稳定表达甲型流感病毒M1蛋白的MDCK细胞系的建立  

Establishment of a stable MDCK cell line expressing M1 protein of influenza A virus

作　　者：张旭[1,2] 滕巧泱[2] 周洁文[2] 徐大伟[2] 颜丕熙[2] 姬希文[2] 闫丽萍[2] 戴晓光[2] 张树梅[2] 王洪斌[1] 李泽君[2] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室,上海200241

出　　处：《中国兽医科学》2011年第7期672-676,共5页Chinese Veterinary Science

基　　金：浦江人才计划项目(09PJ1411900);农业部动物转基因专项(2009ZX08010-022B);公益性行业(农业)专项(201003012)

摘　　要：为建立稳定表达流感病毒A/Puerto Rico/8/34(PR8)株M1蛋白的MDCK细胞系,将流感病毒PR8株M基因28～1 009bp序列插入逆转录病毒载体pMX,构建了重组质粒pMX-PR8M,并与pCI-NF-KB、pMDSV和MDSV质粒共转染293T细胞,制备逆转录病毒样颗粒。利用逆转录病毒样颗粒感染MD-CK细胞,经嘌呤霉素筛选获得具有抗性的细胞克隆,通过PCR、RT-PCR和间接免疫荧光试验筛选的鉴定方法。获得1株稳定表达M1蛋白的MDCK细胞系,命名为MDCK-PR8M1。本研究建立的表达M1蛋白的细胞系,为研究流感病毒M1蛋白生物学功能以及扩增含有外源基因的流感病毒提供了有利的工具。In order to establish stable MDCK cell line expressing M1 gene of influenza virus（PR8）,pMX-PR8M was constructed by inserting M gene（28-1009 bp）between the restriction enzyme sites of NotⅠ and XhoⅠ on pMX vector.To produce retrovirus-like particles expressing M1 protein for PR8,293T cells were transfected with pMX-PR8M together with the plasmids of pCI-NF-KB,pMDSV and MDSV.Then the retrovirus-like particles were used to infect MDCK cells,and the cells were cultured in DMEM with puromycin and 100 mL/L fetal bovine serum.The expression of M1 protein in the survival cells was identified by PCR,RT-PCR and indirect immunofluorescence assay.A cell line that stably expressed the M1 protein was obtained,and designated as MDCK-PR8M1.The MDCK-PR8M1 cell line provides a useful tool for study on the function of M1 protein and multiplication of influenza virus with M1 gene replaced by exogenous genes.

关 键 词：甲型流感病毒 M1蛋白 MDCK 稳定表达 

分 类 号：S852.659.5[农业科学—基础兽医学]