摘要
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Ag85BDNA疫苗的免疫性和免疫效果,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(+)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 riboome 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,48h后提取细胞总蛋白,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-Ag85ARES-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。
Antigen 85A （Ag85A） and antigen 85B （Ag85B） are the two immunodominant antigens of Mycobacteriurn tu- berculosis （Mtb）. In order to construct a eukaryotie 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 in serted 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 ri bosome 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 transfeetion, 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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第8期780-784,共5页
Chinese Journal of Zoonoses
基金
Supported by the National Key Basic Research Program of China(973 Program)(Nos.2012CB126301&2012CB518801)
the National Natural Science Foundation of China(No.31160515)
the Research Fund for the Doctoral Program of Higher Education of China(No.20126401110001)~~
