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 iii iv Nguyễn Thị Minh Hằng iv 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 1.1 Hội chứng rối loạn sinh sản hô hấp lợn 1.1.1 Sơ lược tình hình dịch bệnh 1.1.2 Bệnh tích PRRS 1.1.3 Virus PRRS 1.2 Vaccine phòng PRRS .20 1.2.1 Vaccine sống nhược độc 21 1.2.2 Vaccine vô hoạt 23 1.2.3 Các dạng vaccine thử nghiệm dựa vào kháng nguyên GP5 M chống PRRSV 24 1.3 Biểu tạm thời kháng nguyên PRRSV phương pháp thẩm lọc nhờ Agrobacterium 30 1.3.1 Lợi phương pháp biểu gen tạm thời nhờ Agrobacterium 30 ii iii 1.3.2 Quá trình thẩm lọc nhờ A tumefaciens 33 1.3.3 Một số giải pháp tăng cường biểu gen tạm thời thực vật 35 Chương VẬT LIỆU VÀ PHƯƠNG PHÁP NGHIÊN CỨU 43 2.1 Vật liệu 43 2.1.1 Chủng vi khuẩn 43 2.1.2 Các vector vật liệu thực vật, động vật 43 2.1.3 Các cặp mồi sử dụng nghiên cứu 44 2.1.4 Hoá chất 45 2.1.5 Thiết bị 45 2.2 Phương pháp nghiên cứu 46 2.2.1 Nhân dòng gen mã hố protein GP5, M GP5ectoM virus PRRS 46 2.2.2 Thiết kế cấu trúc vector biểu mang gen m, gp5opt gp5ecto-m phục vụ chuyển gen 47 2.2.3 Biểu tạm thời gen mã hoá kháng nguyên tái tổ hợp thuốc N benthamiana iii iv 49 2.2.4 Đánh giá ảnh hưởng điều kiện biểu gen tạm thời đến mức độ biểu protein M-ELP, GP5-ELP GP5ectoM-ELP thuốc 50 2.2.5 Tách chiết xác định nồng độ protein tổng số 52 2.2.6 Điện di SDS-PAGE lai Western blot 52 2.2.7 Tinh protein M-ELP, GP5-ELP GP5ectoM-ELP 53 2.2.8 Phương pháp đánh giá tính sinh miễn dịch kháng nguyên GP5ELP, GP5ectoM-ELP, M-ELP động vật thí nghiệm 54 Chương KẾT QUẢ NGHIÊN CỨU 61 3.1 Thiết kế vector chuyển gen thực vật mang gen m, gp5, gp5ecto-m tạo chủng A tumefaciens mang vector tương ứng 61 3.1.1 Vector chuyển gen mang gen mã hoá kháng nguyên M dung hợp ELP (MELP) 61 3.1.2 Vector chuyển gen mang gen mã hoá kháng nguyên GP5 dung hợp ELP (GP5-ELP) 65 3.1.3 Vector chuyển gen mang gen mã hoá kháng nguyên GP5ectoM dung hợp ELP (GP5ectoM-ELP) iv v 69 3.2 Tối ưu điều kiện biểu tạm thời gen mã hoá kháng nguyên M -ELP, GP5-ELP, GP5ectoM-ELP thuốc N benthamiana 74 3.2.1 Ảnh hưởng vector hỗ trợ 75 3.2.2 Ảnh hưởng nồng độ AS 76 3.2.3 Ảnh hưởng mật độ vi khuẩn A tumefaciens 78 3.2.4 Ảnh hưởng tuổi 79 3.2.5 Ảnh hưởng tuổi 80 3.2.6 So sánh mức độ biểu gen mã hoá kháng nguyên M-ELP, GP5-ELP, GP5ectoM-ELP điều kiện tối ưu lai miễn dịch 81 3.3 Tinh kháng nguyên M-ELP, GP5-ELP GP5ectoM-ELP 82 3.3.1 Tối ưu nồng độ PEG 8000 cho trình tinh thu nhận kháng nguyên 83 3.3.2 Tinh kháng nguyên phương pháp mITC 86 3.4 Tính sinh miễn dịch dịch thể kháng nguyên tái tổ hợp động vật thí nghiệm 91 v vi 3.4.1 Đáp ứng kháng thể IgG đặc hiệu chuột 91 3.4.2 Đáp ứng kháng thể IgG đặc hiệu lợn 98 Chương BÀN LUẬN KẾT QUẢ NGHIÊN CỨU 108 4.1 Biểu tạm thời gen mã hoá kháng nguyên M-ELP, GP5-ELP GP5ectoM-ELP phương pháp thẩm lọc nhờ Agrobacterium 108 4.1.1 Lựa chọn kháng nguyên thiết kế vector biểu 108 4.1.2 Mức độ biểu tạm thời kháng nguyên tái tổ hợp 109 4.1.3 Cấu trúc vector biểu mức độ biểu gen tạm thời thực vật 110 4.2 Khả kích thích sản sinh kháng thể IgG đặc hiệu chuột 116 4.3 Tính sinh miễn dịch dịch thể lợn .120 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 PHỤ LỤC I vi 149 porcine monocytic cell line supporting porcine 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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. .. dựa vào kháng nguyên GP5 M chống PRRSV 24 1.3 Biểu tạm thời kháng nguyên PRRSV phương pháp thẩm lọc nhờ Agrobacterium 30 1.3.1 Lợi phương pháp biểu gen tạm thời nhờ Agrobacterium