Expression of Major Capsid Protein Gene of Infectious Spleen and Kidney Necrosis Virus of Siniperca chuatsi in Prokaryote

ZHANG Min; BAI Jun-jie; LAO Hai-hua; YE Xing; JIAN Qing; LUO Jian-ren

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April 2004

ZHANG Min, BAI Jun-jie, LAO Hai-hua, YE Xing, JIAN Qing and LUO Jian-ren. Expression of Major Capsid Protein Gene of Infectious Spleen and Kidney Necrosis Virus of Siniperca chuatsi in Prokaryote[J]. Virologica Sinica, 2004, 19(2): 137-140.

Citation: ZHANG Min, BAI Jun-jie, LAO Hai-hua, YE Xing, JIAN Qing, LUO Jian-ren. Expression of Major Capsid Protein Gene of Infectious Spleen and Kidney Necrosis Virus of Siniperca chuatsi in Prokaryote .VIROLOGICA SINICA, 2004, 19(2) : 137-140.

鳜传染性脾肾坏死病毒主要衣壳蛋白基因的原核表达

白俊杰 ^1,2 ,
劳海华 ^1*,, ,
罗建仁 ¹ ,

1.
1. 中国水产科学研究院珠江水产研究所，广东广州 510380
2.
湛江海洋大学水产学院，广东湛江524025

通讯作者： 劳海华, ;

摘要

根据鳜鱼传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus, ISKNV)主要衣壳蛋白（Major Capsid protein, MCP）基因（mcp）序列设计引物，PCR扩增得到一长约1400bp的DNA片段，将其克隆到pGEM-T Easy Vector。氨基酸亲水性分析表明，在150-250位氨基酸之间亲水性很高，可构成主要抗原决定簇及形成跨膜区。mcp基因经PCR改造后克隆至原核表达载体pBV220，构建表达MCP的大肠杆菌基因工程菌，该工程菌经42℃诱导，SDS-PAGE检测，在约50kDa处有一特异蛋白带，含量约为菌体总蛋白的23%。用纯化和复性后的蛋白免疫新西兰大白兔制备抗血清，Western-blotting分析显示，重组MCP制备的抗血清能与ISKNV MCP特异结合，说明表达产物具有与ISKNV MCP相似的抗原特性。
- 鳜鱼传染性脾肾坏死病毒
- , 主要衣壳蛋白
- , 原核表达
- , 免疫印迹

Expression of Major Capsid Protein Gene of Infectious Spleen and Kidney Necrosis Virus of Siniperca chuatsi in Prokaryote

ZHANG Min ^1,2 ,
BAI Jun-jie ^1*,, ,
LAO Hai-hua ¹ ,
YE Xing ¹ ,
JIAN Qing ¹ ,
LUO Jian-ren ¹

1.
1.Pearl River Fisheries Resarch Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China 2. Zhanjiang Ocean University, Zhanjiang 524025, China

Corresponding author: BAI Jun-jie,

Abstract

A pair of primers was designed according to the sequence of ISKNV mcp gene, and a DNA fragment about 1400bp was amplified by PCR using the designed primers. The amplified product was cloned into the pGEM-T Easy Vector. Amino acid hydrophilic analysis indicated that the amino acids residue between 150 and 250 contained a high hydrophilic region, which is possiblely a trans-membrane region and the main epitope of MCP. In order to construct the MCP recombinant expression bacteria, the mcp gene was modified by introducing EcoRⅠ restriction endonuclease sites at both 5’ and 3’ ends and cloned into E.coli expression vector pBV220. After temperature induction and SDS-PAGE analysis, a protein of about 50 kDa in molecular weight was detected. The proportion of expressed MCP in total bacterial protein was about 23%. Two New Zealand rabbits were immunized by purified recombinant MCP to produce anti-serum. Westernblotting showed the recombinant Mcp had some epitopes of the native ISKNV MCP.
- Infectious spleen and kidney necrosis virus(ISKNV)
- , Major capsid protein
- , Prokaryotic expression
- , Western blotting

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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 Major Capsid Protein Gene of Infectious Spleen and Kidney Necrosis Virus of Siniperca chuatsi in Prokaryote

Corresponding author: BAI Jun-jie,

1. 1.Pearl River Fisheries Resarch Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China 2. Zhanjiang Ocean University, Zhanjiang 524025, China

Abstract: A pair of primers was designed according to the sequence of ISKNV mcp gene, and a DNA fragment about 1400bp was amplified by PCR using the designed primers. The amplified product was cloned into the pGEM-T Easy Vector. Amino acid hydrophilic analysis indicated that the amino acids residue between 150 and 250 contained a high hydrophilic region, which is possiblely a trans-membrane region and the main epitope of MCP. In order to construct the MCP recombinant expression bacteria, the mcp gene was modified by introducing EcoRⅠ restriction endonuclease sites at both 5’ and 3’ ends and cloned into E.coli expression vector pBV220. After temperature induction and SDS-PAGE analysis, a protein of about 50 kDa in molecular weight was detected. The proportion of expressed MCP in total bacterial protein was about 23%. Two New Zealand rabbits were immunized by purified recombinant MCP to produce anti-serum. Westernblotting showed the recombinant Mcp had some epitopes of the native ISKNV MCP.