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研究目的是构建HBVS基因和截短C基因融合的胞壁型和分泌型大肠杆菌和分枝杆菌(E.coli-BCG)穿梭质粒。采用PCR方法从结核分枝杆菌MTB毒株H37Rv的基因中扩增出相对分子质量为19000的抗原胞壁区及其上游调控元件基因,克隆入穿梭载体pOLYG中。以含HBV基因组的质粒pCP10序列为模板,PCR扩增获得S基因片段Sw和C基因编码氨基端的部分基因片段Ct,克隆入胞壁型穿梭表达载体pCW和分泌型穿梭表达载体pDE22。经酶切和序列测定证实,胞壁型和分泌型载体pCW-Sw-Ct和pDE22-Sw-Ct构建成功。为进一步研究含S基因和截短C基因融合的重组BCG活疫苗奠定了基础。
The aim of the study was to construct cell-wall and secretory Escherichia coli and mycobacterial (BCG) shuttle plasmids with HBVS gene and truncated C gene fusion. A 19000 molecular weight antigen cell wall region and its upstream regulatory gene were amplified by PCR from the Mycobacterium tuberculosis MTB strain H37Rv and cloned into the shuttle vector pOLYG. The partial Ct fragment encoding the amino terminus of the Sw and C genes of S gene fragment was amplified by PCR using the plasmid pCP10 containing the HBV genome as a template and cloned into the shuttle plasmid pCW and pDE22. After digestion and sequencing, we confirmed that the cell wall and secretory vectors pCW-Sw-Ct and pDE22-Sw-Ct were successfully constructed. Which laid the foundation for further study on the recombinant BCG live vaccine containing S gene and truncated C gene.