整合猪繁殖与呼吸综合征病毒GP5蛋白的重组鼠白血病病毒特性

生物工程学报 Chin J Biotech 2008, May 25; 24(5): 780-785 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2008 Institute of Microbiology, CAS & CSM, All rights reserved

整合猪繁殖与呼吸综合征病毒GP5蛋白的重组鼠白血病病毒特性

党占国1, 夏平安1, 周斌2, 尹彦涛1, 王建举1, 柴春霞1, 崔保安1, 陈溥言2

1 河南农业大学 牧医工程学院微生物实验室, 郑州 450002

2 南京农业大学 农业部动物疫病诊断与免疫重点开放实验室, 南京 210095

摘 要: 通过反转录-聚合酶链式反应(RT-PCR)扩增了猪繁殖与呼吸综合征病毒完整的GP5基因并进行了克隆与鉴定。序列测定结果与已经登陆GenBank 的EF488048高致病性江西株比对分析表明碱基同源性达98.7%。构建含GP5基因的真核表达载体pcDNA-GP5, 通过与鼠白血病病毒(MuLV)假病毒构建体系的两种质粒pHIT60和pHIT111共转染人胚肾细胞293T, 48 h后收集假病毒上清, 超离后通过Western-blot证明GP5蛋白在假型病毒颗粒表面存在, 表明GP5蛋白被整合到此假型病毒粒子表面。通过感染293T、Mark-145不同的靶细胞,证实所构建的假型病毒粒子具有感染性。成功构建了具有感染性的MuLV-GP5假病毒体系, 为研究猪繁殖与呼吸综合征病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。

关键词: 鼠白血病病毒, 猪繁殖与呼吸综合征病毒, GP5基因, 假病毒

Characterization of Murine Leukemia Virus Recombinants Bearing prrsv GP5 Glycoproteins

Zhanguo Dang1, Ping’an Xia1, Bin Zhou2, Yantao Yin1, Jianju Wang1, Chunxia Chai1, Bao’an Cui1, and Puyan Chen2

1 The College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China

2 Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China

Abstract: The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the

GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV. Keywords: murine leukemia virus, prrsv, GP5 gene, pseudotypes

Received: September 29, 2007; Accepted: November 29, 2007

Supported by: the Doctor Foundation of China (No. 20060390944).

Corresponding author: Ping’an Xia. Ter: +86-371-63558516; E-mail: xpa-88@sohu.com 中国博士后基金项目(No. 20060390944)资助。