Expression of Recombinant Envelope Proteins VP19 and VP28 of WSSV and Preparation of Neutralizing Antibody

LI Hong-xia; MENG Xiao-lin*; XU Jin-ping; WANG Jian; LU Wei; CAO Xu

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December 2004

LI Hong-xia, MENG Xiao-lin*, XU Jin-ping, WANG Jian, LU Wei and CAO Xu. Expression of Recombinant Envelope Proteins VP19 and VP28 of WSSV and Preparation of Neutralizing Antibody[J]. Virologica Sinica, 2004, 19(6): 587-590.

Citation: LI Hong-xia, MENG Xiao-lin*, XU Jin-ping, WANG Jian, LU Wei, CAO Xu. Expression of Recombinant Envelope Proteins VP19 and VP28 of WSSV and Preparation of Neutralizing Antibody .VIROLOGICA SINICA, 2004, 19(6) : 587-590.

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

1.
武汉大学病毒研究所，香港绿色生命实验室&
2.
#8226
3.
武汉大学联合研发中心，湖北武汉 430072

摘要

根据GenBank上WSSV囊膜蛋白基因 vp19和vp28的序列，设计并合成两对引物，PCR扩增得到vp19和vp28两基因，大小分别为370bp和630bp。通过EcoRI位点连接两基因，再按正确的阅读框插入表达载体pET-22b（+）中，构建出重组表达载体pET-vp(19+28)并转化大肠杆菌BL21(DE3)。基因工程菌株35℃IPTG诱导，表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带。用Ni2+-柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清，进行螯虾活体中和病毒实验，结果表明抗血清对WSSV的中和效率达到了100%。
- 白斑综合症病毒(WSSV)
- , VP19
- , VP28
- , 中和抗体

Expression of Recombinant Envelope Proteins VP19 and VP28 of WSSV and Preparation of Neutralizing Antibody

1.
Institute of Virology, Wuhan University, Green Life Laboratory•
2.
Wuhan University Joint Research and Development Center, Wuhan 430072, China

Abstract

Two pairs of primers were designed according to the sequences of envelope genes, vp19 and vp28 of White spot syndrome virus (WSSV) in the GenBank. The two DNA fragments about 370bp and 630bp amplified by PCR were linked to the EcoR I restriction endonuclease site and cloned into E.coli expression vector pET- 22b(+) in the proper Open Reading Frame(ORF). With IPTG induction at 35℃, the molecular weight of the engineered protein was about 41kDa, which was identified by SDS-PAGE analysis. Antiserum of the fusion envelope protein VP (19+28) purified by Ni2+- column chromatography was prepared by immunizing rabbit. The VP (19+28) antiserum was used to neutralize the WSSV infection on crayfish by intramuscular injection. The result showed that the antiserum was effective in the neutralization of WSSV on the crayfish which were reared with artificial food at 15-22℃.
- White spot syndrome virus (WSSV)
- , Neutralizing antibody
- , VP19
- , VP28

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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 Recombinant Envelope Proteins VP19 and VP28 of WSSV and Preparation of Neutralizing Antibody

1. Institute of Virology, Wuhan University, Green Life Laboratory•
2. Wuhan University Joint Research and Development Center, Wuhan 430072, China

Abstract: Two pairs of primers were designed according to the sequences of envelope genes, vp19 and vp28 of White spot syndrome virus (WSSV) in the GenBank. The two DNA fragments about 370bp and 630bp amplified by PCR were linked to the EcoR I restriction endonuclease site and cloned into E.coli expression vector pET- 22b(+) in the proper Open Reading Frame(ORF). With IPTG induction at 35℃, the molecular weight of the engineered protein was about 41kDa, which was identified by SDS-PAGE analysis. Antiserum of the fusion envelope protein VP (19+28) purified by Ni2+- column chromatography was prepared by immunizing rabbit. The VP (19+28) antiserum was used to neutralize the WSSV infection on crayfish by intramuscular injection. The result showed that the antiserum was effective in the neutralization of WSSV on the crayfish which were reared with artificial food at 15-22℃.