猪札幌病毒VPg基因的原核表达及免疫原性研究

doi:10.16431/j.cnki.1671-7236.2015.04.007

›› 2015, Vol. 42 ›› Issue (4): 816-822.doi: 10.16431/j.cnki.1671-7236.2015.04.007

猪札幌病毒VPg基因的原核表达及免疫原性研究

单兴娜¹, 杨彬¹, 柳纪省¹, 张韵¹, 杨勃^1,2, 兰喜¹

1. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 草食动物疫病重点开放实验室, 兰州 730046;
2. 甘肃农业大学动物医学院, 兰州 730070

修回日期:2014-12-25 出版日期:2015-04-20 发布日期:2015-05-05
通讯作者: 兰喜 E-mail:lanxi@caas.cn
作者简介:单兴娜(1989-),女,河南洛阳人,硕士,研究方向:动物传染病分子生物学,E-mail:xingnashan@163.com
基金资助:
国家国际科技合作专项(2011DFA31830)

Prokaryotic Expression and Analysis of Immunogenicity of Porcine Sapovirus VPg Gene

SHAN Xing-na¹, YANG Bin¹, LIU Ji-xing¹, ZHANG Yun¹, YANG Bo^1,2, LAN Xi¹

1. Key Laboratory of Grazing Animal Diseases, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Revised:2014-12-25 Online:2015-04-20 Published:2015-05-05

摘要/Abstract

摘要： 为获得猪札幌病毒病毒基因组连接蛋白(viral protein genome-linked,VPg),本试验以中国西北地区猪札幌病毒CH430株RNA为模板,通过RT-PCR扩增VPg基因,将其克隆至pMD19-T Simple Vector,双酶切及基因测序鉴定后,再亚克隆至pET-30a中构建重组质粒,并转化至大肠杆菌BL21(DE3)中进行诱导表达.将纯化的目的蛋白免疫家兔得到超免疫血清.结果显示,获得的VPg基因全长为339 bp,编码113个氨基酸.SDS-PAGE结果显示,重组菌在37 ℃、1.0 mmol/L IPTG诱导表达6 h时重组蛋白表达量最高,表达的目的蛋白主要以包涵体的形式存在,大小为22 ku,与预期结果相符.Western blotting分析结果表明,该重组蛋白与超免疫血清具有良好的反应原性.超免疫血清ELISA效价可达1:12 800,且具有良好的特异性.本试验获得的超免疫血清为研究猪札幌病毒非结构蛋白VPg的结构与功能奠定了基础.

关键词: 猪札幌病毒; 病毒基因组连接蛋白(VPg); 原核表达; 纯化

Abstract: This study was aimed to express viral protein genome-linked (VPg) of porcine sapovirus, using the RNA of the CH430 strain of Northwestern China as a template, the VPg gene was amplified by RT-PCR and cloned into pMD19-T Simple Vector, and then recombinant plasmid was confirmed by double-endonuclease digestion and DNA sequencing. VPg gene was subcloned into a prokaryotic expression vector pET-30a to obtain the prokaryotically expressed plasmid pET30a-VPg. The positive recombinant plasmid was transformed into E. coli BL21 (DE3) cells to induce expression. The fusion purified protein was injected into rabbits to produce hyper-immune serum, the specificity and titer of the hyper-immune serum were determined by Western blotting and ELISA. The results indicated that the full-length of VPg gene was 339 bp, encoding 113 amino acids. The expression of recombination protein reached a maximum level when the recombinant bacteria at the condition of 37 ℃, final concentration of IPTG at 1.0mmol/L and the time of induction at 6 h. The purpose protein at size of 22 ku was consistent with expectation and expressed in the form of inclusion bodies analyzed by SDS-PAGE. Western blotting exhibited that expression products had good reactogenicity with the hyper-immune serum. ELISA result showed hyper-immune serum titer was 1:12 800 and had good specificity. The hyper-immune serum laid the foundation for further investigation on the structure and function of non-structural protein VPg of porcine sapovirus.

Key words: porcine sapovirus; viral protein genome-linked (VPg); prokaryotic expression; purification

中图分类号:

S852.65

单兴娜, 杨彬, 柳纪省, 张韵, 杨勃, 兰喜. 猪札幌病毒VPg基因的原核表达及免疫原性研究[J]. , 2015, 42(4): 816-822.

SHAN Xing-na, YANG Bin, LIU Ji-xing, ZHANG Yun, YANG Bo, LAN Xi. Prokaryotic Expression and Analysis of Immunogenicity of Porcine Sapovirus VPg Gene[J]. , 2015, 42(4): 816-822.

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猪札幌病毒VPg基因的原核表达及免疫原性研究

Prokaryotic Expression and Analysis of Immunogenicity of Porcine Sapovirus VPg Gene

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