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ỦY BAN NHÂN DÂN TP.HCM SỞ KHOA HỌC VÀ CÔNG NGHỆ TP HO CHi MINH ĐẠI HỌC Y DƯỢC TPHCM TT KHOA HỌC CƠNG NGHỆ DƯỢC SÀI GỊN SAPHARCEN Nghiên cứu biểu interferon-alpha gà bề mặt vi khuẩn Bacillus subiilis Chú nhiệm đề tài: Trần Thu Hoa THÀNH PHO HO CHf MINH THANG 10/2010 TOM TAT NỘI DUNG NGHIÊN CỨU Tên đề tài Nghiên cứu biểu interferon-alpha gà bề mặt vi khuẩn Baeillus subfilis Mở đầu: Interferon- gà (ChIFNa) có tiềm hoạt hố thành phần miễn nhằm tạo dịch, cản trở xâm nhập nhân lên virus gà Nghiên cứu dé dang, B subtilis tai t6 hop mang ChIFNa để dùng 1am probiotic, co thé áp dụng rộng rãi nơi chăn nuôi gia cam, tăng hiệu vaccin phòng chồng bệnh virus Phương pháp: Hướng tới tạo ChIENa tái tổ hợp bề mặt bào tử, bề mặt tế phân tử bào tiết mơi trường Ư subtilis, phương pháp sinh học áp dụng RT-PCR đề tổng hop cDNA tir RNA toàn phân chiết từ lách gà; tạo dòng mang đoạn chIFNa va đoạn gen cotB, cwiC amyE với kỹ thuật ly trích DNA nhiễm sắc thể B sưbiilis ly trích DNA plasmid, điện di gel agarose, tỉnh chê DNA, phân đoạn DNA với enzyme cất giới hạn, kêt nôi đoạn DNA chèn vào vectơ, bién nap DNA vao E coli phương pháp xu ly CaCl) tự Western lạnh sốc điện; kiểm tra thẩm định dòng giải trình tế bào blotting Danh giá khả kháng virus Gumboro va Newcastle in vitro trén khang in xơ phôi gà bào tử B subtilis-interferon alpha gà, kha nang thê đặc kháng ovo in vivo sản phẩm gà đến ba tuần tuổi khơng có hiệu Kết quả: Sau thiết kế chiến lược biéu hign chIFNa trén B subtilis, 22 dòng trung gian tạo Bao gồm: pGemT-chlFNacw pUC19-chlENacw, pUC19-chIFNaa pGemT-chIFNaq pGemT-chIFNas; pGemT-chIFNas) pUC19-PewlCvà pUC19-CWCB, pGemT-P.,/c, pUC19-Pawie, pGemT-cw/C chIFNacw, pUC19-Pe-chIFNacw-cwlC, pDG364-Pe-chIFNa-cwlC, _ pGemT- pDG364-cotB 13cotB13, pGemT-cotB13-chIFNas, va pGemT-cotB13-chIFNas3 pUC19-amyE, pUC19chIFNaS1 pDG364-cotB 13-chIFNaS3, pGemT-amyE, chIFNas; va cotB13amyE-chIFNaa, pDG1664- amyE-chIFNa, Cac dong cotB13va pDG364-PcchIFNas3, pDG364-cotB 13-chIFNas1 pDG364-cotB 13-chIFNas3, mặt bào tử bê chIFNa-cwlC duge giải trình tự Sự biểu protein ChIFNa cho phản ứng tiết môi trường nuôi cấy chứng minh bang Western blotting, đặc hiệu với huyết thỏ kháng đặc hiệu ChIENa virus Gumboro Bào tử B sub/ilis biểu ChIEN-ơ có khả phịng bệnh In ovo va Newcastle in vitro, c6 tác dụng ức ché bénh tich té bao hai virus chêt chậm in vivo bao tir tai t6 hop lam tang ty 1é no cua trig ga, gà mắc bệnh SUMMARY OF RESEARCH CONTENT STUDY ON SURFACE DISPLAY OE CHICKEN INTERFERON ALPHA ON BACILLUS SUBTILIS Background: ChIFNa is a powerful antiviral agent and has high Mx promoterinducing activity Bacillus subtilis, a non-pathogenic, spore-forming bacterium, is chosen to design B subtilis—chicken interferon alpha (ChIFNa), a promising recombinant probiotic for chicken against viral, bacterial, and parasitic infections Objectives: To generate subclones for recombinant B subtilis strains expressed ChIFNa on spore Method: Isolate total RNA growing construct subclones coat and vegetative cell surface, as well : from chicken spleen, RT-PCR of chIFNa, cotB, cwiC and amyE by as secreted when to synthesize cDNA; current protocols in molecular biology; sequencing to verify clone, Western blotting to check expression of recombinant protein Determine efficacy of CotB13-ChIFNa protein expressed on B subtilis for protection against infectious bursal disease virus and newcastle disease virus in vitro on chicken embryo fibroblast, in ovo and in vivo on IBDV and NDV challenge in one-to-three-week-old specificpathogen-free chickens Results: The general strategies for the construction of the gene fusions were designed We have cloned successful intermediate E coli clones of pGemTchIFNacw and pUC19-chIFNacw, pGemT-chIFNas;, pGemT-chIFNas3, pGemT- chIFNa,, pGemT-P,c, PUC19-PewlC, pGemT-ew/C and pUC19-CWCB, pUC19PewlC-chIFNacw, pUC19-Pc-chIFNacw-cwlC, pDG364-Pe-chIFNa-cwlC, pGemTcofB13, pGemT-cotB13-chIFNas, and pGemT-cotB13-chIFNas3 pDG364-cotB 13- chIFNaS1, pDG364-cotB13-chIFNaS3 pGemT-amyE, pUC19-amyE, pGemTchIFNa, and pUC19-chIFNaa, pUC19-amyE-chIFNa,, pDG1664- amy-chIFNa, Some of them were checked by sequencing The surface presentation or CotB13ChIFNa on B subtilis spores as well as secreted AmyE-ChIFNa were showed Spore-exposed ChIFNa has biologically activity, such as reduction of cytopathic effect Recombinant spores reduce susceptibility of infection NDV and IBDV protected against Gumboro and Newcastle disease on birds ii Mục lục TÓM TẮT NỘI DUNG NGHIÊN CỨU MUC LUC DANH SACH CAC CHU VIET TAT DANH MUC BANG DANH MỤC HÌNH Vue“ QUT TỐN KINH PHÍ PHẢN MỞ ĐÀU Chương I TONG QUAN DE TAI NGHIEN CỨU 1.1 Tổng quan 1.1.1 Một số bệnh virus gà 1.1.2 Vai trị 1.1.3 Nghiên cứu Tình hình 1.2 1.3 Tình hình interferon gà sử dung bé mat B subtilis nghiên cứu nước nghiên cứu nước VÀ PHƯƠNG Chương II NỘI DUNG rình diện kháng nguyên protein PHÁP NGHIÊN CỨU Nội dung l: Thu thập thông tin va thiết kế phương án biểu IFN Ư subtilis 2.1 2.2.1 Tìm trình tự DNA đích 2.2.2 Thiết kế phương án biểu IFN Ư sưở/ilis 2.2 Nội dung2: Tạo dịng trung gian E coli 2.2.3 2.2.4 2.2.5 2.2.6 Tổng hợp cDNA Phan tg PCR Ly trích DNA nị Ly trích DNA plasmid Dién di trén gel agarose 2.2.8 Phân đoạn DNA 2.2.1 Ly trích RNA tồn phần 2.2.7 Tinh ché DNA với enzyme 2.2.9 Kết nối đoạn DNA chèn vào vector 2.2.10 2.2.11 Biến nạp DNA vào # coli Kiểm tra trình tự san pham tao dong 2.3 Nội dung 3: Tạo B subtilis tái tổ hợp biểu IEN gà bề mặt 2.3.1 Biến nap DNA vao Bacillus subtilis 2.3.2 Kiém tra protein tai tô hợp hợp Nội dung 4: Khảo sát khả kháng virus in vitro in vivo bào tử tái tổ = B subtilis-interferon ga 2.4.1 Nguyên vật liệ 2.4.2 Khảo sát khả kháng virus in v 2.4 2.4.3 Đánh giá hoạt tính kháng virus Newcastle in ovo 2.4.4 Đánh giá hoạt tính kháng virus in vivo ga Nội dung 5: Thử độ ôn định in vivo bào tử tai t6 hop B subtilis-interferon 2.5 Chương III Kết thảo luận 3.1 Nội dung 1: Thu thập thông tin thi kế phương án biểu IFN B subtilis 33 iii 3.1.1 Tìm trình tự DNA đích 3.1.2 Chiến lược thiết kế tạo đòng 3.2 Nội dung2: Tạo dòng trung gian E cø 3.2.1 Dòng pGemT-cø/B13 Dong pGemT-cotB13-chIFNas; Dong pDG364-cotB 13-chIFNas 3.2.5 Dong pGemT-chIFNas; 3.2.6 Dong pGemT-corB13-chIFNas; 3.2.7 Dong pDG364-cotB 13-chIFNas; 3.2.8 Dong pGemT-Pcuc 3.2.9 Dòng pGemT-ew/C 3.2.10 Dòng pGemT-chIFNacw 3.2.11 Dòng pUC19-ch[FNa„v 3.2.12 Dòng pUC19-P¿u¡c-chlFNaev 3.2.13 Dong pUC19-Poyic-chIFNaew- cwlC 3.2.14 Dong pDG364-P.yic-chIFNacw-cwlC 3.2.15 Dong pUC19- amyE 3.2.16 Dong pGemT-chIFNaa 3.2.17 Dong pUC19-chIFNaz 3.2.18 Dong pUC19-amyE-chIFNag 3.2.19 Dong pDG1664- amyE-chIFNa, 3.2.20 Kết giải trình tự đòng trude dua vao B subtilis 3.3 Nội dung 3: Tạo Ö sưởrilis tái tổ hợp biểu IFN-Ø gà 3.3.1 Tao B subtilis tai tổ hợp mang IFN-œ gà bề mặt bào tử 3.3.2 Tạo chủng Ø subzilis tái tổ hợp biểu IFN-œ gà qua hệ thống tiết 3.4 Nội dung 4: Khả kháng virus ¿+ v#rø in wiv0 3.4.1 Tính độc tế bào bảo tử B subtilis biéu hién ChIFN-a di voi CEF, 3.4.2 Kha nang khang virus Newcastel (NDV) in vitro 3.4.3, Kha nang khang virus Newcastel in ovo 3.4.4 Khả kháng NDV ứ vivo 3.4.5 Kha nang khang virus Gumboro (IBDV) in 3.4.6 Kha nang khang virus Gumboro in vivo Nội dung 5: Độ dn định di truyền bào tử B subtilis tai td hop Chương 1V Kết luận TÀI LIỆU THAM KHẢO Phụ lục Thơng tin chỉtiết trình tự Interferon-œ gà chIFNa Phụ lục Trang Gene cla NCBI voi từ khóa “chicken interferon alpha’ Phụ lục Bản đồ enzym cắt giới hạn gen IFN-œ gà Phụ lục Giải trình tu pDG364-cotB 13-chIFNag, Phụ lục Kết Blast đoạn PewIC-ChIFNa-ewlC thực tế với dự kiên Phụ lục Kết Blast đoạn amyE-ch[FNaa thực tế với dự kiế Phụ lục Tài liệu tham khảo Thông tin sử dụng IFN/probiotic thê giới iv DANH SÁCH CÁC CHỮ VIÉT TÁT VIET TAT THUẬT NGỮ Amp Ampicillin Bacillus subtilis biéu hién interferon alpha gà BI BS cDNA CEF chINFa CIS CPE cw CWB DCVBT DNA dNTP ELDso HA IBDV Bacillus subtilis Complementary deoxyribonucleotide acid Chicken embryo fibroblast, té bao xo phôi gà Chicken Interferon-alpha, Interferon-alpha ga Chicken interferon spore, Interferon ga bào tử Cytopathogenicity effect Cell wall, té bao Cell wall binding, gan tế bào Dịch chiết protein vỏ (bào tử) Deoxyribonucleotide acid Deoxyribonucleotide triphosphat Embryo lethal dose 50%, liều gây chết 50% phôi Hemaglutinin (chất ngưng kết hồng cầu) Infectious bursal disease, bệnh Gumboro IFN hoac I Interferon LB NA NDV NST PCR Luria-Bertani RNA rpm SDS Neuraminidase (enzym tan nhay) Newcastel disease virus, bénh Newcastle Nhiễm sắc thẻ Polymerase chain reaction, phan tng chudi polymerase Restriction enzyme, enzym cắt giới hạn Ribonucleic acid Rotation per minute, vong/phut Sodium dodecyl! sulfate DANH MỤC BẢNG Bảng 2.1 Các đoạn môi oligonucleotide sir dung Bang 2.2 Bé tri thi nghiém khao sat hiéu qua in ovo phịng bệnh NDV Bảng 2.3 Quy trình phòng bệnh chung cho gà thử nghiệm Bảng 2.4 Khảo sát khả gây nhiễm virus gà Bảng 2.5 Khảo sát hiệu phòng bệnh IBDV gà Bảng 2.6 Khảo sát hiệu phòng bệnh NDV gà Bảng 3.1 Tóm tắt kết áp dụng Blastn kiểm tra trình tự sản phẩm Bảng 3.2 Định lượng protein phương pháp đo màu Bảng 3.3 Định lượng protein toàn phần DCVBT ặ Bảng 3.4 Đặc tính kháng erythromycin phụ thuộc threonin Bảng 3.5 Tính độc tế bào tế bào CEF DCVBT B Subtilis CIS Bảng 3.6 Tỷ lệ CPE NDV gây theo nồng độ virus môi trường CEF as Bang 3.7 Tinh khang NDV in vitro sinh phẩm xử lý thời điểm Bảng 3.8 Tình trạng trứng phun sinh phẩm lúc gây nhiễm NDV 66 Bảng 3.9 Tình trạng trứng phun sinh phẩm trước gây nhiễm NDV 66 Bảng 3.10 Tỷ lệ gà chết sau nở từ trứng phun sinh phẩm trước : Bảng 3.11 Liều NDV gây chết 50% phôi gà (ELDso) Bảng 3.12 Tỷ lệ gà bảo hộ phòng bệnh NDV -:: ::: -+-++-+ Bảng 3.13 Đáp ứng kháng thể kháng NDV gà sau tuần sử dụng sinh phẩm 68 Bảng 3.14 Tỷ lệ CPE IBDV gây theo nồng độ virus môi trường CEE 69 Bảng 3.15 Tính khang IBDV in vitro cia sinh phẩm xử lý thời điểm 70 Bảng 3.16 Liều IBDV gây chết 50% phôi gà (ELDs) Bảng 3.17 Tỷ lệ gà bảo hộ phòng bệnh IBDV, Bang 3.18 Đáp ứng kháng thẻ kháng IBDV (AGP) vi DANH MỤC HÌNH Hình 1.1 Cơ chế hoạt động IFN-z đáp ứng, miễn dịch Th] Hình 1.2 Cơ chế kháng virus cảm ứng IFN Hình 2.1 Bản đồ vector pGEM®-T Easy Hình 2.2 Bản đồ vector pUC19 Hình 2.3 Bản đồ vector pDG364 vàv pDG1664 Hình 2.4 Quy trình chuẩn bị lớp đơn tế bào CEF Hình 2.5 Quy trình thu nhận huyền dịch virus Hình 2.6 Quy trình cấy chuyên virus Hình 2.7 Quy trình chuẩn độ virus Hình 2.8 Quy trình xác định tính độc tê bào DCVBT Hình 2.9 Lam thạch thực phản ứng AGP Hình 3.1 Trang Nueleotide ba trình tự DNA có liên quan TEN-ơ gà Hình 3.2 Mơ tả tiết trình tự NM 205427 trang Nucleotide Hình 3.3 Chiến lược thiết kế tạo đòng hướng tới tạo IFN tai tổ hợp b mặt bao tir B subtilis Hinh 3.4 Chién lược thiết kế tạo dòng i hướng tới tạo IFN tái tổ hợp bề mat té bao B subtilis hệ Hinh 3.5 Chién luge thiét ké tao dong hướng t tới tạo IFN tái tổ hợp qua thống tiết Ư subilis Hình 3.6 Phương án đưa tổ hợp gen IFN vào n| hiém sac thé B subtilis Hình 3.7 Tạo dịng pGemT-è/B13 Hinh 3.8 Tao dong pGemT-chIFNas; Hinh 3.9 Tao dong pGemt-cofB 13-chIFNag, B chIFNas) Hinh 3.10 Tao dong pDG364-cof13Hinh 3.11 Tao dong pGemT-chIFNasg; Hình 3.12 Kiểm tra plasmid pGemT-corB 13-chIF Nas Hinh 3.13 Tao dong pDG364-cofB 13-chIFNas; Hinh 3.14 Tao dong pGemT- Powic-.- Hinh 3.15 Kiém tra plasmid pGemT- “ewlC bing cắt voi EcoRI va Kpnl Hình 3.16 Kiểm tra plasmid pGemT- chIFNacw cắt với Kpni Hình 3.17 Kiểm tra plasmid pUC19- chIFNaqy cách cất với Kpnl cách cắt với Kpnl Hinh 3.18 Kiém tra plasmid pUC19- PewlC-chIFNaow bang với Sphl & Sall.49 Hình 3.19 Kiểm tra plasmid pUC19-P.„¡c- -chIFNa- ew/C cắt va Sall 50 Hinh 3.20 Kiém tra plasmid pDG364-Pc-chIFNa- ewlC cắt Sphl Hình 3.21 Tạo địng pUC19-amyE Hình 3.22 Tạo dịng pGemT-chIFNaa vii Hinh 3.23 Tao dong pUC19-chIFNaa Hình 3.24 Kiém tra plasmid pUC19-chIFNag bang ct voi Xbal va Hindlll Hinh 3.25 Tao dong plasmid pDG1664-amyE -chÏIENaa Hình 3.26 Chiến lược giải trình tự dịng pDG364-cøB13-ch[FNasy Hình 3.27 Chiến lược giải trình tự dịng pDG364- Pa„c-chlFNacw-cw/C Hình 3.28 Chiến lược giải trình tự dong pDG1664-amyE-chIFNa, Hình 3.29 Đường biểu diễn protein chuẩn (BSA) Hình 3.30 Phân tích protein vỏ bào tử gel polyacrylamide Hình 3.31 Kết phân tích Western blot sơ DCVBT từ CIS Hình 3.32 Chọn lọc B subtilis-amyE-ChIFNaA Hình 3.33 Kiểm tra biểu hién ChIFNa, Hình 3.34 Bệnh tích Hình 3.35 Tác dụng Hình 3.36 Tác dụng phẩm khác tế bào NDV tế bào CEF bảo vệ tế bào CEF DCVBT bảo vệ tế bao CEF sau nhiễm NDV thời điểm xử lý sinh MÀ 4b 1ác0ãu8En41841683413183613ã Hình 3.37 Các triệu chứng biểuhiện 6oga mac ác bệnh ao NDV Hình 3.38 Các bệnh tích gà chết nhiễm NDV : Hình 3.39 Tác dụng bảo vệ tế bào CEF DCVBT IBDV 69 Hình 3.40 Tác dụng bảo vệ tế bào CEF sau nhiễm IBDV thời điểm xử lý sinh phẩm khác Hình 3.41 Các triệu chứng gà bị bệnh đu IBDV Hình 3.42 Một số bệnh tích gà bị bệnh [BDV Hình 3.43 Phân lập CIS từ phân gà thạch có kháng sinh Hình 3.44 Kết khảo sát độ ôn định in vivo CIS . - viii QUYÉT TOÁN KINH PHÍ Đề tài: Nghiên cứu biểu interferon-alpha gà bề mặt vi khuẩn Bacillus subtilis Chủ nhiệm: Trần Thu Hoa Cơ quan chủ trì: Trung tâm khoa học Công nghệ Dược SAPHARCEN Thời gian đăng ký hợp đồng: 18 tháng (Từ tháng 5/2008 đến tháng 11/2009) Thời gian thực giai đoạn 1: (Dùng cho báo cáo giám định) Tổng kinh phí duyệt: 600.000.000 đồng Kinh phí cấp giai đoạn 1: 400.000.000 đồng (Theo thơng báo số: 75/TB-KHCN ngày 13/5/2008) Nội dung TT Trong Ngân sách Kinh phí năm I | Kinh phí cấp 400.000.000 400.000.000 II | Kinh phí tốn 400.000.000 400.000.000 1, | Cơng chất xám | Cơng th khốn | Nguyên, nhiên vật liệu, 9.000.000 123.377.255 246:122.745 9.000.000 123.377.255 249.122.745 18.500.000 18.500.000 0 năm: năm: dung cu, phu ting, VPP | Thiệt bị | Xét duyệt, giám định, nghiém thu | Hội thảo hội nghị | Đánh máy tài liệu Giao thơng liên lạc §._| | Chỉ phí điêu hành 0 0 TV | Kinh phí chuyển sang II | Tiết kiệm 5% năm sau: Nguôn khác Phu luc ‘Signal G:144 A'54 T164 C57 Page 2012 102/010 KB_3130 POP7_BOTV3.mob ‘Spacing: 12.5685472488403 feld MTXF ‘7200 Lane 16 Pos 1022 to 12692 Tan INCE TOMET NOMENON TE TIAN N TACCCEATITTATO T TERRE TEM TOSECTcsIE COE TE CTEC= NET NTTIN 170 KG " m me ” " „ ry ” Bio TRA — ”„ [orem Tene c TH Coan AANCT COCAANGE ToGG CO TOC TOC TOC = Til Te aeA TOTTe CATOG mm = = ™ ” A TMsTT Ota wi TTCTIA TM Trtitccasa ™ CM ieCCCINMAG tccA ) COCEE a GC TÔ TCG ime ATTTAC we CANT TTC3 Trinh ty thu giải với CINIva B1 Model 3100, KBkp BIO, ‘Fle: pOG364-ColB-CIN1-B1.ab1 ‘Signal G257 A227 T:179.C:127 Page tot ?no MT field Points 979 to 12444 ‘Spacing: 12.327712089021 315) POP]_ BDTV3nob IM (6286250-02 628620R0G8256 103-04 6258005-00 Lane sto MW TATAT TUAAANCCG I TCMCANTACE TCAAAG ATCATTTANGTAT FTTATGAATG] TTTITM CATE AAT TO AUCOTITTE REG TS CEG MOTE MCTECE " ww ` " » ° “ ` ACACGAGCCTOATT TOAAAATAGAAGAGCAGACOCCAGT IGGAGTITTG™ AAG! TIAAACACIAITOAGGATOCOGTAGTOTATITTAATATCCAT CAAT CAAAAGTATANGTAN a T „ " " „ a a “ o Ơac26fcZvvv=zseco¿=slboeelte£seoe2/2v2oe^—-C¬C¬ = anniocl at roces NM coN\ sncnoe 6€ €6466€€ GAAGCCATIGAGOOTATC CT IGTAGATAAT TCGTOGAGGTCCO OGOTTTCINTTAA Tite ITTTANGTE IGCTITITANGAGTCTOICTEATANT „ m " om = ™ ™ ™ „ = w TEA AMA ATOM C Ail CAGE MGA ATA TOMT A "„ “ " „ FTTC SA TẾÀ TC T1 we ŒC MTCAC “—————— A ŒÁ06 A TAGE TT TÀI] TRKT wa = 87 Phụ lục "_ Trình tự thu giải với CIN22 cm Model3100 Trle:PDG364-CotB-CiM1-C23.sbt kẽ ben (9208250-02 8288302-01IRE.8810E3-94 0269004-90 ta “8lgnal G144 A54 T84 C67 KB_3130_PO£7_80Tv8.mob 97 na MID tala Polnts 1022 19 12002 88 Phu luc " Nối kết giải từ mồi phần mềm Seqman (In trực tiếp phần mềm Seqman) Blast kết thu với đoạn cotB-chIFNơ dự kiến NCBI: Color key for alignment scores rr 50-80 ea lel|1517 Length=1661 Expect = 0.0 (1602), Score = 2959 bits (99%), Gaps = 19/1675 Identities = 1654/1675 Strand=Plus/Minus (1%) 97 1661 TATAAAAAGAATGATATTGAGCGTTTTGACCGTGAGCCGGATGTGATCTGCGAGTATATT II TIIIIEIIIIIIIJIEIIIIEIEEIIEIEIEIIIEIIIIIIEIIIIIIIIIIIEIIIIEIIIIII TAT-AAAAGAATGATATTGAGCGTTTTGACCGTGAGCCGGATGTGATCTGCGAGTATATT Query 98 AAAAACCGTTCACAATACCTCAANGATCATTTAAGTATTTTATGAATGCGTGAAAATGGG 157 Sbjct 1602 AAAAACCGTTCACAATACCTCAAAGATCATTTAAGTATTTTATGAATGCGTGAAAATGGG 1543 Query 158 217 Sbjct 1542 TATTCGCGGAAAAAGCGACAATTAGGCTATTGAATTAGTTCAACAAATAAATGTGACACG (1III11IIIIIIIIEIIIIIITTITTIIIEIIEITETTIIITITETTITITIIIIIIIIIIILII TATTCGCGGAAAAAGCGACAATTAGGCTATTGAATTAGTTCAACAAATAAATGTGACACG Query 218 TATATATGCAGTATGTTTATCATCTATGTATAAGTGACTAGGAGGAATTTGAATGAGCAA 277 Sbjct 1482 Query 278 Sbjct 1422 Query 338 Sbjct 1362 Query 398 Sbjct 1302 Query Query 38 Sbjct 1I11111|11111111111111111111111111111111111111111)L11111111 EIIIIIIIIIIITIEIIIIIITIITITIEITIIEIEITIITEIITTIIIIIIIIIIIITIIIIII TATATATGCAGTATGTTTATCATCTATGTATAAGTGACTAGGAGGAATTTGAATGAGCAA 1603 1483 1423 GAGGAGAATGAAATATCATTCAAATAATGAAATATCGTATTATAACTTTTTGCACTCAAT I11111111111111111111111111111111111111111111111111111111111 GAGGAGAATGAAATATCATTCAAATAATGAAATATCGTATTATAACTTTTTGCACTCAAT 337 GAAAGATAAAATTGTTACTGTATATCGTGGAGGTCCGGAATCTAAAAAAGGAAAATTAAC J111111111111111111111111111111111111111111111111111111111|1 GAAAGATAAAATTGTTACTGTATATCGTGGAGGTCCGGAATCTAAAAAAGGAAAATTAAC 397 AGCTGTAAAATCAGATTATATAGCTTTACAAGCTGaaaaaaaaaTAATTTATTATCAGTT 457 1243 458 AGCTGTAAAATCAGATTATATAGCTTTACAAGCTGAAAAAAAAATAATTTATTATCAGTT GGAGCATGTGARAAGTATTACTGAGGATACCAATAATAGCACCACAACAATTGAGACTGA Sbjct 1242 GGAGCATGTGAAAAGTATTACTGAGGATACCAATAATAGCACCACAACAATTGAGACTGA 1183 Query 518 577 Sbjct 1182 GGAAATGCTCGATGCTGATGATTTTCATAGCTTAATCGGACATTTAATAAACCAATCAGT 1111111111111111111111111111111111111111111J1111111111111111 GGAAATGCTCGATGCTGATGATTTTCATAGCTTAATCGGACATTTAATAAACCAATCAGT Query 578 - TCAATTTAACCAAGGGGGTCCGGAATCTAAAAAAGGAAGATTGGTCTGGCTGGGAGATGA II111I11111111111111111111111111111111111111111111111111111 TCAATTTAACCAAGGGGGTCCGGAATCTAAAAAAGGAAGATTGGTCTGGCTGGGAGATGA 1122 637 1111111111111111111111111111111111111111111111111111111111 I11111111111111111111111111111111111111111111111111111111111 1363 1303 517 1123 1063 89 Phụ lục Query 638 TTACGCTGCGTTAARCACAAATGAGGATGGGGTAGTGTATTTTAATATCCATCACATCAA 697 Sbjct 1062 TTACGCTGCGTTAARCACARATGAGGATGGGGTRGTGTATTTTAATATCCATCACATCAA 1003 Query 698 757 Sbjct 1002 AAGTATAAGTAAACACGAGCCTGATTTGARRATAGAAGAGCAGACGCCAGTTGGAGTTTT 111111111111111111111111111111111111111111111111111111111111 AAGTATAAGTAAACACGAGCCTGATTTGAAAATAGAAGAGCAGACGCCAGTTGGAGTTIT Query 758 817 Sbjct 942 GGAAGCTGATGATTTAAGCGAGGTTTTTAAGAGTCTGACTCATAAATGGGTTTCAATTAA TIIIITIIITIIETEEIEITIITIEIIIIIIIIITIITTEIETIEIIIIIIIIIIIIIIEIEIIIIIII GGAAGCTGATGATTTAAGCGAGGTTTTTARGAGTCTGACTCATAAATGGGTTTCAATTAA Query 818 TCGTGGAGGTCCGGAAGCCATTGAGGGTATCCTTGTAGATARTGCCGACGGCCATTATAC 877 Sbjct 882 TCGTGGAGGTCCGGAAGCCATTGAGGGTATCCTIGTAGATAATGCCGACGGCCATTATAC 823 Query 878 TATAGTGAAAAATCAAGAGGTGCTTCGCATCTATCCTTTTCACATAARAAGCATCAGCTT 937 Sbjct B22 TATAGTGARAAATCAAGAGGTGCTTCGCATCTATCCTTTTCACATAARANGCATCAGCTT 763 Query 938 997 Sbjct 762 AGGTCCRAAAGGGTCGTACAAAAAAGAGGATCaaasa3aaTGACAAAACCAGGAAGACAA IITIIIIIIITITEIIIEIIJIIIIIIEIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII RGGTCCAAAAGGGTCGTACARAAAAGAGGATCAAAAAAATGAACAARACCNGGAAGACAA Query 998 Sbjct II1IIIIIIIIIIIIIIIIIIIIIITIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII TUE EEE POPPE EEO Pee Eee eee Ee eee ee IIIIITIIIIIIIIIEIEIIIIEIIIIIEIIEIITIETIEIIIEIIEIEIIEIIIIEIIIEIEIIEIII 943 883 703 702 TAATGATAAGGACAGCAATTCGTTCRTTTCTTCAARATCATATAGCTCATCARAATCATC IEITIIIIIIIIIIIIIIIIIEITIIIIEIEITIIIIIIIIIIIIEITIIIIIIIIIIIIIII TARTGATAAGGACASCAATTCGTTCATTTCTTCAAAATCATATAGCTCATCAARATCATC 643 Query 1058 TAAACGATCACTAAAATCTTCAGATGATCAATCATCCAGATCTCGCACCACCACCGAGCC 1117 Sbjct 642 TAAACGATCACTAAAATCTTC A-G -AT AGATCTCGCACCACCACCGAGCC 595 Query 1118 CCACCRGGCTCCTGCCCAGCACAACGCGAGTCCCACCATGGCTGTGCCTGCARGCCCACA 1177 Sbjct 594 CCACCAGGCTCCTGCCCAGCRCAACGCGAGTCCCACCATGGCTGTGCCTGCAAGCCCACA 535 Query 1178 GCACCCACGGGGGTACGGCATCCTGCTGCTCACGCTCCTTCTGAAAGCTCTCGCCACCAC 1237 Sbjct 534 GCACCCACGGGGGTACGGCATCCTGCTGCTCACGCTCCTTCTGRAAGCTCUCGCCACCAC 475 Query 1238 CGCCTCCGCCTGCAACCACCTTCGCCCCCAGGATGCCACCTICTCTCACGACAGCCTCCA 1297 Sbjct 474 CGCCTCCGCCTGCAACCACCTTCGCCCCCAGGATGCCACCTTCTCTCACGACAGCCTCCA 415 Query 1298 GCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTG 1357 Sbjct 414 GCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTG 355 Query 1358 CTCCTTCAACGACACCATCCTGGACACCAGCRACACCCGGCAAGCCGACAAAACCACCCA 1417 Sbjct 354 CTCCTTCAACGACAGCATCCTGGACACCAGCAACACCCGGCAAGCCGACARAACCACCCA 295 Query 1418 CGACATCCTTCAGCACCTCTTCAAARTCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAA 1477 Sbjct 294 CGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAA 235 Query 1478 CGACAGCCAACGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCA 1537 Sbjct 234 CGACAGCCAACGCCAAAGCCTCCTCAACCGGSATCCACCGCTACACCCAGCACCTCGAGCA 175 Query 1538 ATGCTTGGACAGCAGCGACACGCGCTCCCGGACGCGATGGCCTCGCAACCTTCACCTCAC 1597 Sbjct 174 ATGCTTGGACAGCAGCGRCACGCGCTCCCGGACGCGATGGCCTCGCAACCTTCACCTCAC 115 Query 1598 CATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAACGATTACAGCGCCTG 1657 Sbjct 114 CATCRAAAAACRCTTCAGCTGCCTCCACRCCTTCCTCCRAGTCG-CGATTACAGCGCCTC Query 1658 Sbjct 55 IIIIIIIIIIIIIIIIIIIEIE 1 II IIIIIITIIIIEIIIIIIIIIEII IIIIIIIEIIIEIEIIEIIIIIEIEIEIIEIEPIIIEIIIIIEIEIIETITIIIIIIIEIIEIIII THTTTEIETITITEIETIEJIIEIEIIEITIEITIEIIITIEIIIIEITEIEIIIIEIIEIIIIEIIEITIIIIT TIIIIIIIIIEIIEIIIITITIIIEIEIIIIEIIEIIIIITIIEIIIIIIIIIIIIIIIIIIIIIII IIITIIIITIITIIEITIEIIEITIITIITIIIITITITIIIIEITIIIIIIETTI*EIEIIIII TIITIIIIIITEITITIIIEIIITIITIIIITIEITITITETIIIITIIEIIITIEIEIIIII TTITIIIIIIIIIIITIEIEIIITIIIITITIIIITIIIEIEIIIIITIIIIIIIIIIEIIII TTIIIIIIEITIIIIIIIEIIIIIEIIIIIIIIIITIEIIEIIIIIIITIIIIIIIIIIEIIIEII TIEIIIIIIIIIIITIIITIIIIIEIEIIITIIIIIIIIEIIIITIEIIIEIEIIIIIIIII IIIIIIIIIIEIIIIIIIIIIIIIIIIIIIIIIIIIIITIIITÔ TIIIIIIIEIIIEIII CGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAAC 1IIIIIIIIIIIIIIIEIIIIIIIIIIIIEIIIIIIII ee tee IỊỊ CGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTG-T-CC-GCAC-TCCA-AAC 1057 = 56 1712 90 Phụ lục " Trinh ty codon dung hop cotB va chIFNa Bang phan mém Seqbuilder, thu từ kết giải trình tự khung đọc mở (ORF) từ ba khởi đầu ATG gen mã hóa CotB dịch mã liên tục hết gen mã hóa ChIENd Trình tự codon CotB ChIFNơ dung hợp không bị lệch khung so với dự kiến Chỉ bị acid amin cuối CotB TAABAACCGTTC TATAARAGAAT GATATIGASCGTTI TGACCGI GA GCC GGATGTGAT CIGCGAGTATAT † † † † + † † † + + † + † CA CT CSSCCTACACTA GACGCTCATATAATTITIGGCAAS ATATITICTIACTATAACTCGCABAACTGS 79 ° ° CACAÄTACCTCAAÄGXTCATTTÀÃGTATTTTÀTGÀÄTGCGTG144⁄4TG2GTATTCGCGGAXkAXÄGCGACAX +† ‡† 4† +† +† +† ‡† ñ† + +1 + † † + CSCIGIT AAGCSCCTITIT 2% RTTCRTRARA TACT TACGCACT TT TA CCCAT 440 ARR TGIGACACGTATATAT GCAGIATGITTATCATCTATG CARAT GITCAA TIAGGCTATIGRATIA 4† + ‡ †n † †+ † †} † †+ † † † † † RGATACATA AAT AGT CATACA CGT TCAAGTT GIT TATTT ACACTGT GCATATATA AR CTT ART COGATAR 210 ATT CAARTARTGARATATCGIATT ARGIGACT AGGAGGAATT TGAA TGR GCARGAGGA GAA TGAAAT: † † + † † † † + + + † + † † GI ITATTACTITATAGCAT AS ACE CGTICTCCT CIT ACTITATAGIAA TICACEGATCCTCCTTARACTT 280 ° ° SN “om ATRACTITTTGCACT CAA TGAAAGATAAAATT GT TACTGTAT AT CGTGGAGGT CCG GAATCTARABBA GS c5 † † † † † † † + † † † † † TCC GCCT TAGATTITI TATIGAARAACGT GAGIT ACTI TCE AT TIT AACAATGACATATAGCACCTCCAG ARARTTARCAGCTGTARZAT CAGAT TATAT AGCI TIA CAAGCTGARABAAABATARTTTATTATCAGE + 4† 4† +† †4 † † † t † + + † † GTCAAC IABATASTA TITTTITAT TITTAATT GICGACATIT TAGI CTAAT ATATCGAAAT GI TCGACTI (See ° ICAATTI AR CCALSGSGET CC ATGCIGATGATTTICATAGCTT AAT CSGAC TTTARTARACCARTCAGT }† ++ + + + + +† '+ + + † † + + † + + + GSITCCCCCAGS CGAATTA GCCIGIAAATIATTIGGIIAGICAAGTIIAAATI TACGACTACTAAAAGTAT AZ2GCI GGGA GAT GATTACGCT GCGTT AAACA CRAATGAGSATGGS AA GGAAGAT TGGICIG GGAATCTA + + † + + + † + + + + + + + ACCGA GCGACGCAATTTIGIGITTACTCCTACCT BAT ACCAG CCCT CTACT CCTTAGATTITTTCCTICTA m8 “om ro somo Homey ° GAGCAT GI GAARA GT ATT ACTGAGGAT ACCAATAATA GCACCACAA CATT GAGACTGAGGAAAT + GCI CE ñ† ‡† Sai† AI ‡ +† L† † ++ + T ITAA' GGITA IAT CGIGGIGTIG TCGTACA CITTT CATAATGACTCCTAT CA CGAGCCT GATTI GAABATAGAAGAGC TCACE AA GT AAA ATCCA TCABAAGTAT GTAGIGTATITTAAT +† + + }† 1† † + ++ } + † + CS TATCITCI TARACTTI CAT CACAT ALAATTATAGGT AGIGLAGITI ICATATI CATIT GI GCICGGAC 700 STGAT TT AAGCGAGGTT TT TAA GAGT CTGACTCATABATGGGI \† ‡+ ++ +† + +} † + +4 † ATTTACCCA ATT CCAALR CI CAGACTGAGI TCT GOGGT CAA CCTCARR ACCT ICGACTACTARATICGCT ° | aaa 770 91 Phu luc os om o Yom to m= 3a † +tửêrrrrrrrrmrrrrrrmrirrrrrrrErrrErrrxt † † AARGTTAAT TAG CA CCTCCAGGCCTT CGGTAACTCCCA TA GGAACAT CATT ACGECTGCCGSTARTATGA ATAGIGAASAATCARGAGGI GCTTCGCATCTATCCTT TT CACAT AA BAA GOAT CAG CT TAG GT CCARBAG 4† + †+ † †+ † † †+ † † † +4 TAT CACTITITAGTI CICCACGRAGCGTAGAT AGGAA BA GIGTATI TIT CGTAGICGRATCCAGSTITIC GGT CGTACAAAARAGAGGAT CAAA LR ATGAACAARA CCAGGAR GA CARTAAT GAT AAGGACAGCAATTC oe† † †+ † † + + † † † † + CCRGCATGTITITICTOCTAGITIT TI TACT GI TIT GGTCCTI CL GITAT TACTATT CCT GICGTTAAG sau ay I | GIT CATTTCIT CAAAATCATATAGCTCATCAA AATCATCTARACGATCACT AABAT CI TCAGATAGATCT ++ 1† Tử† † + † † +t † † + †‡ + CAAGIAAAGAAGITT TAGTATATOGAGTAGTI TT AGTAGATT TGCTAGIGATTITAGAAGI CAT CIAGA CGECACCACCACCGAGCOCCA CCAGSCT CCT GCCCAGCACAACGCGA GICCCACCATGGCIGTECCTGCAR ‘t ‡ }† † †+ + + t + †+ +} t4 †+ GCGTGGTGGTGGCTCGGGGTGGTCCGAGGÁCGGGTCGTGTTGCGCTCAÀGGGTGGT/.CCGACACGGACGTT 8+ a GECCCACAG CACCCACGSS ESTA CEG CA TCCTGCT SCT CA CGCTCCI TCT GABA GCT CT CGCCACCACE tt rf + 4+ ++ +† +† + + + +4 † + † CESGIGTCETEGSTECCCCCAT GCCET AGGACGA CGA GT GCGAGGAAGACITT CGA GA SCGGT GETESS: CTCCGCCTGCAACCACCTTCGCCCCCAGG1TGCCACCTTCTCTCACGACASCCTCCAGCTCCTCCGGGAC +† +ttt 4† t+ + + + +t + + + + GAGGOGGA CGT IGGT GGAAG CGGGGET CCTACGGTGGAA GAGAGTGCIGICGGAGSICGAGGAGECCCTS ATGGCT CCCACACTA CCC CA GCTGT GCCCA CA GCACAAC GOGTCTT SCT CCTTCAACGACACCAT CCT GS + + + + + + + +t + + † † + ; TACCGAGGGIGTGAT GGGGT CGACACGGGT GT CGTGT TGCGCAGAA CGAGGAAGIT GCIGIGSTAGGACC 1340 ACACCAGCAACACCCGGCAR TOGA CABRA CCACOCA CGACATCCT TCAGCACCECTT CAABATCCICAG + + + + + + + + + TGLGGICGTIGIGGSCCSTT CGGCE GI TIT GSIGGST GC’ 1sng a a Ễ a CAG COCCA SCA CT CCAGCCCACTGGAA CGA CA GC CAR CGCCRAR GC CTCCT CAACCGGATCCAGK + + + + + + + + + + GICGSEGT CGTGAGSTCGGSTGACCTT GCI GI CGGTT GCGGT TT CGGAGGAGT TGSCCTAGGI GECGATS om on OMY oc Momo “ome o Momo Momo Komp Lo ° omy ce Som to Komen TICAAT TAATCGT GGAGETCCGGAA GCCAT IGAGGGT AT CCT TGTA GAT AATGCCGACGSCCATTATACT ACCCAGCA CCT CGAG CAA TG CT IGSACAGCAGCGACA CS CEC TOCCEGACS CGATEGOCICGCARCCIIC t + † † † † † † † † † + + † TGGGTCGTGGAGCTCGTTACG1ÄCCTGTCGTCGCTGTGCGCGAGGSCCTGCGCTACCSGAGCGTTGSAAG Phụ lục Phu lục Kết Blast đoạn PcwlC-ChIENa-ew/C thực tế với dự kiến >1c1|16645 Length=1363 Score = 2464 bits (1334), Expect = 0.0 Identities = 1340/1343 (99%), Gaps = 0/1343 (08) Strand=Plus/Minus GTTRACCGAACCCATAGTACATACAAACATATGGCGTATGCACAGATTTCATGTCGGRCC Query 25 EIEEIEIEEEEEEEETEEIEHEEEEETEETTEHTEETTETEITTETITTETTTITTTTII Sbjct 1363 GTTAACCGAACCCATAGTACATACAAACATATGGCGTATGCACAGATITCATGTCGGACC 1204 Query 85 — GTATCTGTTGTCGCTTCATGTTGANTTGTGCGCTTTCCCGAGAAATAATTTCTGGATGTG 144 Sbjct 1303 GTATCTGTTGTCGCTTCATGTTGAATTGTGCGCTTTCCCGAGRAATAATTTCTGGATGTG 1244 Query 145 AAAGGGTTATTTCCTCATTTTTCAGTTGARTGATAATCGTACAAGCAGAAGCCGTGTTTT 204 Sbjct 1243 AAAGGGTTATTTCCTCATTTTTCAGTTGARTGATAATCGTACAAGCAGAAGCCGTGTTTT 1184 Query 205 TTCATATCCTGTRATGAGGTGATGARRAATGTCTAGACGCACCACCACCGAGCCCCACCA 264 Sbjct 1183 TTCATATCCTGTAATGRGGTGATGAAAAATGTCTAGACGCACCACCACCGASCCCCRCCA 1124 Query 265 324 Sbjct 1123 GGCTCCTGCCCAGCACRACGCGAGTCCCACCATGGCTGTGCCTGCAAGCCCACAGCACCC (I11111ITTEEIEIETEEEEITIEEITTTETETETTTTTIETTEIETETTIEEIETTTITTITTITELII GGCTCCTGCCCAGCACARCGCGAGTCCCACCATGGCTGTGCCTGCAAGCCCACASCACCC Query 325 ACGGGGGTACGGCATCCTGCTGCTCACGCTCCTTCTGARAGCTCTCGCCACCACCGCCTC 384 Sbict 1063 ACGGGGGTACGGCATCCTGCTGCTCACGCTCCTTCTGRAAGCTCTCGCCACCACCGCCTC 1004 Query 385 444 Sbjct 1003 CGCCTGCAACCACCTTCGCCCCCAGGATGCCACCTTCTCTCACGRCRGCCTCCAGCTCCT 00000 000000000000000000000000000ƠÚĨƠ GZẽẽẽẽaẽ II CTTCTCTCACGACAGCCTCCAGCTCCT CAGGATGCCAC CGCCTGCAACCACCTTCGCCCC Query 445 CCGGGACATGGCTCCCRCACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTGCTCCTT 504 (1111111111111111111111111111111111111111111111111111Ð1LLDII, IEEIEEEIEIEIHEEIIEITTEIEITTTETEIEETTEIEIETTTIEIIITTTTIITTIEETTTFTITTTTITIT PCCP ECE EETEETTTTTEETTEEIETITEEELEEHEHTIEEEELEEETEITTTEEEEEELHTTTTTEIT PEEP EPEC EEE EEE Pee Eee eee 1064 344 884 Sbjct 943 CCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTTGCTCCTT Query 505 CARCGACACCATCCTGGACACCAGCAACACCCGGCRAGCCGACAAAACCACCCACGACAT 564 Sbjct Query 883 565 CAACGACACCATCCTGGACACCAGCAACACCCGGCARGCCGACARAACCACCCACGACAT CCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGANCGACAG 824 624 EIIEEEEIEIEIEITIITITEIIIIEEEITITIEIEETETITTEEITITTTETTEITITIIEIIII II Sbjct Query Sbjct 823 625 763 fIEIEIEIEIETIEEEEIIEIEETTTEJEEEEITIETIITIVTEITIEEEITIETITITEHEIEHEII CCTTCNGCACCTCTTCRAAATCCTCAGCAGCCCCAGCACTCCAGCCCACTGGAACGACHG CCAACGCCRRAGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCARTGCTT (111111111 1EEEEEEPIETETTTETEIĐIETETITTIETEITFIETETTITTTTTTTTTETI CCAACGCCRARGCCTCCTCAACCGGATCCACCGCTACACCCAGCACCTCGAGCARTGCTT 74 684 704 Phu luc T44 Query 685 GGACAGCAGCGACACGCGCTCCCGGACGCGATGGCCTCGCAACCTTCACCTCACCATCAR Sbict 703 GGACAGCAGCGACACGCGCTCCCGGACSCGATGGCCTCGCAACCTTCACCTCACCATCAA s44 Query 745 AAARACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAACGATTACAGCGCCTGCGCCTS 804 Sbjct 643 AARACACTTCAGCTGCCTCCACACCTICCTCCAAGACRACGATTACAGCECCTGCGCCTS 584 Query 805 GGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAR s64 Sbjct 583 GGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAR 524 Query 865 'CACGCGCRCTTASCCCCAAACGCACCTCCCACCCTTGTC 924 Sbjct 523 CACGCGCACTTAGCCCCAAACGCACC CCCACCCTIGTCCTATTTATCTATTTGTTCAAC 464 Query 925 see Sbjct 463 CAGACRARATAR ee eee eee eee E CE HUE CAAARTAR CTATITATTCAGA ICTATTTATTCTI GUGTATTTATTCT TATTTATACAAAC Query 985 1044 Sbjct 403 AGCTCTCCTTTTCAACACTGAAGGTACCCTTARAAAGACTTCCAGCTCAGGGTTATATAA PEPE TEEPE AGCTCTCCTTTACAACACTGAAGGTACCCTTARRARGACTTCCAGCTCAGGGTTATATAA Query 1045 1104 Sbjct 343 GGTTCAAATCGGCGCATTTARAGTCARAGCGAATGCCGACTCGCTCGCARGTAATGCCGA IIIIEITIIIIIIIEIIIIITEIEIITIITEEEETITIITIEIEIETEITITIEEEETTITITIIIEIIEIII 'GGTTCAAATCGGCGCATTTARAGTCARRGCGRRTGCCGACTCGCTCGCARGTRATGCCGB 284 Query 1105 AGCCAAAGGTTTTGACTCGATIGTCCTTTTARAGGACGGATTATACAAAGTGCAGATTGS 1164 CGCATTTTCATCCARAGACAATGCAGACACCCTCGCTGCCAGAGCGRAAAATGCCGGCTT TOPE PEEP eee eee eee CGCATTTTCATCCRAAGACAATGCAGACACCCTCGCTGCCAGAGCGRAARATGCCGGCTT 1224 eC 1284 IỊ11TIIIĐFIEIIIETEIEIIEIIIETIEITIETIIEITIEIIIEIITITIITIEETIEIT[TIEITEITIITIIT THEIIEIIIETETTTITEETEIEEEHEEEEITTTEIEEEEETETEETTEHETIIIIEIEIEILIETIEEITII EII1IIIIEITEIIIEIEIEIITITEIEEITTEIEITIIEITIIEEIITIIIIIIIITEITITITTIIII EEEETEEEIETETEITIETTTEEEEEIIEEEHTTEEHEIETEEELEEEIEITTEIEIEI Query Sbjct 1225 TGACGCTATTGTGATCCTAGAATCATAGCCGAGACGGGGACGAGCGTCTCAT 1H EEIEIIIEEEIEIHEIEIEITEIEEIIETIEIIEIEEIEIEETEIEETEETEIEETTTIIIIETIITTI 'TGACGCTATTGTGATCCTAGAATCATAGCCGAGACGGGGACGAGCGTCTCATAARARAC Sbjct 163 Query Sbjct ATTCAAGRATATCAACRACAAACTGTG 1285 CCGGCTCCTCATCGCGASaRACCGGG-= EIITTTEITIETEEIIEITTTTTTIEIETETTTTIEITTTEIEETTEEETTETTTTTITTIEETTITTI 'CCGGCTCCTCRTCGCGAG/ AACCGGGTTTTTTTATTCAAGBATATCARCAACARACTGTG 103 Query 1345 Sbjct 404 HACE eee eee RGCCRRAGGTTTTGACTCGATTGTCCTTTTARAGGACGGATTATACRAAGTGCAGATTGG Sbjct Query Eee 43 RCCATGTTTCCAGACGGGTCGAC POPPE ACCATGTTTCCAGACGGGTCGAC 164 104 1344 44 1367 21 94 Phụ lục Phụ lục Kết Blast đoạn amyE-chIFNa, thực tế với dự kiến >1c1|19887 Length=1171 Score = 2130 Identities bits = (1153), 1172/1172 Expect (100%), Gaps = 0.0 = 0/1172 (0%) Strand=Plus/Plus Query TTGTGARGCTGGCTTACAGARGAGCGGTARRAGAAGAAATAAAAAAGAAATCATCTTTTT 64 THIETITIIIIIIIIITITIIIITIIIIIIIEITIIIRIIIEIIIIIIIEIIIIIIIIFIIIIII Sbjct TTGTGAAGC?GGCTTACAGARAGAGCGGTAAAAGBAGAAATAARRAAGAARTCATCTTTTT 60 Query 65 TGTTTGGAAAGCGAGGGAAGCGTTCACAGTTTCGGGCAGCrrrrrrrRTAGGAACATTGR 124 Sbjct 61 VTEC EERO TGTTTGGAAAGCGAGGGAAGCGTTCACAGTTTCGGGCAGCTTTTTTTATAGGAACATTGA 120 Query 125 Sbjct 121 Query 185 TTTGTATTCACTCTGCCAAGTTGTTTTGATAGAGTGATTGTGATAATTTTAAATGTAAGC EIEIIIITIIEIIIIIEIITITEETEIETITIITIEIEITIEEIIIIIEITIEITEIITITIIIIIIITEII TTTGTATTCACTCTGCCARGTTGTTTTGATAGAGTGATTGTGRTAATTTTAAATGTAAGC 184 GTTAACAAAATTCTCCAGTCTTCACATCGGTTTGAAAGGAGGAAGCGGAAGAATGAAGTA 244 TUTE 180 ‘Sbjct 181 GTTAACKAAATTCTCCAGTCTICACATCGGTTTGAAAGGAGGAAGCGGAAGAATGAAGTA 240, Query 265 AGAGGGATTTTTGACTCCGAAGTAAGTCTTCAAAAAATCAAATAAGGAGTGTCAAGAATG 304 THTEIITIITEIITIIITIIIIEIITIEIITTTITEIIEIIEIITEIIEITIEITTIITIII Sbjct 241 AGAGGGATTTTTGACTCCGAAGTAAGTCTTCAARAAATCLAATANGGAGTGTCAAGAATG 300 Query 305 TTTGCAAAACGATTCAAAACCTCTTTACTGCCGTTATTCGCTGGATTTTTATTGCTGTTT 364 Sbjct 201 Query TEIIITEIIIIIITIEIIEIIIIIIEIEIEIIIIITIEIIIIIEIIIIIIIIIIIEIIIIIIIIII TTTGCAAAACGATTCAAARCCTCTTTACTGCCGTTATTCSCTGGATTTTTATTGCTGTTT 360 365 CATTTGGTTCTGGCAGGSACCGGCGGCTGCGAGTGCTGARACGGCGRACARATCGAATGAG 424 Sbjct 361 POUCA EEE Pee eee eee CATTTGGTTCTGGCAGGACCGGCGGCTGCGAGTGCTGAAACGGCGAACAAATCGAATGAG 420 Query 425 Sbjct 421 TCTAGACGCACCACCACCGAGCCCCACCAGGCTCCTGCCCAGCACAACGCGAGTCCCACC 1I1111111111111111111111111111111111111111111111111111111111 TCTAGACGCACCACCACCGAGCCCCACCAGGCTCCTGCCCAGCACRACGCGAGTCCCACC 480 Query 485 ATGGCTGTGCCTGCAAGCCCACAGCACCCACGGGGGTACGGCATCCTGCTGCTCACGCTC 544 Sbjct 481 EHTTEETETEFEEIIEITTEETTEEEEITEHTTIIEETTTTEETTTEEEITTIEIETTITIEEEIIEIII ATGGCTGTGCCTGCAAGCCCACAGCACCCACGGGGGTACGGCATCCTGCTGCTCACGCTC 540 Query 545 Sbjct 541 CÝTCTGARAGCTCTCGCCRCCACCGCCTCCGCCTGCAACCACCTTCGCCCCCAGGATGCC TTC Eee CTTCTGARAGCTCTCGCCACCACCGCCTCCGCCTGCAACCACCTTCGCCCCCAGGATGCC 484 604 600 Phu luc Query 605 Sbjct 601 Query 665 Sbjct 661 Query Sbjct Query 785 Sbjct 781 Query 845 Sbjct 841 Query 905 Sbjct ACCTTCTCTCACGACAGCCTCCAGCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTG TT TTTETTTTEETEEEHEEEHITEEEHEIEEHTIETEEIHETEEEEEHEIELTTEEEITTII RCCTTCTCTCACGRCAGCCTCCAGCTCCTCCGGGACATGGCTCCCACACTACCCCAGCTG 664 TGCCCACAGCACARCGCGTCTTGCTCCTICAACGACACCATCCTGGACACCAGCAACACC HE COPE eee TECCCACAGCACAGCGCGTCTTGCTCCTTCAACGACACCATCCTGGACACCAGCAACACC 124 (CGGCAAGCCGACARAACCACCCACGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGC T84 EHTTTTEEHITEIEIHIETIEIHTEEETHEEETETHTTLEETEEEEHEEITTEEEEHTITETTEITITETTTIHI (CGGCAAGCCGACARAACCACCCACGACATCCTTCAGCACCTCTTCARAATCCTCAGCAGC T80 660 720 CCCAGCACTCCAGCCCACTGGAACGACAGCCAACGCCAAAGCCTCCTCAACCGGATCCAC LIEITITTTTITEIEEEEEEITTTTTEIETTTTTTITTTETEITETTIEIEETTITTTIITLTEETI 'CCCAGCACTCCAGCCCACTGGAACGACAGCCARCGCCAARGCCTCCTCARCCGGATCCAC B44 'CGCTACACCCAGCACCTCGAGCAATGCTTGGACAGCAGCGACRCGCGCTCCCGGACGCGA OA eee TOCC 'CGCTACACCCAGCACCTCGAGCRATGCTTGGACAGCAGCGRCACGCGCTCCCGGACGCGA 904 'TGGCCTCGCAACCTTCACCTCACCATChARRRRCACTTCAGCTGCCTCCACACCTTCCTC JEEITTTTTIEITIIIIITEIEITTTEITEIEEIEIEITITITTTITTTIITITTITIEITIITIIII 'TTGGCCTCGCAACCTTCACCTCACCATCARRAAACACTTCAGCTGCCTCCACRCCTTCCTC 964 840 900 959 Query 965 CRAGACAACGATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGG 1024 Sbjct s60 TOCCOA CAAGACAACGATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCETGCCTGS 1019 Query 1025 Sbjct 1084 1020 TTCCTGCACATCCACAACCTCACAGGCAACACGCGCACTTAGCCCCARACGCACCTCCCA LEEIITTIIIEITIIIIEIEIEITITEITTETIETTEIITIHTTTEIETETETEIEEEETETITITTTIEEIH TICCTGCACATCCACAACCTCAC.\GGCAACACGCGCACTTAGCCCCRAACGCACCTCCCA Query 1085 'CCCTTGTCCTATTTATCTATTTATTCAACTATTTATACAAACGCCTATTTATTCTTCTAT 1144 Sbjct 1080 Query 1145 Sbjct 1140 COCO 'CCCTTGTCCTATTTATCTATTTATTCRACTATTTATACAAACGCCTATTTATTCTTCTAT TTATTCTICTATTTATTCAGACAAAATAAAGC 1I1111111111/1111111111111111111 TTATTCTTCTATTTATTCAGACAAAATARAGC 1079 1139 1176 1171 96 ae Phụ lục Tài liệu tham khao Théng tin sir dung IFN/probiotic giới 1- Beliavskaia VA, Cherdyntseva NV, Bondarenko VM, Litviakov NV Biological effects of interferon, produced by recombinant bacteria of the probiotic preparation subalin Zh Mikrobiol Epidemiol Immunobiol 2003 Mar-Apr;(2):102-9 [Article in Russian] Abstract The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host 2- Beliavskaia VA, Ignat'ev GG, Cherdyntseva NV, Litviakov NV Adjuvant properties of subalin, a recombinant interferon-producing probiotic Zh Mikrobiol Epidemiol Immunobiol 2001 Nov-Dec:(6):77-82 [Article in Russian] Abstract The adjuvant properties of subalin, a recombinant probiotic prepared from live bacteria Bacillus subtilis producing human alpha 2-interferon were studied in the scheme of its use with vaccines against parvovirus enteritis and distemper Subalin was shown to be capable of preventing immunosuppression caused by the injection of vaccines, accelerating the formation of the antigen-specific clone of memory cells and enhancing antigen-specific immune response The mechanisms of the adjuvant effect of subalin were considered; this effect was shown to be due to the action of interferon excreted by bacteria of B subtilis into the lumen of the intestine The advantages of this method of interferon supply and the prospects of using subalin preparation as adjuvant are discussed Malik 3- Beliavskaia VA, Kashperova TA, Bondarenko VM, I'ichey AA, Sorokulova IB, a NI Experimental evaluation of the biological safety of gene-engineered bacteria using model strain Bacillus subtilis interferon-producing strain Zh Mikrobiol Epidemiol Immunobiol 2001 Mar-Apr:(2):16-20 [Article in Russian] Abstract The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants The present work is the first experimental evaluation of the biological safety of genetically modified for microorganisms, used as the component of Subalin, a probiotic preparation intended use in veterinary practice Sử Phụ lục 4- Cherdyntseva NV, Litviakov NV, Smol'ianinov ES, Beliavskaia VA, Masycheva VI Modulation of the antitumor effect of cyclophosphamide by the recombinant probiotic Subalin Vopr Onkol 1997;43(3):313-6 [Article in Russian] Abstract A new preparation "subalin" developed on the basis of a recombinant saprophyte strain of Bacillus subtilis 2135/105 producing human interferon alpha-2 has been tested for its ability of increasing the effectiveness of antitumor therapy The study used Lewis lung carcinoma bearing mice C57B1/6 treated with injections of 60 mg/kg body cyclophosphamide, intraperitoneally, on days and of tumor growth Subalin was administered per as on days 1-14 after tumor transplantation Combined treatment with cyclophosphamide and subalin resulted in a significantly higher inhibition of primary and metastatic spreading as compared with control receiving tumor growth cyclophosphamide alone Also, there were fewer animals with metastasis; the average number of these lesions per animal and the total affected area were less Subalin proved to exert a moderate antitumor and antimetastatic effect The cytostatic activity of peritoneal macrophages was higher in mice treated with subalin It is suggested that the chemosensitizing and antitumor effects of subalin are due to its ability to induce endogenous interferon production 5- Furzikova TM SergeAchuk MG, Sorokulova IB, Smirnov VV The effect of the cultivation conditions on the properties of bacilli comprising the basis of probiotics Mikrobiol Z 1999 Sep-Oct;61(5):19-27 [Article in Russian] Abstract Such biological factors as bile, gastric juice, blood serum, amino acids and pH of the medium have been studied for their effect on the growth intensity and antagonistic activity of bacilli being the basis of biosporin and subalin It has been established that pH of the medium within 7.0-9.0 as well as certain concentrations of the gastric juice, bile, blood serum and most of amino acids did not affect the growth of Bacillus subtilis and Bacillus subtilis 2335/105 A regular decrease of growth intensity in bacilli under the increase of the gastric juice concentration or decrease of pH of the medium to 5.0-2.0 is registered The mentioned biological factors affect differently the antagonistic properties of the studied cultures 6- Furzikova TM, Sorokulova IB, SerhiAchuk MH, Sichkar SV, Smirnov VV, The effect of antibiotic preparations and their combinations with probiotics on the intestinal microflora of mice Mikrobiol Z 2000 May-Jun;62(3):26-35 [Article in Ukrainian] Abstract Antibiotic drugs (biseptol, polymyxin, canamycin) as well as their combinations with probiotics biosporin and subalin have been studied for their effect on mice intestine microflora It has been established that under peroral administration of the above drugs dysbacteriosis of the 2-3rd degrees arises in animals; under their cornbined use with probiotics one can prevent the development of dysbacteriosis to considerable extent It has been shown that dysbacteriosis of the 2-3rd degrees can be liquidated for 5-7 days, using probiotics biosporin and subalin No self-recovery of normal intestinal microbiocenosis is observed in this period Inconsiderabie variations in the composition of normoflora under combined use of antibiotics with probiotics are renewed on the 3-5th day after stopping the drugs intake 98 Phu luc 7- Litviakov NV Cherdyntseva NV, Beliavskaia VA, Malinoyskaia EA, Ilinykh IS, Smol'ianinov ES Role of macrophages in the antitumor action of the recombinant probiotic Subalin Vopr Onkol 2001;47(1):86-9 [Article in Russian] Abstract Course administration of the recombinant probiotic subalin was shown to significantly potentiate the cytotoxic action of macrophages from healthy and tumor-bearing mice against mastocytoma P-815 cells and syngeneic target cells of lung sarcoma of Lewis Carrageenan, an inhibitor of macrophage activity, significantly cut down the antimetastaic effect of subalin The crucial role of macrophages in the mechanism of this function of the drug is discussed 8- Osipova IG, Sorokulova IB Tereshkina NV, Grigor'eva LV Safety of bacteria of the genus Bacillus, forming the base of some probiotics Zh Mikrobiol Epidemiol Immunobiol 1998 Nov-Dec:(6):68-70 [Article in Russian] Abstract The study of the safety of bacillary strains forming the base of new probiotics (biosporin and subalin) was made For control, the safety of Bacillus sp IP5832 (the base of the preparation of bactisubtil) was studied The results obtained in this investigation demonstrated that the strains contained in biosporin and subalin were safe when injected intravenously and intraperitoneally into animals in dose of x 10(9) cells per 0.5 ml of physiological saline The introduction of Bacillus sp IP5832 in the same dose produced 100% lethal effect 9- Potebnia HP Safronova LA Cheremshenko NL, Lisovenko HS, Sorokulova IB probiotic Prykhod'ko VO, Trokhymenko NV, Tanasiienko OA, Bombin AV Influence of subalin on efficiency [Article in Ukrainian] Abstract of antitumor vaccine Mikrobiol Z 2006 Nov-Dec:68(6):51-8 Préparation subalin created on the basis of a recombinant strain of B subtilis 2335/105 containing the gene of synthesis of human a-2-interferon has been tested in the experiment for its ability of increasing the efficiency of antitumor vaccine prepared from syngenic tumor cells and cytotoxic lectin-the metabolism product of the strain of B subtilis B- 7025 On the models of Lewis lung carcinoma of C57B1 mice and sarcoma-37 of Balb/c for the mice it was shown that the complex use of the antitumor vaccine and subalin makes more efficient tumor growth suppression and survival of treated animals as compared with the separate use of autovaccine or subalin The prospects and expediency of the complex use of vaccine and subalin at malignant tumors immunotherapy were established 10- Sirokvasha EA Paran'ko SI, Kozitskaia SN Vinnikov Al Investigation of subalin effect on urogenital microflora of pregnant women Mikrobiol Z 2002 Jan-Feb;64(1):2730 [Article in Russian] Abstract Bacteriological examination of 30 women with incomplete pregnancy has been conducted Representatives of 18 genera of aerobic and anaerobic bacteria as well as yeast-like fungi of Candida genus were found It has been established that spore-forming bacteria of the Bacillus genus from subalin preparation have antagonistic activity in respect of the strains of of pathogenic and opportunistic bacteria with characteristic zones of growth depression 12-15 mm This demonstrates that the agents of infectional urogenital process are effectively suppressed by subalin 89 Phụ lục 11- Smirnov VV, Rudenko AV, Samgorodskaia NV, Sorokulova IB, Reznik SR, SergeAchuk TM Susceptibility to antimicrobial drugs of strains of bacilli used as a basis for various probiotics Antibiot Khimioter 1994 Apr:39(4):23-8 [Article in Russian] Abstract Five strains of the genus Bacillus used as components of biological preparations i.e bacterin SL, biosporin, bactisubtil and subalin were tested for their susceptibility to 38 antimicrobial agents, The strains were simultaneously resistant to antibiotics: astreonam, colistin, penicillin, ceftizoxime and cefuroxime High susceptibility of the strains to some penicillins, cephalosporins and aminoglycosides was recorded The gene engineered strain B subtilis 2335(105) marked by the kanamycin resistance gene showed cross resistance to amikacin and tobramycin Trimetoprim, sulfotrimetoprim and norfloxacin proved to be active against the strains 12- Sorokulova IB Beliavskaia VA, Masycheva VA, Smirnov VV Recombinant probiotics: problems and prospects of their use for medicine and veterinary practice Vestn Ross Akad Med Nauk 1997:(3):46-9 [Article in Russian] Abstract Approaches to designing a new probiotic class based on recombinant strains of bacteria that produce the predetermined therapeutic proteins are dealt with The prospects of the approach are shown via studies of the biological properties of Bacillus subtilis 2335 strain transformed by the plasmid encoding the synthesis of human interferon alpha-2 The recombinant strain was demonstrated to preserve the high antagonistic activity of the parent culture (the bases of the probiotic biosporine) and to acquire marked antiviral properties due to interferon synthesis The antiviral activity of the designed strain was shown by in vitro and in vivo experiments on experimental viral infections By using this strain, the authors designed the new probiotic subaline, a promising biological agent for medicine and veterinary practice Subalin has a number of advantages: it combines antibacterial and antiviral properties, is easy to use and prepare 13- Sorokulova IB, Kirik DL, Pinchuk IV Probiotics against Campylobacter Pathogens J Travel Med 1997 Dec 1:4(4):167-170 Abstract Background: The subject matter of this study was to investigate, for the first time, the immediate-preventive effect of probiotics, composed of bacillus species, in a murine model of Campylobacter infection Methods: An established model of Campylobacter infection in mice with a defined LD50 was utilized to assess the protective effect of probiotics Results: The results obtained demonstrate that the level of animal protection, after a single administration of the new probiotics biosporin and subalin, reached 90-100% at LD50 and 80% at LD100 Conclusions: Such efficacy of probiotics is considered to be due to their high antagonistic activity against those pathogens registered in vitro Antagonistic activity of other tested probiotics (bactisubtil and cereobiogen) to different cultures of Campylobacter was not manifested 14- Sorokulova IB Effect of probiotics from bacilli on macrophage functional activity Antibiot Khimioter 1998:43(2):20-3, [Article in Russian] Abstract The influence of new probiotics from bacilli ie biosporin and subalin on the functional activity of murine peritoneal macrophages was studied After a single oral administration of the probiotics the maximum activation of the macrophages was observed in hours The activation level depended on both the dose and the probiotic The highest value of the 100 Phụ lục stimulation index was recorded with subalin Analogous regularities were stated with parenteral administration of the probiotics Some mechanisms of activation of peritoneal macrophages after oral administration of probiotics from live microbial cultures are discussed 15- Sorokulova IB Outlook for using bacteria of the genus Bacillus for the design of new biopreparations Antibiot Khimioter 1996;41(10):13-5 [Article in Russian] Abstract The data evident of the promising use of Bacillus as a basis of new probiotics are presented The criteria of the Bacillus screening among the other representatives of the exogenic microflora provided the design of a probiotic, named biosporin, which markedly differed from the other biological preparations based on aerobic sporulating bacteria Biosporin proved to be an efficient agent in the treatment of acute intestinal infection due to pathogenic and opportunistic bacteria as well as in the treatment of dysbacteriosis of various etiology A principally new probiotic, named subalin, with antibacterial and antiviral properties was designed on the basis of biosporin by the gene engineering The data on the promising use of Bacillus in the composition of complex probiotics are also presented 16- Sorokulova IB The safety and reactogenicity of the new probiotic subalin for volunteers Mikrobiol Z 1998 Jan-Feb:60(1):43-6 [Article in Russian] Abstract Data are presented on the study of innocuousness and reactogenicity of a new recombinant probiotic subalin characterized by antibacterial and antiviral activity Volunteers who took subalin (one dose twice a day for 10 days) were under observation at Kyiv ScientificResearch Institute of Epidemiology and Infectious Diseases of the Ministry of Public Health of Ukraine They carried out clinical and bacteriological investigations during administration and after termination of the course of oral administration of subalin testified to its innocuousness as well as to the absence of any side-effects 101
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