三重融合PCR法构建感染性辛德毕斯嵌合病毒cDNA克隆  

Construction of Infectious Chimeric cDNA Clone of Sindbis Virus by Triple Fusion PCR

作　　者：王力华[1] 付士红[1] 唐青[1] 杨益良[1] 梁国栋[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2006年第2期107-113,共7页Chinese Journal of Virology

基　　金：国家自然科学基金(No.30470083)

摘　　要：利用三重融合PCR法,即将3个DNA片段放在同一个反应里进行长片段PCR扩增法,融合得到了包含辛德毕斯病毒XJ-160株结构基因E3、E2、6K、3′UTR和辛德毕斯病毒YN87448株结构基因E1的融合DNA片段E3E26KE13′UTR。通过此融合片段两端引入的XbaI和XhoI酶切位点将其连接到XJ-160株的感染性全基因组cDNA克隆骨架上,成功构建了辛德毕斯病毒株XJ-160与YN87448外膜糖蛋白基因E1相互替换的嵌合病毒cD-NA克隆,命名为pBR-XJ160YE1。该克隆线性化后经体外转录,RNA转录体脂质体法转染BHK-21细胞,36h后细胞发生病变。间接免疫荧光检测到病毒蛋白的表达。提取5次传代后细胞上清中病毒RNA,RT-PCR法检测证明病毒来源于嵌合病毒cDNA克隆。此感染性辛德毕斯嵌合病毒cDNA克隆可以作为研究辛德毕斯病毒E1糖蛋白基因相关功能及XJ-160病毒和YN87448病毒存在单方向血清学反应的分子机理的分子生物学工具。Using triple fusion PCR method, i.e. long PCR-based fusion of three DNA fragments simultaneously, we assembled DNA fragment E3E26KE1＇3＇UTR which included E3, E2, 6K, 3＇UTR of XJ-160 virus and E1 of YN87448 virus. Ligating the fusion fragment to the backbone of infectious full length cDNA clone of XJ-160 virus through Xba I and Xho I enzyme sites, we constructed the chimeric viral cDNA clone which exchanged the E1 glycoprotein of XJ-160 virus and YN87448 virus successfully, and named it pBR-XJ160Y E1. After linearizing pBR-XJ160Y E1 and in vitro transcripting, RNA transcript was transfected into BHK-21 cell via liposomes. Cytopathic effect （CPE） was commonly observed 36h after transfection. The protein expression of chimeric virus was identified by indirect immunofluorescence analysis. Isolated RNA of the chimeric virus in the culture media after passaged five times in BHK-21 cell proved that the virus came from pBR-XJ160YE1 by RT-PCR and DNA sequencing. The infectious chimeric viral cDNA clone can act as a molecular biology tool for studying the relative function of E1 glycoprotein of Sindbis virus and the molecular bases of one way serology reaction between XJ-160 virus and YN87448 virus.

关 键 词：辛德毕斯病毒 融合PCR 嵌合病毒 CDNA克隆 E1糖蛋白 

分 类 号：R373.3[医药卫生—病原生物学] Q78[医药卫生—基础医学]