携带变形链球菌表面蛋白、葡聚糖结合蛋白及信号肽基因的真核表达质粒的构建

Construction and Identification of Recombinant Anticaries DNA Vaccine with Signal Tide,Surface Protein and Glucan-binding Protein Antigen Genes of Streptococcus Mutans

目的通过构建带有变形链球菌表面蛋白SpaP的P区、葡聚糖结合蛋白A葡聚糖结合区(glucan bind-ing domain,GBD)及一段信号肽基因的嵌和体真核表达质粒pSSG,为基因疫苗的制备提供实验基础。方法通过PCR扩增,获得分别编码SpaP的P区和GBD的两个基因片段sp和gg,以及人白介素2信号肽基因(signal)。将它们分别定向插入pMD20-T克隆载体中,转化感受态大肠杆菌DH5α,挑取阳性克隆,初步鉴定后测序。将获得的3个基因片段分别双酶切,胶回收纯化后将同样进行双酶切的pVAX1质粒在T4 DNA连接酶作用下进行连接反应,获得真核表达质粒pSSG,并转化DH5α,提取纯化pSSG,进行质粒RCR,酶切电泳鉴定并测序。结果真核表达质粒pSSG构建正确,目的基因sp、gg和signal定向插入至真核表达载体。结论真核表达质粒pSSG包含了SpaP的P区和GBD以及人白介素2信号肽的编码基因,为基因疫苗研究奠定基础。

Objective This study is aimed to clone the gene encoding GbpA functional area（glucan-binding domain,GBD） and P region of SpaP,together with a signal sequence into the eukaryotic expression vector pVAX1 and to construct an eukaryotic expression plasmid called pSSG used as anti-caries DNA vaccine.Methods By the method of PCR,the gene fragment named gg encoding GbpA functional area（GBD） and sp encoding P region of SpaP of Streptococcus mutans UA159 were amplified.And then,a length of human InterleukinsⅡ gene signal was designed and amplified by overlap extension PCR.The three fragments obtained were then inserted directionally into a pMD20-T vector,which were transformed into E.coli DH5α.After that,the positive bacteria clones were selected,identified and sequenced.The gene fragments signal,gg and sp were digested with two restriction enzymes.Then the digested fragments were recollected with agar and purified.And ligation reaction is conducted with the pVAX1 plasmid（an eukaryotic expression vector） digested with the same two restriction enzymes and the target fragments under the action of T4 DNA ligation enzymes.And then the fusional anti-caries DNA vaccine pSSG was constructed,which was transformed into E.coli DH5α.Afterwards,pSSG was purified and identified through colony PCR,electrophoresis after digestion with the two enzymes and sequencing.ResultsThe gene gg and sp,as well as a signal gene were cloned utilizing PCR technique and the result of gene sequence analysis accorded with the gene sequences designed.Through ligation reaction after digestion with the two restriction enzymes,the three target genes were directly cloned into the vector pVAX1 plasmid.The chimera of eukaryotic plasmid pSSG was successfully constructed.Conclusion The plasmids pSSG consist of genes of gg,sp and signal may act as gene vaccines for further study.