含风疹病毒部分核酸序列的耐核糖核酸酶病毒样颗粒的构建和表达  

Construction and expression of RNase-resisting virus-like particles containing rubella virus RNA

作　　者：彭建明[1] 李金明[1] 徐克前[2] 王忠芳[1] 王露楠[1] 邓巍[1] 

机构地区：[1]卫生部北京医院卫生部临床检验中心,100730 [2]中南大学医学技术与情报学院

出　　处：《中华检验医学杂志》2005年第3期302-304,共3页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学研究基金资助项目 (30371365 );首都医学发展科研基金资助项目(2002 3041)

摘　　要：目的　构建原核表达系统,表达内含风疹病毒部分核酸序列的由噬菌体包膜蛋白构成的病毒样颗粒,并包被重组RNA,使其具耐核糖核酸酶 (RNase)的特性。方法　用逆转录聚合酶链反应(RT PCR)扩增风疹减毒活疫苗中风疹病毒糖蛋白E1的部分保守区域,进行TA克隆后,用限制性内切酶HindⅢ酶切,获得所需要的目的片段,并与用HindⅢ酶切的表达载体pNCCL1相连接,构建一新的表达载体pNCCL1 Ru。再转化E ColiBL21 DE3,用IPTG诱导表达噬菌体MS2包膜蛋白后,再自我组装成病毒样颗粒,并将风疹病毒部分序列包裹到病毒样颗粒内。结果　成功构建了新的表达载体pNCCL1 Ru,其原核表达产物病毒样颗粒中所包裹的风疹病毒RNA序列与风疹BRDⅡ株同源性高达98%,所包裹的RNA具有耐RNase消化的特性以及良好的稳定性。结论　我们构建的表达载体pNCCL1 Ru及原核表达系统,可作为构建和制备耐RNase的风疹病毒核酸标准品和质控品的平台,可为实验室进行风疹病毒核酸的检测提供稳定的无传染性的标准品和质控品。Objective To construct an expression system to produce the virus like particles containing a part of the sequence of rubella virus genome RNA, which are ribonuclease resistant due to the encapsulation of the RNA by bacteriophage MS2 coat proteins Methods The RT PCR products of rubella virus RNA fragments were cloned to TA vector pBS T, then targeted segments could be obtained when the recombinant plasmids pBS T Ru were digested by restriction endonuclease HindⅢ and cloned to prokaryocytic expression vector pNCCL1 The recombinant plasmids named pNCCL1 Ru were transfected into E Coli BL21 DE3 and induced to express with IPTG Results The recombinant plasmids were successfully constructed The bacteriophage MS2 coat protein that express in BL21 can self assembly to form ribonuclease resistant virus like particles and the rubella virus RNA was encapsulated into virus like particles The sequence of rubella virus RNA in virus like particles show high homology(98%)with rubella virus BRDⅡstrain The encapsulated RNA can resist the digestion of RNase A and have good stability Conclusion The virus like particles which containing rubella virus RNA can be expressed in prokaryocyte and it could be used as standard and control in detecting rubella virus RNA, it provide a new, stable and noninfectious RNA standard in RNA detection

关 键 词：风疹病毒 病毒样颗粒 表达载体 包膜蛋白 质控品 核糖核酸酶 标准品 核酸序列 酶切 IPTG 

分 类 号：R346[医药卫生—基础医学]