结核分枝杆菌抗原融合蛋白Ag85B-ESAT6和Rv2031c-Rv2626c重组腺病毒的构建及鉴定

Construction and identification of a recombinant adenovirus encoding fusion proteins of multi-stage antigens of Mycobacterium tuberculosis

目的构建表达结核分枝杆菌多阶段抗原融合蛋白Ag85B-ESAT6(AE)和Rv2031c-Rv2626c(R2)的重组腺病毒,为其用于结核病新型疫苗的研究奠定基础。方法体外合成人延长因子1α(EF1α)启动子DNA序列,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV中,构建含有CMV、EF1α双启动子的腺病毒穿梭载体。PCR扩增课题组前期构建的AE、R2融合蛋白基因,并依次亚克隆至上述腺病毒穿梭载体的CMV和EF1启动子下游,阳性重组穿梭质粒命名为pAd-AE-R2。将pAd-AE-R2与骨架质粒共转染HEK293细胞,包装获得重组腺病毒,命名为Ad-AE-R2。RT-PCR法和间接免疫荧光法(IFA)对重组腺病毒表达的AE、R2融合蛋白的基因和蛋白表达水平进行鉴定。结果Ad-AE-R2瞬时转染的HEK293细胞中可转录表达AE、R2融合蛋白基因。IFA法采用Ag85B、ESAT-6、Rv2626c和Rv2031c 4种抗原的单克隆抗体可分别检测到Ad-AE-R2感染的巨噬细胞中具有特异性的绿色荧光,表明Ad-AE-R2能够在宿主细胞内表达AE、R2融合蛋白。结论成功构建并包装获得表达结核分枝杆菌多阶段抗原融合蛋白AE、R2的重组腺病毒,为其用于结核病新型疫苗研究奠定基础。

This study aimed to construct a recombinant adenovirus encoding two fusion proteins,Ag85B-ESAT6(AE)and Rv2031c-Rv2626c(R2),from multi-stage antigens of Mycobacterium tuberculosis to facilitate research on a new vaccine against tuberculosis.The human Elongation Factor 1α(EF1α)promoter DNA sequence was synthesized in vitro and cloned into the adenovirus shuttle vector pAdTrack-CMV to construct an adenovirus shuttle vector containing CMV and EF1αdouble promoters.Previously constructed fusion protein genes of AE and R2 were amplified by PCR and cloned downstream of the CMV and EF1αpromoters in the above adenovirus shuttle vector.The positive recombinant shuttle plasmid was named pAd-AE-R2.pAd-AE-R2 and the skeleton plasmid were co-transfected into HEK293 cells to obtain a recombinant adenovirus,denoted Ad-AE-R2.The gene and protein of AE and R2 expressed by Ad-AE-R2 were identified by RT-PCR and indirect immunofluorescence.IFA results showed that AE and R2 fusion protein genes were transcribed specifically in HEK293 cells,and specific green fluorescence was detected by monoclonal antibodies against Ag85B,ESAT-6,Rv2626c or Rv2031c in macrophages infected with Ad-AE-R2 by IFA,thus indicating that Ad-AE-R2 enabled correct expression of AE and R2 fusion proteins in host cells.In conclusion,the recombinant adenovirus encoding fusion proteins of AE and R2 from multi-stage antigens of Mycobacterium tuberculosis was successfully constructed,thus providing a research foundation for its use as a new TB vaccine.