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目的:构建含鼠疫杆菌F1抗原结构基因caf1的重组载体,并在酿酒酵母中表达。方法:将鼠疫杆菌的F1抗原结构基因caf1插入到pHSS6mTn3xHA/lacZ质粒中,构建重组载体pHSS6mTn3xHA/lacZcaf1。将重组载体经NotI酶切后,以醋酸锂法转化酿酒酵母,将转化的酿酒酵母接种于SCUra平板上筛选阳性克隆,表达产物用SDSPAGE和Westernblot进行鉴定。结果:经SDSPAGE鉴定和Westernblot分析表明,转化的酿酒酵母可表达鼠疫杆菌F1表面抗原蛋白。结论:成功地构建了重组载体pHSS6mTn3xHA/lacZcaf1,并在酿酒酵母中稳定表达鼠疫杆菌F1表面抗原,为制备可经消化道途径免疫的鼠疫杆菌基因疫苗奠定了基础。
OBJECTIVE: To construct a recombinant vector containing caf1 gene of F1 antigen of Yersinia pestis and express it in Saccharomyces cerevisiae. Methods: The F1 gene of Y. pestis caf1 was inserted into pHSS6mTn3xHA / lacZ plasmid to construct recombinant vector pHSS6mTn3xHA / lacZcaf1. After the recombinant vector was digested with NotI, Saccharomyces cerevisiae was transformed by lithium acetate, and the transformed Saccharomyces cerevisiae was inoculated on SCUra plate to screen positive clones. The expressed product was identified by SDSPAGE and Western blot. Results: The identification of SDSPAGE and Western blot analysis showed that the transformed Saccharomyces cerevisiae could express the surface antigen protein of Y. pestis F1. Conclusion: The recombinant vector pHSS6mTn3xHA / lacZcaf1 was successfully constructed and stably expressed the surface antigens of S.foetii F1 in Saccharomyces cerevisiae, which laid the foundation for the preparation of the gene vaccine of Yersinia pestis that can be immunized by the digestive tract.