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目的在减毒炭疽芽孢杆菌AP422中表达BslA蛋白,并分析其生物学活性。方法利用PCR方法从炭疽芽孢杆菌中扩增出bslA基因,克隆至穿梭质粒pDG148,采用电转化方法将表达载体转入减毒炭疽芽孢杆菌AP422后诱导表达重组BslA。并利用SDS-PAGE电泳、Western印迹、间接免疫荧光实验和流式细胞术分析目标蛋白的表达,并对其进行空间定位,最后用细菌黏附实验分析其生物学功能。结果 BslA蛋白在AP422中获得成功表达,且目标蛋白主要表达于菌体表面。表达BslA蛋白的AP422(pDG-BslA)菌株具有黏附宿主细胞表面的能力,该黏附能力与阳性对照A16R菌株基本一致。结论在减毒炭疽芽孢杆菌AP422中实现了BslA蛋白的功能性表达,为进一步研究该蛋白的功能和构建新的疫苗候选株奠定了实验基础。
Objective To express BslA protein in attenuated Bacillus anthracis AP422 and analyze its biological activity. Methods The bslA gene was amplified from Bacillus anthracis by PCR and cloned into the shuttle plasmid pDG148. The recombinant plasmid was transformed into attenuated Bacillus anthracis AP422 by electroporation and then induced to express recombinant BslA. SDS-PAGE electrophoresis, Western blotting, indirect immunofluorescence assay and flow cytometry were used to analyze the expression of the target protein. The biological function of the target protein was analyzed by bacterial adhesion assay. Results The BslA protein was successfully expressed in AP422, and the target protein was mainly expressed on the cell surface. The AP422 (pDG-BslA) strain expressing the BslA protein has the ability to adhere to the host cell surface in a manner that is substantially consistent with the positive control A16R strain. Conclusion The functional expression of BslA protein was achieved in attenuated Bacillus anthracis AP422, which laid the foundation for further study on the function of this protein and construction of a new vaccine candidate strain.