结核DNA疫苗质粒pVAX1-Rv3407的构建及体外表达

Construction and in vitro expression of plasmid pVAX1-Rv3407 as a DNA vaccine against tuberculosis

目的构建结核DNA疫苗质粒pVAX1-Rv3407,并检测其在哺乳动物细胞293T中的表达。方法以基因重组卡介苗(rBCG)AERAS-422基因组为模板,PCR扩增Rv3407基因,构建真核表达质粒pVAX1-Rv3407,转染293T细胞,采用免疫荧光法及Western blot法检测Rv3407蛋白的表达。结果 PCR扩增获得长300 bp的Rv3407基因片段;重组真核表达质粒pVAX1-Rv3407经双酶切及DNA测序证明构建正确;转染48 h后,Rv3407蛋白在293T细胞中有效表达,且主要分布在细胞质中。结论成功构建了结核DNA疫苗质粒pVAX1-Rv3407,为新型结核病疫苗的研制奠定了基础。

Object ive To construct plasmid pVAX1-Rv3407 as a DNA vaccine against tuberculosis and determine its expression in mammal cells 293T. Methods Rv3407 gene was amplified by PCR using the AERAS-422 genome of recombinant BCG （rBCG） as a template, based on which recombinant plasmid pVAX1-Rv3407 was constructed and transfected to 293T cells. The expression of Rv3407 protein was identified by IFA and Western blot. Results Rv3407 gene fragment at a length of 300 bp was amplified by PCR. Restriction analysis and DNA sequencing proved that recombinant plasmid pVAX1-Rv3407 was constructed correctly. Rv3407 protein was effectively expressed in 293T cells 48 h after transfection, which was mainly distributed in cytoplasm. Conclusion Recombinant plasmid pVAX1-Rv3407 as a DNA vaccine against tuberctdosis was successfully constructed, which laid a foundation of develoDinz novel tuberculosis vaccine.