博尔纳病病毒p24和p40基因星形胶质细胞特异表达重组质粒的构建  

Establishment on specific expression of GFAP recombinant plasmids by astrocyte from p24 and p40 gene in Borna disease virus

作　　者：李文娟[2] 张亮[1,2] 邓婧[1,2] 金戈[1,2] 黄荣忠[1,2] 房亮[2] 谢鹏[1,2] 

机构地区：[1]重庆医科大学附属第一医院神经内科,重庆400016 [2]重庆医科大学神经科学研究中心,重庆市神经生物学重点实验室,重庆400016

出　　处：《中国人兽共患病学报》2012年第1期1-5,共5页Chinese Journal of Zoonoses

基　　金：国家重大科学研究计划(973计划)项目(2009CB918302);重庆市卫生局医学科学技术研究项目(2010-2-102);重庆市自然科学基金计划(CSTC,2010BB5393)

摘　　要：目的构建星形胶质细胞特异性表达的博尔纳病病毒磷蛋白和核蛋白基因重组质粒,以供后续实验研究使用。方法用PCR方法扩增BDV p24和p40基因完整序列同时加入Nhe I和EcoR I酶切位点,将所得目的片段克隆至pMD18-T载体,再通过酶切连接将其连接至pCMvie-GFAP质粒中的GFAP启动子的下游,分别得到p24和p40重组质粒。用酶切、PCR以及测序3种方法鉴定重组质粒,并将质粒转染人胶质瘤细胞株(U251),采用免疫细胞化学方法检测目的蛋白表达。结果 pCMvie-GFAP-BDVp24和pCMvie-GFAP-BDVp40重组质粒经酶切、PCR鉴定可见目的条带位置与预期相符,经测序可见插入序列正确,质粒转染U251细胞后,经免疫细胞化学检测到目的蛋白表达。结论成功构建了pCMvie-GFAP-BDVp24和pCMvie-GFAP-BDVp40星形胶质细胞特异性表达质粒,为研究这两种病毒蛋白的致病机制及其对星形胶质细胞的影响提供了工具。In order to establish specific expression of GFAP recombinant plasmids by astrocyte from p24 and p40 gene in Borna disease virus the target DNA fragments of BDV p40 and p24 genes were replicated by PCR and added to endonuclease cutting sites Nhe I and EcoR I.Then these two obtained fragments were respectively cloned into the pMD18-T vector.At the last step,BDVp40/p24 fragments were obtained by Nhe I and EcoR I double digestion to connect with pCMvie-GFAP plasmid,Finally,the p24 and p40 recombinant plasmids were established and confirmed by Nhe I and EcoR I double digestion,PCR and sequencing.In addition,the p24 and p40 recombinant plasmids were transfected into U251 cell line and their expressed proteins were detected by immunocytochemistry assay.The results indicated that the target bands were also observed and the sequences were correct in detections,and immunocytochemistry detection showed p24 and p40 proteins were expressed in U251 cell.In conclusion the pCMvie-GFAP-BDVp24p40 plasmids were successfully established,which are useful tools for studying the pathogenesis of these 2 virus proteins and their effects on astrocyte.

关 键 词：博尔纳病病毒 核蛋白 磷蛋白 星形胶质细胞 质粒 

分 类 号：R373.9[医药卫生—病原生物学]