结核杆菌保护性抗原-遍在蛋白质系统的建立

Construction of Mycobacterium tuberculosis protective antigens-ubiquitin system

目的 :将结核杆菌 4种保护性抗原基因与小鼠遍在蛋白质 (泛素 )基因融合 ,建立抗原 -遍在蛋白质系统。方法 :通过RT- PCR技术从小鼠睾丸组织中克隆遍在蛋白质 c DNA,同时培养结核杆菌并从基因组中克隆出 4种保护性抗原编码区 ,然后通过柔性接头将遍在蛋白质与 4种抗原的基因分别融合在一起 ,并插入到 p VAX质粒中。结果 :扩增出来的 4种抗原基因大小分别约为 0 .3、0 .7、1.0、1.6 5 kb,遍在蛋白质 PCR产物约为 0 .2 kb,均与 Gen Bank中登录的相符 ;p VAX重组质粒酶切结果显示融合基因正确地插入到 p VAX载体中 ,全自动测序显示抗原基因和遍在蛋白质基因均为所需基因 ,柔性接头序列也完全正确。 结论 :成功地构建了中国株结核杆菌的 4种保护性抗原基因 -遍在蛋白质融合系统 。

Objective: To fuse Mycobacterium tuberculosis protective antigen gene with mice ubiquitin gene, constructing antigen ubiquitin system. Methods: Mice ubiquitin cDNA was amplified by RT PCR from mice testicle,and 4 antigen genes were obtained by PCR from cultured Mycobacterium tuberculosis . Ubiquitin and 4 antigen genes were linked by flexible adaptor respectively and the fusing genes were cloned into pVAX vector.The recombinant plasmids were digested with endonuclease and sequenced.Then the recombinant plasmids were transfected into COS7 cells and the expression was assayed by ELISA. Results:Ubiquitin and 4 antigen genes were 0.2,0.3,0.7,1.0,1.65 kb in length by agarose electrophoresis. Endonuclease digestion of the recombinant plasmids indicated that the fusion genes were correctly inserted into pVAX vector. Sequencing results of fusion nucleic acid vaccines were identical to those in GenBank.The recombinant plasmids expressed in COS7 cells. Conclusion: Four Chinese Mycobacterium tuberculosis protective antigens ubiquitin systems are successfully constructed and can be expressed in eukaryotic cells. This may provide a basis for diagnosis and therapy of tuberculosis.