汉坦病毒A9株M基因片段全长cDNA克隆及其在痘苗病毒中的瞬时表达  

Cloning and Transient Expression of M Genome Segment cDNA of Hantaan Virus Chinese Isolate A9

作　　者：邱建明[1] 石晓宏[1] 杭长寿[1] 宋干[1] 姚树元[1] 

机构地区：[1]北京中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1995年第4期332-336,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：采取反转录一聚合酶链反应（ＰＣＲ），分３个片段扩增Ｉ型汉坦病毒（野鼠型）中国毒株Ａ９株Ｍ基因片段，分别克隆入ＰＧＥＭ－Ｔ载体中。选择３个正向插入的ＴＡ９ＩＢ、ＴＡｇＣＤ、ＴＡｇＥＡ克隆，选用ＣｌａＩ、ＥｃｏＲＶ进行酶切、连接，获得了１个在ＰＧＥＭ－Ｔ载体中插入３．６ｋｂ的Ａｇ株Ｍ基因片段ｃＤＮＡ克隆，酶切图谱分析证实播入片段正确。将Ａ９Ｍ片段在痘苗病毒／Ｔ７噬菌体ＲＮＡ聚合酶瞬时（Ｔｒａｎｓｉｅｎｔ）表达系统中进行表达，用抗汉坦病毒糖蛋白单克隆抗体进行免疫荧光检测，观察到很强的特异性荧光，证明ＡｇＭ基因ｃＤＮＡ能进行表达。To provide M genome segment cDNA of Hantaan virus Chinese isolate for the development of genetic engineering vaccine against hemorrhagic fever with remal syndrome (HFRS),the M genome segment of A9 virus was amplified into three fragments by three pairs of primer with the method of reverse transcription and polymerase chain reaction (RT-PCR),AND THE PCR products wree cloned into PGEM-T vector.The full length M genome segment cDNA clone(TA9M) was constructed through ligating the three PCR product clones together at the same direction, and identified with mest-PCR and restriction endonuclease analysis. The A9 M cDNA could be expressed in the transient transient expression system of vaccinia virus/T7 phage RNA polymerase, and verified with immunoflourescense assay using monoclonal antibodies directed against glycoprotins of Hantaan virus.

关 键 词：汉坦病毒 聚合酶链反应 基因表达 DNA 痘苗病毒 

分 类 号：R373.32[医药卫生—病原生物学]