Expression of vp4 Gene from Porcine Rotavirus in E. coli

SONG Yan; LI Yi-jing*; WEI Li-li; YU Xiao-long; FAN Chen; SHI Dong-fang

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October 2004

SONG Yan, LI Yi-jing*, WEI Li-li, YU Xiao-long, FAN Chen and SHI Dong-fang. Expression of vp4 Gene from Porcine Rotavirus in E. coli[J]. Virologica Sinica, 2004, 19(5): 462-466.

Citation: SONG Yan, LI Yi-jing*, WEI Li-li, YU Xiao-long, FAN Chen, SHI Dong-fang. Expression of vp4 Gene from Porcine Rotavirus in E. coli .VIROLOGICA SINICA, 2004, 19(5) : 462-466.

猪轮状病毒vp4基因在大肠杆菌中的表达

1.
东北农业大学动医学院病原分子生物学实验室, 黑龙江哈尔滨 150030

摘要

以猪轮状病毒JL94株核酸为模板扩增该病毒vp4全基因，对扩增产物进行测序及序列比较；根据VP4的5’端(1bp～750bp)特异片段主要决定其活性的观点，再设计一对引物扩增该主要抗原位点基因，将此主要抗原位点基因同pGEX-6P-1载体连接并转化入E.coli.BL21(DE3)plays，经IPTG诱导表达出蛋白质；对表达的蛋白进行Western blot分析、纯化和血清中和抗体试验。结果表明：JL94株与国外分离株CRW-8株、BEN-307株vp4全基因片段氨基酸同源性分别为96.98%和98.05%，说明JL94株与CRW-8株、BEN-307株属于同一VP4血清型；经IPTG诱导VP4主要抗原位点基因获得了高效表达，表达量占菌体蛋白的26%；Western blot结果和所表达的融合蛋白免疫小鼠产生的中和抗体能阻断JL94在MA104细胞上引起的细胞病变，说明所表达蛋白有良好的生物学活性。
- 猪轮状病毒
- , vp4基因
- , VP4主要抗原位点基因
- , 原核表达

Expression of vp4 Gene from Porcine Rotavirus in E. coli

1.
Molecular Virology Veterinary Medicine College, Northeast Agricultural University, Harbin 150030, China

Abstract

A pair of primers was used in cloning and sequence analysis of vp4 gene from Porcine rotavirus JL94 isolated in China. According to the idea that 5’ segments of VP4 (1bp～750bp) principally control the activity of vp4 gene, a pair of another primers was used in cloning and obtained major antigen sites. The gene was cloned into the expression plasmid pGEX-6P-1. The recombinant plasmid VP4-pGEX-6P-1 was transformed into E. coli BL21(DE3)plays and induced with IPTG. Homology of amino acids between CRW-8 strain and BEN-307 strain is 96.98% and 98.05% respectively. The product of the VP4 gene was 26% of total bacterial protein of BL21. Western blot test and neutralization test circumstantiate the protein of expression has biological activity.
- Porcine rotavirus(PRV)
- , vp4 gene
- , Major antigen site
- , Prokaryotic 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

Expression of vp4 Gene from Porcine Rotavirus in E. coli

1. Molecular Virology Veterinary Medicine College, Northeast Agricultural University, Harbin 150030, China

Abstract: A pair of primers was used in cloning and sequence analysis of vp4 gene from Porcine rotavirus JL94 isolated in China. According to the idea that 5’ segments of VP4 (1bp～750bp) principally control the activity of vp4 gene, a pair of another primers was used in cloning and obtained major antigen sites. The gene was cloned into the expression plasmid pGEX-6P-1. The recombinant plasmid VP4-pGEX-6P-1 was transformed into E. coli BL21(DE3)plays and induced with IPTG. Homology of amino acids between CRW-8 strain and BEN-307 strain is 96.98% and 98.05% respectively. The product of the VP4 gene was 26% of total bacterial protein of BL21. Western blot test and neutralization test circumstantiate the protein of expression has biological activity.