突变型HBV前C-C基因疫苗的构建及真核细胞表达  

Construction and expression of mutated HBV precore-core DNA vaccines

作　　者：张敏[1] 辛绍杰[2] 胡燕[2] 侯俊[2] 沈宏辉[2] 王志杰[2] 貌盼勇[2] 

机构地区：[1]解放军军医进修学院,北京100853 [2]解放军第三○二医院,北京100039

出　　处：《传染病信息》2008年第5期294-297,共4页Infectious Disease Information

基　　金：解放军第三○二医院院长创新基金资助项目(2004001)

摘　　要：目的采用点突变技术对HBV前C-C基因中的蛋白酶切位点进行4处点突变,构建突变型HBV前C-C/ENHⅡ重组基因疫苗,转染HepG2细胞,研究其在真核细胞内的表达情况。方法采用单引物二次PCR法对质粒VEC依次进行基因点突变,得到151+154(VE2)、151+154+164+167点突变(VE4)2种突变型疫苗,转染HepG2细胞,通过细胞免疫化学、酶联免疫分析、免疫印迹等方法检测其在HepG2细胞内的表达情况及表达产物的分子量。结果VEC、VE2、VE4转染HepG2细胞均胞浆表达目的蛋白,产物分子量分别为18、20、22 kD,细胞裂解液的HBeAg含量VE4、VE2高于VEC,上清中则反之。结论突变型HBV前C-C基因疫苗VE2、VE4构建成功并表达预期病毒前C蛋白前体。Objective To construct mutated HBV precore -core/ENH Ⅱ recombinant DNA vaccines by introducing point mutations into 4 protease cleavage sites of HBV precore -core gene and transfeet them into HepG2 cell line to observe their expression in eukaryotic cells. Methods Single premier twice PCR method was established and point mutation was sequentially introduced into plasmid VEC, obtaining two mutated vaccines, VE2 （151＋154 mutations） and VE4 （151＋154＋164＋167 mutations）, which were then transfected into HepG2 cells in vitro. Immunohistochemistry, ELISA and West blot measurement were employed to detect the expression of the two mutated vaccines and molecular weight of their products. Results Target proteins were expressed in cytoplasm after transfection of VEC, VEC2 and VEC4 into HepG2 cells, with the molecular weight of 18, 20 and 22 kD, respectively. HBeAg concentration was higher in cell lysate but lower in superuatant in VE4 and VE2 than that in VEC. Conclusions The mutated HBV precore -core DNA vaccines VE2 and VE4 are successfully constructed, and HBV precore proteins arc expressed.

关 键 词：乙型肝炎 DNA疫苗 点突变 Pre—C抗原 增强子Ⅱ 

分 类 号：R514.03[医药卫生—内科学] R392.11[医药卫生—临床医学]