132 TÓM TẮT LUẬN ÁN BẰNG TIẾNG ANH 133 TÀI LIỆU THAM KHẢO 140 DANH MỤC CÁC TỪ VIẾT TẮT AS aa 2b CMV Acetosyringone Amino acid (axit amin) Cucumber mosaic virus (CMV) 2b protein (Protein 2b virus ii iii bp BSA CaMV Cs DNA ER ELISA ELP et al Fc GFP GP5 GP5ecto HC-Pro HP-PRRSV khảm dưa chuột) Base pair (Cặp base) Bovine serum albumin Cauliflower mosaic virus (Virus khảm súp lơ) Cộng Deoxyribonucleic acid Endoplasmic reticulum (Lưới nội chất) Enzyme linked immunosorbent assay Elastin-like polypeptide And co-workers (và cộng sự) Fragment crystallizable Green fluorescent protein (Protein huỳnh quang xanh) Glycoprotein Ectodomain of glycoprotein (Vùng ngoại bào protein GP5) Helper component protease (Protein hỗ trợ) Highly pathogenic Porcine reproductive and respiratory syndrome virus (Virus gây hội chứng rối loạn sinh sản hô hấp thể độc lực HRP IPMA cao) Horseradish peroxidase Immunoperoxidase monolayer assay (Xét nghiệm miễn dịch đơn lớp IgG kb kDa LB cộng hợp enzyme peroxidase) Immunoglobulin G Kilobase Kilodalton Luria-Bertani (Môi trường nuôi khuẩn Luria-Bertani) iii iv LV M MES mg MLV mITC Lelystad virus Matrix/ Membrance protein 2-(N- Morpholino)ethanesulfonic acid Milligram Modified-live virus (Virus sống làm biến đổi = virus nhược độc) Membrane-based Inverse transition cycling (Chu trình chuyển pha nptII nghịch đảo qua màng) Gene mã hoá Neomycin phosphotransferase (Gen kháng Nos nsp OD kanamycin) Nopaline synthase terminator Non-structural protein Optical density (Mật độ quang) ORF Open reading frame (Khung đọc mở) PBS Phosphate-buffered saline PCR Polymerase chain reaction (Phản ứng chuỗi trùng hợp) Fc Stabilizing domain - "Fragment of crystallizable chain of IgG" PRRSV (Vùng định kháng thể IgG) Porcine Reproductive and Respiratory Syndrome virus (Virus gây RNA hội chứng rối loạn sinh sản hô hấp lợn) Ribonucleic acid ScFv Single chain variable fragment SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis TSP Total soluble protein (Protein tổng hợp) 35S Ter 35S Terminator (Trình tự kết thúc phiên mã) VLP Virus-like particle WT Wild type (Cây không chuyển gen) DANH MỤC BẢNG Bảng 1.1 Tình hình dịch PRRS giai đoạn 2015 – 2016 ……………… Bảng 2.1 Trình tự mồi sử dụng nghiên cứu gen gp5opt, gp5ecto- 44 iv v m m ……………………………………………………… Bảng 2.2 Thành phần phản ứng ghép nối gen m, gp5opt với vector pRTRA 35S-Histag-Cmyc-100xELP ……………………… Bảng 2.3 Thành phần phản ứng ghép nối đoạn gp5ecto với gen m với vector pRTRA 35S-Histag-Cmyc-100xEL ……………… Bảng 2.4 47 47 Thành phần phản ứng ghép nối cassette gen với vector pCB301 ……………………………………………………… 48 Bảng 3.1 So sánh mức độ biểu tạm thời gen mã hoá kháng nguyên tái tổ hợp (M-ELP, GP5-ELP, GP5ectoM-ELP) thuốc N benthamiana ………………………………… 82 Bảng 3.2 Hiệu suất thu hồi mức độ tinh protein M-ELP, GP5-ELP, GP5ectoM-ELP …………………………… 91 DANH MỤC CÁC HÌNH v Hình 1.1 Hình 1.2 Hình 1.3 Hình 1.4 Hình 1.5 Hình 1.6 Đặc điểm hình thái virus PRRS ………………………… Ảnh hiển vi điện tử hạt Arterivirus ………………… Mơ hình cấu trúc hạt virus PRRS khung đọc mở vi hệ gen PRRS ………………………………………………… Đặc điểm GP5 ………………………………… Cấu trúc Arterivirus ………………………………………… Sơ đồ mô tả biểu gen tạm thời sử dụng vector 9 Hình 1.7 Hình 1.8 Hình 2.1 pEAQ phương pháp thẩm lọc A tumefaciens ………… 34 Thẩm lọc nhờ A tumefaciens vào N benthamiana ……… 34 Sơ đồ chu kỳ chuyển pha nghịch đảo qua màng ……… 41 Chuyển gen tạm thời vào thuốc nhờ A tumefaciens Hình 2.2 Hình 2.3 Hình 3.1 phương pháp hút chân khơng ………………………… 50 Sơ đồ thí nghiệm gây miễn dịch chuột ………………… 55 Sơ đồ thí nghiệm gây miễn dịch lợn …………………… 56 Thiết kế vector tách dòng pRTRA 35S-m-Histag-Cmyc- Hình 3.2 100xELP …………………………………………………… 61 Sơ đồ cấu trúc cassette gen m mã hố protein M dung hợp 62 Hình 3.3 ELP Kết thiết kế vector chuyển gen pCB301 35S-m-HistagCmyc-100xELP ……………………………………………… Phân tích sản phẩm Colony-PCR kiểm tra A tumefaciens 63 Hình 3.4 65 Hình 3.5 mang vector pCB301 35S-m-Histag-Cmyc-100xELP ……… Thiết kế vector tách dòng pRTRA 35S-gp5opt-Histag-Cmyc- 66 Hình 3.6 100xELP …………………………… Sơ đồ cấu trúc cassette gen gp5opt mã hoá protein GP5 dung hợp ELP …………………………………………………… Kết thiết kế vector chuyển gen pCB301 35S-gp5opt- 67 Hình 3.7 Hình 3.8 Histag-Cmyc-100xELP ……………………………………… 68 Sản phẩm Colony-PCR kiểm tra A.tumefaciens mang vector Hình 3.9 pCB301 35S-gp5opt-Histag-Cmyc-100xELP ……………… 69 Thiết kế vector nhân dòng pRTRA 35S-gp5ecto-m-Histag- 12 13 14 Cmyc-100xELP ……………… ………… Hình 3.10 Sơ đồ cấu trúc cassette gen gp5ecto-m mã hoá protein 70 GP5ectoM dung hợp ELP vector pRTRA ……………… Hình 3.11 Kết phân tích kiểm tra vector chuyển gen mang gen 72 gp5ecto-m mã hóa kháng nguyên GP5ecto-M dung hợp ELP… ……………… ……………… ……………… …… Hình 3.12 Sản phẩm Colony-PCR kiểm tra A.tumefaciens mang vector 72 chuyển gen pCB301 35S-gp5ecto-m-Histag-CmycHình 3.13 100xELP… ……………… ……………… ……………… 73 Ảnh hưởng vector hỗ trợ pBI-35S-2bCMV pIBT-35SHC-Pro PVY đến mức độ biểu kháng nguyên tái tổ hợp thông qua kết lai Western sử dụng 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HỌC VÀ CÔNG NGHỆ VIỆT NAM VIỆN CÔNG NGHỆ SINH HỌC NGUYỄN THỊ MINH HẰNG NGHIÊN CỨU BIỂU HIỆN KHÁNG NGUYÊN (M, GP5, GP5ectoM) CỦA VIRUS GÂY HỘI CHỨNG RỐI LOẠN SINH SẢN VÀ HÔ HẤP Ở LỢN TRONG CÂY THUỐC... rối loạn sinh sản hô hấp lợn thuốc phương pháp thẩm lọc nhờ Agrobacterium với mục đích tạo sở khoa học thực nghiệm để biểu gen mã hoá kháng nguyên chủng virus PRRS gây hội chứng rối loạn sinh sản. .. phương pháp biểu gen tạm thời Hệ thống biểu gen tạm thời thực vật phương pháp thẩm lọc nhờ Agrobacterium nhằm sản xuất kháng nguyên có nhiều ưu điểm như: Mức độ biểu kháng nguyên cao, sản xuất