HPV16E7致瘤相关位点的定向突变及其表达纯化

Study on the site-directed mutagenesis,expression and purification of HPV16E7 oncogenic sites

目的研究定点突变HPV16E7基因中与转化有关的位点,原核细胞表达突变后的HPV16E7的表达及其免疫原性。方法在引物内设计突变位点,T载体克隆全部与转化有关的全长HPV16E7基因,然后亚克隆构建pET-28a-HPV16E7质粒(命名为pET-28a-HPVm16E7),转化E.Coli BL21细菌,在异丙基硫代半乳糖苷(IPTG)诱导下表达HPVm16E7蛋白,用HPV16E7单克隆抗体对其进行Western Blot分析。结果 DNA测序证明了构建的pET-28a-HPVm16E7是正确的,转化细菌后能够表达HPVm16E7蛋白,并能与不同的HPV16E7单抗抗体反应。结论在引物中设计突变位点能够定向突变HPV16E7与转化有关的位点,原核表达的HPVm16E7不影响其免疫原性,增加了基于HPV16E7的疫苗的安全性。

Objective To explore the related sites of transformation in site‐directed mutagenesis of HPV16E7 gene and to study the expression and immunogenicity after the mutation of HPV 16E7 in prokaryotic cell .Methods Mutated sites were designed in the primer .Full length HPV16E7 gene were cloned by T carrier .Then the plasmid of pET‐28a‐HPV16E7 was subcloned （named pET‐28a‐HPVm16E7） ,and the bacteria of E .Coli BL21 were transformed and induced by isopropyl thiogalactoside （ITPG） to ex‐press HPVm16E7 protein .The protein was analyzed by Western Blot using monoclonal antibody of HPV 16E7 .Results DNA se‐quencing showed that pET‐28a‐HPVm16E7 was direct .The bacteria after transformation could express HPVm16E7 protein and re‐act with different kinds of HPV16E7 monoclonal antibodies .Conclusion Mutated sites designed in the primer can make the muta‐tion and transformation of HPV16E7 gene directionally .Expression of HPVm16E7 in prokaryotic cells would not influence the im‐munogenicity while increase the security of HPV16E7 vaccine .