Tạo dòng, biểu hiện, tinh sạch và đánh giá khả năng tương tác của 30 amino acid vùng đầu C của độc tố vi khuẩn Clostridium perfringens với thụ thể Claudin-4=Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

0 Người đánh giá. Xếp hạng trung bình 0

Tác giả: Kiến Quang Huỳnh, Hoàng An Nguyễn, Phước Khải Hoàn Nguyễn, Thị Mộng Quỳnh Phạm, Văn Hiếu Trần

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Phát triển Khoa học & Công nghệ: Chuyên san Khoa học Tự nhiên, 2018

Mô tả vật lý:

Bộ sưu tập: Báo, Tạp chí

ID: 473496

Thêm vào giỏ

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivoVaccine đường uống là chiến lược đang được quan tâm nhất hiện nay trong việc điều trị các bệnh về đường tiêu hóa do nhiễm khuẩn với nhiều ưu điểm nổi bật hơn hẳn so với vaccine truyền thống ở dạng tiêm. Nhằm giải quyết tình trạng dung nạp miễn dịch và sự phân tán kháng nguyên ở ruột, cũng như có thể kích thích đáp ứng miễn dịch hiệu quả, tế bào M hiện diện trong đường ruột là mục tiêu cần nhắm trúng đích cho việc vận chuyển kháng nguyên. Nhiều nghiên cứu cho thấy, 30 amino acid của vùng đầu C độc tố loài Clostridium perfringens (CPE30) có khả năng bám với thụ thể Claudin-4 trên bề mặt tế bào M. Để tạo nguồn nguyên liệu cho việc đánh giá khả năng bám dính của CPE30, gene cpe30 được dòng hóa vào vector pET-gfp bởi hai enzyme cắt giới hạn BamHI và NdeI trên chủng E. coli DH5α. Kết quả biểu hiện và xác nhận sự biểu hiện của protein dung hợp CPE30-GFP được cảm ứng bằng IPTG trong tế bào chủ E. coli BL21(DE3) bằng phương pháp SDS PAGE và lai Western với kháng thể kháng 6xHis Tag, cho thấy khả năng biểu hiện vượt mức protein CPE30-GFP trong tế bào chất và chủ yếu ở dạng tan. Đồng thời, CPE30-GFP đã bước đầu được tinh sạch với độ tinh sạch khoảng 92,3%. Thử nghiệm tương tác in vitro trên chip bán dẫn silicon đo điện trường (SiNW FETs) cho thấy CPE30 GFP tương tác tốt với thụ thể Claudin-4 (R4). Kết quả này tạo tiền đề cho việc nghiên cứu tiếp theo sự tương tác CPE30 với tế bào M in vivo

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