结核分枝杆菌Ag85A和Ag85B基因真核共表达载体的构建与表达

结核分枝杆菌Ag85A和Ag85B基因真核共表达载体的构建
与表达
李武;邓光存;刘晓明;王玉炯
【摘要】Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the two immunodominant antigens of Mycobacterium tuberculosis (Mtb).In order to construct a eukaryotic expression vector pcDNA-Ag85A IRES-Ag85B which can co express Ag85A and Ag85B genes of Mtb,the target gene fragments encoding the Ag85A and Ag85B antigens were amplified and inserted into the multicloning site (MCS) of the shuttle plasmid vector pcDNA3.1(+),in a position between the CMV promoter and the BGH poly (A) signal in orientation.For the sake of co expression of the two genes,they were connected by internal ribosome entry site (IRES) sequence.After verifying the correctness of the construct by restriction and sequencing analysis,the recombinant plasmid was then transfected into Human Embryonic Kidney (HEK) 293T cells and the cell lysates were harvested and separated by SDS-PAGE 48 hours after transfection,and subjected to Western blot analysis.Western blot analysis revealed that both of the antigens could be expressed in 293T cells respectively.In conclusion,we have successfully constructed the eukaryotic co-expression vector pcDNA-Ag85A IRES-Ag85B,which provides a basis for the further study of the immunogenicity and protective activity of the two antigens.%目的 Ag85A和Ag85B是结核分枝杆菌的主要优势保护抗原,为提高编码Ag85A 和Ag85B DNA疫苗的免疫原性和免疫效果,特构建真核共表达Ag85A和Ag85B
抗原的真核表达载体pcDNA-Ag85A IRES-Ag85B,并利用293T细胞对其表达进行检测.方法 PCR扩增Ag85A和Ag85B基因,逐步将Ag85A和Ag85B基因定向插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加A信号BGH poly(A)之间,并将Ag85A和Ag85B基因以内部核糖体进入位点(internal ribo some entry site,IRES)序列连接,酶切和测序验证后将重组质粒转染至293T细胞中进行真核表达,48 h后提取细胞总蛋白,表达产物经SDS-PAGE分离和Western blot分析.结果限制性内切酶酶切及测序结果表明,重组质粒pcDNA-Ag85AIRES-Ag85B基因序列和阅读框架正确.将重组质粒转入293T细胞后,利用Ag85A和Ag85B特异性抗体Western blot检测证实两种抗原可在同一载体中各自独立表达.结论成功构建了能够同时独立表达结核分枝杆菌Ag85A和Ag85B抗原基因的真核表达载体pcDNA-Ag85A IRES-Ag85B,为下一步研究它们的免疫原性和免疫效果奠定了基础.
【期刊名称】《中国人兽共患病学报》
【年(卷),期】2013(029)008
【总页数】5页(P780-784)
【关键词】结核分枝杆菌;Ag85A;Ag85B;共表达;Western blot
【作者】李武;邓光存;刘晓明;王玉炯
【作者单位】宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川750021;宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川 750021;宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川 750021;宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川 750021
【正文语种】中文【中图分类】R378.91。