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为了解析cagT蛋白的结构,进而了解cagT基因在幽门螺杆菌致病中的作用,应用PCR技术从幽门螺杆菌26695菌株基因组中扩增cagT基因,T/A法克隆,连接至pET-28a(+)载体,转化宿主细胞E.coliBL21(DE3),IPTG诱导目的蛋白表达,亲和层析法纯化目的蛋白,免疫家兔制备抗体并鉴定其抗原性.成功克隆cagT基因,重组cagT基因片段的测序结果与GenBank上公布的序列相同.重组cagT蛋白以可溶和包涵体两种形式表达,免疫家兔所得的抗体滴度为1∶32.结果构建了高效表达cagT蛋白的重组载体pET-28a(+)-cagT,表达的蛋白具有良好的免疫原性,为后续的结构和功能研究奠定了基础.
In order to analyze the structure of cagT protein and further understand the role of cagT gene in the pathogenesis of H. pylori, cagT gene was amplified from the genome of H. pylori strain 26695 by PCR and cloned into pET-28a (+ ) Was transformed into host E.coli BL21 (DE3), IPTG was used to induce the expression of the target protein, and the recombinant protein was purified by affinity chromatography and immunized to prepare the antibody and identify the antigenicity of the recombinant plasmid. Sequencing of cagT gene and recombinant cagT gene fragment The result was the same as the sequence published in GenBank.The recombinant cagT protein was expressed in both soluble and inclusion bodies and the antibody titers of immunized rabbits were 1:32.Results The recombinant vector pET-28a +) - cagT, the expressed protein has good immunogenicity, which lays the foundation for the subsequent study of structure and function.