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【目的】克隆表达炭疽芽胞杆菌BlsA的功能区片段并对其生物学功能进行鉴定。【方法】以炭疽芽胞杆菌A16R基因组DNA为模板PCR扩增bslA(260-652)基因片段,克隆至pET-28a(+)载体。将成功构建的重组质粒转化入大肠杆菌Rosetta(DE3)中,诱导表达后收集菌体经超声破碎后,对可溶表达部分用镍柱进行亲和层析纯化。以纯化后的蛋白为抗原,免疫BALB/c小鼠制备该蛋白的多抗,用ELISA和Western blot检测抗血清;使用间接免疫荧光实验和细菌黏附实验研究目标蛋白及其抗体的生物学功能。【结果】BslA(260-652)获得了可溶性表达,纯化后纯度约为87.4%。以纯化蛋白为抗原,免疫BALB/c小鼠制备的抗血清ELISA效价可达1∶20000。将BslA(260-652)蛋白与Hela细胞共孵育后,能够直接和Hela的细胞膜结合。细菌黏附实验表明BslA(260-652)蛋白及其相应的多抗血清都能够显著地抑制炭疽芽胞杆菌A16R对Hela细胞的黏附。【结论】大肠杆菌表达得到的炭疽芽胞杆菌BslA(260-652)蛋白具有与天然蛋白相似的生物活性,为深入研究BslA蛋白在炭疽芽胞杆菌致病过程中的作用奠定实验基础。
【Objective】 The objective of this study is to clone and express the functional region of Bacillus anthracis BlsA and to identify its biological function. 【Method】 bslA (260-652) gene fragment was amplified by PCR from Bacillus anthracis A16R genomic DNA and cloned into pET-28a (+) vector. The successfully constructed recombinant plasmid was transformed into Escherichia coli Rosetta (DE3), and after the expression was induced, the collected bacterial cells were sonicated, and the soluble expression part was purified by affinity chromatography with a nickel column. The purified protein was used as antigen to immunize BALB / c mice to prepare polyclonal antibody. The antiserum was detected by ELISA and Western blot. The biological function of target protein and its antibody was studied by indirect immunofluorescence assay and bacterial adhesion assay. 【Result】 Soluble expression of BslA (260-652) was obtained. Purity was about 87.4% after purification. The antiserum ELISA titer prepared by immunizing BALB / c mice with the purified protein as antigen was up to 1: 20000. After BslA (260-652) protein is incubated with Hela cells, it can directly bind Hela cell membrane. Bacterial adhesion experiments showed that BslA (260-652) protein and its corresponding multi-antiserum could significantly inhibit the adhesion of Bacillus anthracis A16R to Hela cells. 【Conclusion】 Bacillus anthracis BslA (260-652) expressed in Escherichia coli has biological activity similar to that of natural protein, which lays an experimental foundation for further study on the role of BslA protein in the pathogenesis of Bacillus anthracis.