稳定表达流感病毒PR8 HA蛋白MDCK细胞系的建立及鉴定  

Establishment and identification of stable PR8 HA gene expression MDCK cell line

作　　者：黄海碧[1,2] 滕巧泱[2] 王朝霞[2] 戴晓光[2] 张树梅[2] 张旭[2] 申之义[1] 李泽君[2] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]中国农业科学院上海兽医研究所农业部寄生虫学重点开放实验室,上海200241

出　　处：《中国预防兽医学报》2010年第7期517-520,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：上海市浦江人才计划(09PJ1411900)

摘　　要：为建立稳定表达流感病毒(IV)HA蛋白的MDCK细胞系,本研究利用含有嘌呤霉素抗性基因的pMX载体,在启动子下游的AgeⅠ和NotⅠ两个限制性内切酶位点之间插入PR8株HA基因的开放阅读框,构建重组质粒pMX-PR8HA,将pMX-PR8 HA与3个分别表达VSVG、gag/pol和NF-KB的重组质粒共转染293T细胞,获得逆转录病毒样颗粒。用含有逆转录病毒样颗粒的细胞培养上清液感染MDCK细胞,利用嘌呤霉素进行筛选、纯化得到稳定表达PR8株HA蛋白的细胞系。连续传10代后,进行PCR、RT-PCR、IFA和western blot鉴定,结果表明,获得了能够稳定表达IV PR8株HA蛋白的MDCK细胞系。该细胞系的建立有望为IV表达外源基因提供可靠的培养细胞系。In order to establish stable influenza virus（IV） HA gene expression MDCK cell line,recombinant plasmid pMX-PR8HA was constructed by inserting the IV PR8 strain HA gene into plasmid pMX,and co-transfected the 293T cells with the recombinant pCI-gag-pol,pCI-NF-KB and pCI-VSVG（expressing vesicular stomatitis virus G protein）.72 hour post transfection,the retroviruses-like particals from the supernatant of transfected 293T cell culture was used to infect MDCK cell.MDCK cell clones expressing HA were screened by using the culture medium in the presence of puromycin.The HA gene and protein in the cells were detectable by IFA,PCR,RT-PCR and western blot tests after ten passages.The stable HA gene expression MDCK cell line could be useful to study IV as a viral vector for expressing exogenous gene.

关 键 词：流感病毒 MDCK细胞系 HA基因 稳定表达 

分 类 号：S852.65[农业科学—基础兽医学]