中国汉坦病毒H8205株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达  

Cloning and Transient Expression of the Gene Encoding Human Hantavirus H8205 Strain G2 Glycoprotein in Mammalian Cells

作　　者：王小红[1] 黄汉菊[1] 任刚 李钟铎[2] 陈秀珠 

机构地区：[1]华中科技大学同济医学院基础医学院病原生物学系,武汉430030 [2]军事医学科学院微生物与流行病研究所,北京100850

出　　处：《华中科技大学学报（医学版）》2002年第3期230-233,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目 (No. 30 170 819)

摘　　要：从感染病毒乳鼠脑组织提取总 RNA,采用 RT- PCR和分子克隆技术将扩增到的 G2糖蛋白基因插入含 CMV启动子的 pc DNA3.1/ His质粒载体中 ,通过脂质体介导转染 COS- 7细胞 ,用 SDS- PAGE、 Western- blot及 IFIA方法分别测定表达产物的相对分子量及特异性。结果证明获得正向插入的 G2 - pc DNA3.1/ His重组表达质粒 ,表达产物的相对分子量为 5 6 ku,与理论预期大小一致 ,并且可与汉坦病毒 H82 0 5株的腹水抗体起特异反应。表明构建的 G2 - pc DNA3.1/His重组质粒所表达的蛋白为中国汉坦病毒株特有 ,能在哺乳动物细胞中表达并具有抗原性 ,重组质粒可应用于汉坦病毒的Total RNA from the brain tissue of the young mice infected with virus was extracted. The G2 glycoprotein gene was amplified and cloned into the plasmid vector pCDNA3 1/His containing CMV promoter by using RT PCR and molecular cloning technique,the recombinant plasmid G2 pCDNA3.1/His was transfected into eukaryotic COS 7 cells by lipofectine mediated transfection. SDS PAGE, Western blot and indirective FIA analysis were used to analyze the molecular weight and specificity of the expressed protein respectively. The results showed that the positive recombinant plasmid harbouring the right inserted G2 glycoprotein gene fragment was abtained. The protein expressed in mammalian cells COS 7 was about 56 ku, corresponding to the estimated molecular size of the G2 glycoprotein, and could react with the Hantavirus H8205 strain hyoerimmune mouse ascites. It was suggested that the G2 glycoprotein gene could be expressed in mammalian cells transiently and reserved Chinese specific antigenicity, indicating that the recombinant plasmid G2 pCDNA3 1/His could be used to develope DNA vaccine against Hantavirus.

关 键 词：中国 汉坦病毒 H8205株 G2糖蛋白 DNA疫苗 基因表达 基因克隆 真核细胞 

分 类 号：R373.32[医药卫生—病原生物学] Q785[医药卫生—基础医学]