HPV16l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础。本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞。转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符。表达产物经Western blotting分析:能与HPV16L1单克隆抗体特异结合。真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础。

Gene Cloning and Eukaryotic Expression of HPV 16l1 Gene

To clone and express in vitro Human papillomavirus type 16 l1 (HPV16L1) gene ,and provide a good basis for further research DNA vaccine against HPV16 infection and human cervical cancer, the HPV16l1 gene fragment was amplified from genome of p16L1BN1 by PCR and cloned into eukaryotic expression vector pcDNA3.0.Sequencing showed the plasmid pcDNA3-HPV16L1 was constructed successfully. Then the recombinant plasmid pcDNA3-HPV16L1 transfected 7721 human liver cancer cells using Lipofectamine 2000 reagent. The molecular weight of the expressed protein, was 55kDa, analyzing by SDS-PAGE, as same as the molecular weight of HPV16L1. The Western blotting result show that the expressed protein could be detected by HPV16L1 specific monoclonal antibody. This study provided a good basis for further research on HPV16L1 DNA vaccine.