PstS1-卡介苗重组疫苗的构建

目的：构建结核分支杆菌分泌蛋白磷酸盐特异转运系统1（PstS1）的重组卡介苗。方法:以结核分支杆菌H37Rv基因组为模板，通过PCR扩增获得PstS1基因序列；将PstS1基因与大肠杆菌-卡介苗穿梭表达质粒PMD31重组，得到S-PMD31重组质粒，对S-PMD31重组质粒进行HindⅢ、BanHⅠ双酶切及测序鉴定，转化大肠杆菌，用IPTG对工程菌株进行诱导表达，并通过电穿孔技术将S-PMD31重组质粒导入卡介苗中构建重组卡介苗。结果：用引物P1、P2对结核分枝杆菌H37R_V基因组进行PCR扩增获得长为1 100 bp的PstS1基因序列，S-PMD31重组质粒的HindⅢ、BanHⅠ双酶切及测序鉴定证实：PstS1基因正确插入载体PMD31中，并能在大肠杆菌中诱导表达。通过质粒提取及PCR扩增表明重组质粒正确导入卡介苗中。结论：成功地构建了PstS1重组卡介苗，预期能提高卡介苗的免疫原性和保护力。

Construction of PstS1 -BCG recombinant vaccine

Objective To construct phosphate-specific transport system 1(PstS1)-BCG recombinant vaccine.Methods PstS1 gene was amplified from the genome of mycobacterium tuberculosis H37Rv and was cloned into E.coli-M.bovis BCG shuttle plasmid PMD31 to get recombinant plasmid S-PMD31.The S-PMD31 was introduced into E.coli and PstS1 expressed in high level induced by IPTG.S-PMD31 was then introduced into BCG by transformation or electroporation.Results 1 100 bp PstS1 gene was amplified from the genome of mycobacterium tuberculosis H37Rv,and then was cloned into E.coli-M.Bovis BCG shuttle plasmid PMD31.The PstS1 gene was correcctly inserted into shuttle plasmid PMD31 confirmed by restriction endonuclease digestion analyzing and DNA sequencing.PstS1 protein could be expressed in E.coli and then was introduced into M.bovis BCG by electroporation.Conclusion The recombinant PstS1-BCG vaccine is successfully constructed.It is anticipate that the PstS1-BCG vaccine can improve the immunogenicity and protective efficacy of BCG vaccine.