痘苗病毒天坛株非必需区TA25R…TA36L的两步重组表达载体的构建  

CONSTRUCTION OF TWO-STEP RECOMBINANT VECTOR TO EXPRESS FOREIGN GENE IN NON-ESSENTIAL REGION TA25R-TA36L OF VACCINIA VIRUS TIANTAN STRAIN

作　　者：邹艳[1] 阮力[1] 娄元梅[1] 朱诚[1] 陆柔剑[1] 孙朝晖[1] 朱既明[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》1998年第2期97-103,共7页Chinese Journal of Virology

基　　金：国家863高科技生物领域及国家自然科学基金

摘　　要：针对痘苗病毒天坛株非必需区ＴＡ２５Ｒ－ＴＡ３６Ｌ，构建了可去除ＴＡ２６Ｒ至ＴＡ３５Ｌ的１０个ＯＲＦｓ，并同时表达ＬａｃＺ基因的两步重组表达载体ｐＡ２４２７ＬａｃＺ。通过两种方法（一步重组和两步重组）获得了在该区域表达ＬａｃＺ基因的重组痘苗病毒ＲＶＡ２４２７ＬａｃＺ。实验表明，一步重组法获得重组病毒的机率大约为１‰，两步重组法的机率可达６２．５‰。由此可见，两步重组法可显著提高重组病毒的筛选机率。ＬａｃＺ基因可以在该区域中稳定表达，重组病毒与野毒株在繁殖能力上没有明显差别，表明ＴＡ２５Ｒ－ＴＡ３６Ｌ是较稳定的外源基因插入区域。上述结果对于深入研究该非必需区的功能特点。Two-step recombinant vector pA2427LacZ has been constructed to express foreign genes in non-essential region TA25R-TA36L of vaccinia virus Tiantan Strain.This vector can be used in deletion of 10 ORFs of TA26R-TA35L and expresses LacZ gene in the region simultaneously.Through two methods of one-step and two-step recombination,we obtained recombinant vaccinia virus RVA2427LacZ expressing LacZ gene in this region.Results showed that the probability of screening the recombinant virus through one-step method was about 1‰ while the probability through two-step method could reach 62.5‰,that can increase greatly the screening probability of the recombinant viruses.In addition,we proved that LacZ gene could be expressed stably in this region with no distinct difference between recombinant virus and wild virus on the aspect of virus propagation ability.All these results proved that TA25R-TA36L would be a stable insertion region to express foreign genes.This experiment lays a foundation for studing the function and properties of this non-essential region and,further,the exploitment for expression of foreign genes and construction of vaccine strain.

关 键 词：痘苗病毒 天坛株 非必需区 重组 表达载体 

分 类 号：Q784[生物学—分子生物学] R392.7[医药卫生—免疫学]