Tạo dòng và biểu hiện gien mã hóa kháng nguyên Glyceraldehyde - 3- phosphate dehydrogenase (GAPDH) của Edwardsiella ictaluri trong E.ColiBL21 (DE3)

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Tác giả: Thị Bích Lân Đinh, Hữu Rin Đồng, Viết Quân Lê, Viết Tuấn Khanh Lê, Thăng Long Phùng

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Khoa học kỹ thuật Thú y, 2023

Mô tả vật lý: tr.32

Bộ sưu tập: Metadata

ID: 346577

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In this study, we successfully cloned and expressed the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antigen protein of Edwardsiella ictaluri isolated from tissue samples of bacillary necrosis diseased striped catfish (Pangasianodon hypophthalmus) in An Giang province, Viet Nam. The gene encoding GAPDH antigen was cloned into vector pGEM®-T Easy and expressed with vector pET200/D-TOPO in Escherichia coli BL21 (DE3) cells. The results of sequencing showed that the nucleotide sequence of GAPDH gene was 996 bp in length, encoding a polypeptide with 331 amino acid residues, and 99% similarity to the polypeptide chain of recorded GAPDH antigen in GenBank (accession number: AVZ80933.1). The result of expressed protein analysis on 15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel showed that molecular mass of fusion protein was approximately 39 kDa, and direct ELISA determined that expressed protein was the 6xHisGAPDH recombinant protein.Trong nghiên cứu này, chúng tôi đã tạo dòng và biểu hiện thành công gen mã hóa protein kháng nguyên Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) của Edwardsiella ictaluri phân lập từ mẫu cá tra (Pangasianodon hypophthalmus) bị nhiễm bệnh gan thận mủ ở tỉnh An Giang, Việt Nam. Gen mã hóa cho kháng nguyên GAPDH được tạo dòng vào vector pGEM®-T Easy, sau đó được biểu hiện bởi vector pET200/D-TOPO trong tế bào Escherichia coli BL21 (DE3). Kết quả giải trình tự cho thấy gen GAPDH của Edwardsiella ictaluri có chiều dài 996 bp, mã hóa một chuỗi polypeptide dài 331 amino acid, liên tục, và tương đồng 99% so với trình tự chuỗi polypeptide củakháng nguyên GAPDH đã được công bố trên Ngân hàng Gen (GenBank) (mã số AVZ80933.1). Kết quả điện di protein được biểu hiện trên gel sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) 15% cho thấy protein dung hợp trong thể vùi có khối lượng phân tử khoảng 39 kDa, và ELISA trực tiếp khẳng định rằng protein được biểu hiện là protein kháng nguyên tái tổ hợp 6xHis-GAPDH.

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