融合表达Ag85BESAT6的结核分枝杆菌真核表达载体的构建及其免疫原性
分枝杆菌,,分枝杆菌,结核;Ag85B;ESAT6;融合表达;遗传载体,0引言,1材料和方法,2结果,3讨论,【参考文
Construction of Mycobacterium tuberculosis eukaryotic expression vector fused expression Ag85BESAT6 and its immunological propertiesSHI ChangHong, FAN XiongLin, XU ZhiKai, LI Yuan, BAI YinLan, XUE Ying
Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xian 710033, China
【Abstract】 AIM: To construct eukaryotic expression vector expressing secreted protein Ag85BESAT6 of Mycobacterium tuberculosis (MTB), and to investigate the expression in vitro and the production of humoral immunity response immunized with those recombinant plasmids. METHODS: The gene encoding Ag85B and ESAT6 protein were amplified by polymerase chain reaction (PCR) from genome of MTB H37Rv strain, and inserted into cloning vector pGEMTeasy. After sequence analyzed, digested by restriction endonuclease and cloned Ag85BESAT6 into corresponding sites of the eukaryotic expression vector pcDNA3, and named AZpcDNA3EF and EZpcDNA3AF respectively. Each plasmid was transfected into CHO cells by electroporation, expression was analyzed by RTPCR at the level of mRNA, by indirect immunofluorescence technique (IIFT)at the level of protein.The specific antibody titers in the sera of BALB/c mice immunized with two recombinant plasmid were detected by ELISA. RESULTS: Sequences of Ag85B and ESAT6 by PCR amplified were identical with that of GenBank reported. The specific mRNA of the fused protein could be detected by RTPCR. CHO cell transfected with two recombinant plasmids were both observed fluorescence by IIFT. Recombinant plasmid could induce higher titer specific antibody in BALB/c mice. The sera specific antibody titer of mice immunized with AZpcDNA3EF is 1∶1000, and that of mouse immunized with EZpcDNA3AF is 1∶1500. CONCLUSION: Secreted protein Ag85B and ESAT6 of MTB was successfully fusion expressed in eukaryotic expression vector. It may be a promising preventive DNA vaccine against TB. ......
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