表达PRRSV GP2a/GP2b、GP3、GP4、GP5和M蛋白重组腺病毒的构建

陈振海; 林天龙; 俞伏松; 宋铁英; 龚晖; 方勤美; 杨金先

表达PRRSV GP2a/GP2b、GP3、GP4、GP5和M蛋白重组腺病毒的构建

1.
福建省农业科学院生物技术研究所, 福建, 福州, 350003;
2.
福建省畜禽疫病防治工程技术中心, 福建, 福州, 350013

基金项目:

福建省科技重大专项(2006NZ0003-2)

详细信息

通讯作者:
林天龙(1955- ),男,研究员,主要从事动物免疫学、动物疾病学研究(E-mail:lint05@163.com).

中图分类号: S858.28;S855.3;Q784
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出版历程
- 收稿日期: 2007-07-20
- 修回日期: 2007-08-13
- 刊出日期: 2007-09-15

Establishment of recombinant adenoviruses for expressing PRRSV GP2a/GP2b,GP3,GP4,GP5,and M proteins

1.
Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China;
2.
Fujian Animal Diseases Control Technology Development Center, Fuzhou, Fujian 350013, China

摘要

摘要: 以PRRSV FJ-1a株为模板,采用RT-PCR法分别扩增出GP2a/GP2b-3、GP3、GP4、GP5和M蛋白基因片段,双酶切后插入穿梭质粒pDC316。利用AdMaxTM法,将5个重组穿梭质粒分别与腺病毒辅助质粒共转染293细胞,得到重组腺病毒Ad-GP2a/GP2b-3、Ad-GP3、Ad-GP4、Ad-GP5和Ad-M。重组腺病毒滴度为107.7～109.0TCID50·mL-1,而以Ad-GP5滴度最低,仅为107.7TCID50·mL-1。PCR检测第3、6、9代重组腺病毒稳定性,确证重组病毒均能稳定携带目的基因。对目的基因转录的RT-PCR鉴定结果显示,重组腺病毒分别感染293细胞后能转录产生目的基因mRNA。表达PRRSV主要囊膜蛋白基因重组腺病毒的获得,为进一步研制PRRSV基因工程疫苗,解析主要囊膜蛋白功能奠定基础。
- 猪繁殖与呼吸综合征病毒 /
- 重组腺病毒 /
- 囊膜蛋白
Abstract: GP2a/GP2b-3,GP3,GP4,GP5,and M protein gene fragments of porcine reproductive and respiratory syndrome virus FJ-1a strain were amplified by RT-PCR and cloned onto shuttle vector pDC316.After co-transfection of one of the five recombinant shuttle vectors and the helper plasmid into 293 cells,recombinant adenoviruses Ad-GP2a/GP2b-3,Ad-GP3,Ad-GP4,Ad-GP5 and Ad-M were obtained.The titers of the five adenoviurses were 107.7-109.0 TCID50·mL-1.Among them,the Ad-GP5 titer was the lowest,at 107.7 TCID50·mL-1.The genome stability was determined by PCR detection of heterologous gene fragments in recombinant adenoviruses at 3rd,6th and 9th passages.In addition,when the recombinant adenoviruses were infected with the 293 cells,mRNA transcription of heterologous genes were confirmed by RT-PCR test.The establishment of recombinant adenoviruses for expressing PRRSV envelope proteins would provide a basic for further study on PRRSV genetic vaccine and the functions of the proteins.
- PRRSV /
- recombinant adenovirus /
- envelope protein

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参考文献(4)

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