动物RNA病毒检测关键试剂—反转录酶(M-MLV RTase)的制备  

Preparations of the M-MLV RTase as the key reagent for the detection of animal RNA viruses

作　　者：牛建蕊 高志强[2] 蒲静[2] 张伟[2] 刘环[2] 刘文晓 张鹤晓[2] NIU Jian-rui GAO Zhi-qiang PU Jing ZHANG Wei LIU Huan LIU Wen-xiao ZHANG He-xiao(Beijing Senkang Biotech Development Co. , Ltd, Beijing 101400, China Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100026, China)

机构地区：[1]北京森康生物技术开发有限公司,北京怀柔101400 [2]北京出入境检验检疫局,北京朝阳100026

出　　处：《中国兽医杂志》2016年第11期26-28,共3页Chinese Journal of Veterinary Medicine

基　　金：国家质检总局科技计划项目(2014IK232)

摘　　要：反转录酶是动物RNA病毒分子诊断用到的关键试剂之一。为获得纯度高、活性高、制备简单经济的M-MLV RTase,本试验将M-MLV RTase的基因序列进行密码子优化,合成并构建表达载体。将表达载体转化到大肠杆菌E.coli Rosetta宿主菌种,经IPTG诱导后实现M-MLV RTase的可溶性表达。利用Ni2+柱亲和层析纯化载有6x His标记的重组MMLV RTase,对重组酶进行Western Blotting试验,鉴定为M-MLV RTase。通过对动物流感病毒RNA的普通RT-PCR和荧光RT-PCR检测来评价重组酶的活性并估测其活性单位。将活性单位相当的重组酶和商品酶进行比较,结果显示,制备的重组酶活性高,对不同浓度病毒RNA的检测结果与商品酶相当。研制的M-MLV RTase可以用于动物RNA病毒的分子诊断。Reverse Transcriptase was used as one of the key reagents for Animal RNA virus molecular diagnosis.In order to obtain high purity,high activity,economy and easy preparation of M-MLV RTase,the codons of the sequence of M-MLV RTase were optimized,and the expression vector was constructed and transformed into E.coli Rosetta which was induced by IPTG.Soluble M-MLV RTase expression was achieved,and the M-MLV RTase with a tag of 6 x His was purified by Ni^2＋ affinity chromatograph and identified through Western blotting.The activity and activity unit of the recombinant enzyme were evaluated through the detection of animal influenza virus by ordinary RT-PCR and real time RT-PCR.Compared to commercial Enzyme,the recombinant M-MLV RTase has the same efficasy in testing the influenza virus in different dilutions.The recombinant M-MLV RTase can be used in the diagnosis of animal RNA viruses.

关 键 词：M-MLV RTase 表达 纯化 RT-PCR 

分 类 号：S852.659.3[农业科学—基础兽医学]