Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex

YU Jie-mei; He Jin-sheng*; LI Dong-liang; YANG Bing; YUAN Yuan

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June 2006

YU Jie-mei, He Jin-sheng*, LI Dong-liang, YANG Bing and YUAN Yuan. Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex[J]. Virologica Sinica, 2006, 21(3): 213-216.

Citation: YU Jie-mei, He Jin-sheng*, LI Dong-liang, YANG Bing, YUAN Yuan. Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex .VIROLOGICA SINICA, 2006, 21(3) : 213-216.

人呼吸道合胞病毒RNA聚合酶复合体蛋白N、P的表达鉴定

1.
安徽医科大学免疫学教研室，安徽合肥 230032

摘要

本研究根据编码A亚型人呼吸道合胞病毒（Human Respiratory Syncytial Virus，HRSV）核壳体蛋白（Nucleocapsid protein, N）和磷蛋白（phosphoprotein, P）的基因序列，各设计一对特异性的引物，应用RT-PCR技术，从感染HRSV的HEp-2细胞中扩增获得n和p的基因片断，克隆至真核表达载体pcDNA3.1(+)。获得的重组质粒通过脂质体Lipofectamine 2000转染COS-7细胞，72 h后再用Western blot鉴定蛋白的表达。结果显示真核表达载体pcDNA3.1(+)/N和pcDNA3.1(+)/P的限制性内切酶分析结果与预期一致，基因序列分析显示没有发生无义突变，利用蛋白印记方法也检测到了N和P的特异性条带。于是我们认为成功构建了含有HRSV N和P编码基因的真核表达载体，在真核细胞内能顺利表达。为进一步开展HRSV反向遗传学等研究奠定了基础。
- 人呼吸道合胞病毒（HRSV）
- , 核壳体蛋白
- , 磷蛋白
- , 真核表达

Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex

1.
Department of Immunology, Anh.ui Medical University, Hefei 230032, China

Abstract

The n and p genes from subgroup A human respiratory syncytial virus (HRSV), were amplified by RT-PCR. The n product was cloned into pGEM-T easy vector and that of p into pcDNAII vector and the two genes were subcloned into the eukaryotic expression vector pcDNA3.1(+). The resulting recombinant plasmids pcDNA3.1(+)/N and pcDNA3.1(+)/P were checked by restriction enzymes and DNA sequencing and were tranfected into COS-7 cells with the aid of Lipofectamine 2000. Expression of N and P were detected by western blots at 72 hours post transfections. Expressed N and P in COS-7 cells was confirmed by western blot. The constructed expression vectors will be used for the further reverse genetics experimentations on HRSV.
- Human respiratory syncytial virus(HRSV)
- , Nucleocapsid protein
- , Phosphoprotein
- , Eukaryotic expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expression of N and P of Human Respiratory Syncytial Virus RNA Polymerasa Complex

1. Department of Immunology, Anh.ui Medical University, Hefei 230032, China

Abstract: The n and p genes from subgroup A human respiratory syncytial virus (HRSV), were amplified by RT-PCR. The n product was cloned into pGEM-T easy vector and that of p into pcDNAII vector and the two genes were subcloned into the eukaryotic expression vector pcDNA3.1(+). The resulting recombinant plasmids pcDNA3.1(+)/N and pcDNA3.1(+)/P were checked by restriction enzymes and DNA sequencing and were tranfected into COS-7 cells with the aid of Lipofectamine 2000. Expression of N and P were detected by western blots at 72 hours post transfections. Expressed N and P in COS-7 cells was confirmed by western blot. The constructed expression vectors will be used for the further reverse genetics experimentations on HRSV.