摘要
目的构建结核分枝杆菌抗原ESAT-6的真核表达质粒,并研究其免疫原性。方法采用聚合酶链反应(PCR)方法自结核分枝杆菌H37Rv基因组DNA中扩增esat-6基因片段,定向亚克隆入真核表达载体pVAC,构建pVAC-esat-6重组质粒;利用脂质体介导的转染技术,将其导入Ve-roE6细胞,RT-PCR法检测mRNA表达情况,Western-blot法鉴定表达产物;重组质粒经基因枪免疫小鼠后,采用酶联免疫吸附试验(ELISA)检测小鼠血清抗ESAT-6特异性抗体以及小鼠脾淋巴细胞培养上清液干扰素-γ(IFN-γ)含量。结果成功构建了真核表达重组质粒pVAC-esat-6,并可在VeroE6细胞中表达;重组质粒免疫小鼠3次后,抗体滴度达到了1∶1600,重组质粒组(pVAC-esat-6组)小鼠脾淋巴细胞培养上清液IFN-γ水平明显高于对照组(pVAC组)(P<0.01)。结论成功构建结核分枝杆菌抗原ESAT-6的真核表达质粒pVAC-esat-6,免疫小鼠后诱导产生了特异的体液免疫应答和细胞免疫应答。
Objective To construct the eukaryotic expression plasmids of Mycobacterium tuberculosis ESAT6 antigen and to investigate its immunogenicity in mice. Methods The sequence encooding ESAT-6 was amplified by PCR from Mycobacteriurn tuberculosis H37Rv genomic dNA. Then, the amplified fragment was sub cloned into pVAC expression vector. The eukaryotic expression plas mid pVAC ESAT 6 was transfeeted into Veto E6 cells with liposome. The mRNA expression of pVAC-csat-6 in Veto E6 cells was detected by RT-PCR and its expression product was analyzed by Western-blot. The plasmid pVAC esat-6 was introduced into the mice by gene gun injection. The spe cific antibody to ESAT-6 and the level of IFN-γ in supernatant of spleno-lymphocyte cultures were detected by ELISA methods. Results The recombinant plasmid pVAC-esat-6 was successfully construeted, and the expression of ESAT-6 was also detected in vitro. After vaccinated three times, the mice produced specific antibody and the level of IFN-γ in supernatant of spleno-lymphocyte cultures in group of pVAC-esat-6 was significantly higher than group of vector alone （P 〈 0.01）. Conclusions The eukaryotic expression plasmids of Mycobacteriurn tuberculosis ESAT-6 antigen were successfully constructed, and the recombinants pVAC-esat-6 could induce specific humoral immunity and cellular immunity in immunized mice,
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2006年第1期7-11,共5页
Chinese Journal of Infectious Diseases
