结核分枝杆菌Hsp65-Esat6与hGM-CSF联合DNA疫苗的构建与体外研究  

Construction and primary study of DNA vaccine containing Hsp65-Esat6 of mycobacterium tuberculosis and h GM-CSF in vitro

作　　者：王森林[1] 张梦媛[1] 王学坤[1] 周曙光[1] 江振友[1] 

机构地区：[1]暨南大学医学院微生物学与免疫学教研室,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2016年第1期29-35,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家"十二五"科技重大专项(2014ZX10003002;2012ZX10004903)

摘　　要：目的:应用分子生物学技术,构建p IHsp65-Esat6GM共表达质粒,并在真核细胞中表达.方法:以p EGHsp65Esat-6为模板经聚合酶链反应(PCR)扩增出Hsp65-Esat6基因,同时从质粒p ORF-h GM-CSF中扩增出基因佐剂h GM-CSF,分别克隆到真核表达载体p IRES的多克隆位点A(MCSA)和B(MCSB)中,构建p IHsp65-Esat6GM共表达质粒,并转染Hep G-2细胞中表达和检测.结果:酶切鉴定提示插入的基因片段大小分别为1.93 kb和0.47kb,测序分析表明克隆的Hsp65-Esat6和人粒细胞-巨噬细胞集落刺激因子(h GM-CSF)序列与Gen Bank上公布序列完全一致;Western-bolt检测到Hsp65-Esat6融合蛋白相对分子质量(Mr)为75 000 k Da;RT-PCR方法检测出p IGM,p IHsp65GM和p IHsp65-Esat6GM转染组细胞均有h GM-CSF mRNA表达,三组细胞中h GM-CSF基因mRNA的表达量差异无统计学意义(P>0.05).ELISA方法检测到p IHsp65-Esat6GM转染组细胞培养上清中h GM-CSF的表达,且与空载体对照组比较差异有统计学意义(P<0.01).结论:成功构建和表达了p IHsp65-Esat6GM质粒,为研制优于卡介苗(BCG)的新型抗结核病的DNA疫苗奠定了基础.Aim：To construct and express the co-expression plasmid pIHsp65-Esat6GM by molecular biological methods. Methods： Recombined plasmid pEGHsp65Esat-6 as template to amplify Hsp65-Es- at6 gene and used plasmid pORF-hGM-CSF as template to amplify gene adjuvant hGM-CSF gene （granu- locyte-macrophage colony-stimulating factor） by PCR were used, and then recombined them with the two multiple cloning sites （ MCSA and MCSB） of pIRES vector to construct a co-expression plasmid pIHsp65- Esat6GM. The plasmid was finally transfected into HepG-2 cells to detect the expression of Hsp65-Esat6 and hGM-CSF proteins by Western-bolt and ELISA respectively. Results： Restriction enzyme digestion demonstrated that the inserted gene fragments had 1.93 kb and 0. 47 kb, DNA sequencing revealed that the cloned Hsp65-Esat6 and hGM-CSF sequences were consistent with the GenBank reported. TheHsp65-Esat6 protein detected by Western-boh had 75 000 kDa. The hGM-CSF mRNA expression of HepG-2 cells transfected with pIGM, pIHsp65GM and pIHsp65-Esat6GM were detected by RT-PCR, the differences among three groups weren＇t statistically significant （ P 〉 0. 05 ）. hGM-CSF expression from pIHsp65-Esat6GM transfected cells was detected by ELISA, the difference between the plHsp65-Es- at6GM group and the plRES blank vector group was statistically significant （P 〈 0.01 ）. Conclusion：The successful construction and expression of a co-expression vector containing Hsp65-Esat6 gene and gene adjuvant hGM-CSF are fulfilled which lays a foundation for the further research on a novel DNA vaccine that is better than BCG vaccine in the p＇revention of tuberculosis.

关 键 词：热休克蛋白65kd-早期分泌性抗原靶6kd 人粒细胞-巨噬细胞集落刺激因子 双顺反子 基因佐剂 

分 类 号：R378.911[医药卫生—病原生物学]