天津医药

• 细胞与分子生物学 • 上一篇    下一篇

基于慢病毒载体系统构建重组JSRV－NM假病毒

龚淑敏1,李光明2,吴志敏3,董莉真3,程彬3,张斌3,朱泽4   

  1. 1. 天津市第四医院
    2. 医大微生物教研室
    3. 天津医科大学基础医学院
    4. 天津医科大学 基础医学院 病原生物教研室
  • 收稿日期:2013-12-31 修回日期:2014-04-01 出版日期:2014-08-15 发布日期:2014-08-15
  • 通讯作者: 龚淑敏

Construction of JSRV-NM Pseudovirions by High Eficiency Packaging System of the Lentivirus

  • Received:2013-12-31 Revised:2014-04-01 Published:2014-08-15 Online:2014-08-15
  • Contact: Shu-Min GONG

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摘要：

【摘要】目的  采用慢病毒质粒结构重组JSRV-NM假病毒，克服JSRV-NM病毒只能通过绵羊支气管穿刺接种扩增，不能通过体外细胞培养扩增的问题。方法 采用慢病毒高效包装系统pMD.G、pCMV-HIV△8.2和pHIVeGFP，以JSRV-NM病毒的env包膜质粒pCMVJSRV-NM替代VSV-G病毒的包膜质粒pMD.G，转染293T细胞，复制、包装并产出重组的JSRV-NM假病毒。用real-time PCR检测WPRE表达来测定假病毒的滴度，用接种24孔板的293T细胞检测假病毒感染力。结果  成功重组了基于慢病毒质粒结构的JSRV-NM假病毒，可超速离心浓缩成高滴度（1×108TU/mL）的病毒，Lv-JSRV-NM包膜与Lv-VSV-G包膜按感染复数（MOI）=3的比例感染Hela细胞具有相同的感染能力。结论  基于慢病毒质粒构建的JSRV-NM假病毒，能够在293T细胞中复制和扩增，产出的假病毒具有稳定性、感染力和安全性，符合二级生物实验室安全的要求。

关键词: JSRV-NM病毒, 假病毒, 慢病毒载体, env基因, 病毒包装

Abstract:

[Abstract] Objective   To overcome the fact that SRV- NM virus can only multiple and amplify through partially pu?
rified jaagsiekte retrovirus inoculated intratracheally in sheep but it cannot be augmented usingin vitro cell culture, we con?structed JSRV-NM pseudovirions based on high efficiency packing system of lentivirus.Methods   Lentivirus of three highefficiency packing plasmids system pMD.G, pCMV-HIV8.2and pHIV-eGFP was developed, and JSRV-NM-env coatedplasmid pCMVJSRV-NM was used to substitute VSV-G virus coated plasmid pMD.G then co-transfected into293T cells toreplicate, package and produce restructured JSRV-NM pseudovirions.Gene expressionofpseudovirion was determinedthrough WPRE using real time PCR; Virus infectivity was detected through inoculating JSRV- NM pseudovirions into 24pore plates.Results   We construct JSRV-NM pseudovirions successfully based on the lentivirus system. JSRV-NM pseudo?virions can also be concentrated to higher titer (108TU/mL detected by real time PCR by ultracentrifugation without signifi?ant loss of activity. JSRV-NM and VSV-G pseudovirions infected on Hela cells (both MOI =3) respectively and no obvi?ous difference were shown on their infection efficiency detected by real time PCR.Conclusion   Based on lentivirus system,JSRV-NM pseudovirions can be multipled and amplified in293T cell culturein vitro. JSRV-NM pseudovirions is stablewithout loss its infection activity and the requirements of biological laboratory safety II was also met. JSRV-NM pseudoviri?ons will provide a useful tool for further study of JSRV-NM–env infection across species or its induction of lung adenocarci?noma.

Key words: JSRV-NM viruses, pseudovirions, lentiviral vector, env gene, virus packageing