HPV16 E6E7与C3d3融合基因真核表达质粒的构建及表达  

Construction and expression of HPV16E6E7 and C3d3 fusion gene eukaryotic expression vector

作　　者：郑秀惠[1,2] 李力[1] 郭建新[1] 郑英如[1] 李军果[1] 林爽[1] 陈竹钦[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所妇产科,重庆400042 [2]第三军医大学大坪医院妇产科

出　　处：《重庆医学》2010年第1期40-42,共3页Chongqing medicine

基　　金：第三军医大学青年基金资助项目(06XG051)

摘　　要：目的构建可作为DNA疫苗的包含人乳头瘤病毒16型(HPV16)E6E7与C3d3融合基因真核表达质粒,并在体外进行表达和鉴定。方法采用PCR技术扩增HPV16 E6E7片段,将该片段插入到pMDT-18载体中后,亚克隆至真核表达载体pSG.SS.C3d3.YL和pSG.SS.YL,构建E6E7与C3d3融合基因及E6E7表达质粒pSG.SS.E6E7.C3d3.YL和pSG.SS.E6E7.YL。经限制性酶切鉴定和DNA序列测定后,将重组质粒pSG.SS.E6E7.C3d3.YL、pSG.SS.E6E7.YL转染COS-7细胞,用Western blot及免疫细胞化学技术检测其表达。结果经酶切、DNA测序及转染真核细胞后表达产物的鉴定结果显示,重组质粒构建成功。结论构建的质粒可在真核细胞内正确表达,为其作为DNA疫苗诱导免疫效应的动物实验打下了基础。Objective To construct plasmid pSG. SS. E6E7. C3d3. YL and pSG. SS. E6E7. YL which can respectively express Flk-IECD-C3d3 fusion protein and Flk-IECD protein in eukaryotie cell. Methods PCR was applied to amplifiy E6E7 eDNA with Bgl Ⅱ and BamH Ⅰ restriction endonuclease site at two ends respectively. Then the cDNA fragment was linkaged into pMDT-]8 and was subcloned into eukaryotic expression vector pSG. SS. C3d3. YL and pSG. SS. YL, which leading to pSG. SS. E6E7. C3d3. YL and pSG. SS. E6E7. YL. After identification by endonuclease digestion and DNA sequence analysis, the recombinant plasmids were transfected into COS-7 cells and the expressed product was detected by Western blot and immunocytochemistry. Results The con structions of eukaryotic expression vector pSG. SS. E6E7. C3d3. YL and pSG. SS. E6E7. YL were successful and the two plasmids could express recombinant E6E7-C3d3 fusion protein and Flk-1ECD protein respectively. Conclusion The success in constructions of pSG. SS. E6E7. C3d3. YL and pSG. SS. E6E7. YL do the groundwork for further studies in animals.

关 键 词：人乳头瘤病毒16型 C3D3 DNA疫苗 

分 类 号：R737.33[医药卫生—肿瘤]