羊口疮病毒ORF129基因的原核表达、纯化及鉴定

doi:10.16431/j.cnki.1671-7236.2018.07.009

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (7): 1798-1803.doi: 10.16431/j.cnki.1671-7236.2018.07.009

羊口疮病毒ORF129基因的原核表达、纯化及鉴定

杜国玉^1,2, 刘永杰², 吴锦艳², 王光祥², 尚佑军², 张勇¹

1. 甘肃农业大学动物医学院, 兰州 730070;
2. 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, 国家口蹄疫参考实验室, 兰州 730046

收稿日期:2017-12-28 出版日期:2018-07-20 发布日期:2018-07-20
通讯作者: 尚佑军, 张勇 E-mail:shangyoujun@caas.cn;zhychy@163.com
作者简介:杜国玉(1992-),女,安徽宿州人,硕士生,研究方向:动物疫病诊断及免疫防治技术,E-mail:779270108@qq.com
基金资助:
国家重点研发计划课题（2017YFD0500903）；国家现代肉羊产业技术体系项目（CARS-39-04B）

Prokaryotic Expression, Purification and Identification of ORF129 Gene of Orf Virus

DU Guoyu^1,2, LIU Yongjie², WU Jinyan², WANG Guangxiang², SHANG Youjun², ZHANG Yong¹

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Veterinary Etiological Biology, Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Lanzhou 730046, China

Received:2017-12-28 Online:2018-07-20 Published:2018-07-20

摘要/Abstract

摘要：

试验旨在克隆羊口疮病毒ORF129基因，并对其编码蛋白进行表达及纯化。参照GenBank已发表羊口疮病毒OV-IA82株ORF129基因序列（登录号：AY386263.1），用Primer Premier 5.0软件设计合成1对特异性引物，利用PCR方法扩增ORF129全基因序列，将其与质粒pET-28a （+）经BamHⅠ和Hind Ⅲ双酶切后连接，构建重组质粒pET-28a （+）-ORF129。重组质粒经双酶切和测序鉴定后转化大肠杆菌Rosttta感受态细胞，利用IPTG诱导ORF129基因的表达，经SDS-PAGE分析后，将表达产物超声破碎、洗涤、溶解，然后采用尿素梯度透析方法进行复性，经亲和层析法纯化目的蛋白并通过Western blotting对其进行鉴定。双酶切鉴定和测序结果表明，本试验成功构建了重组质粒，读码框正确，菌液起始D_{600 nm}值为0.4~0.8，37℃、220 r/min、1 mmol/L IPTG诱导3 h时能得到ORF129包涵体蛋白，蛋白大小为58 ku，在加入精氨酸、甘油的复性液中，蛋白复性率较高，将复性后蛋白与Ni柱结合，在咪唑浓度为500 mmol/L时，能将蛋白洗脱，纯化的蛋白经Western blotting鉴定正确，证明蛋白成功表达。本研究成功构建了羊口疮病毒ORF129基因原核表达重组质粒，并成功表达和纯化了ORF129蛋白，为后续羊口疮病毒检测方法的建立奠定基础。

关键词: 羊口疮病毒; ORF129基因; 原核表达

Abstract:

The aim of the experiment was to clone the ORF129 gene of orf virus,express and purify its encoded protein.According to the published ORF129 gene sequence of OV-IA82 strain in GenBank (Accession No.:AY386263.1),a pair of specific primers was designed and synthesized using Primer Premier 5.0 software,and the ORF129 gene was amplified by PCR.The whole gene sequence was ligated with the plasmid pET-28a(+) by BamH Ⅰ and Hind Ⅲ,and the recombinant plasmid pET-28a(+)-ORF129 was constructed.After identified by double digestion and sequencing,the recombinant plasmids were transformed into E.coli Rosttta,and the expression of ORF129 gene was induced by IPTG.After SDS-PAGE analysis,the expression products were disrupted,washed and dissolved by ultrasound,and then urea gradient dialysis method was used.After renaturation,the target protein was purified by affinity chromatography and identified by Western blotting.Double restriction enzyme digestion and sequencing results showed that the recombinant plasmid was successfully constructed with the correct reading frame.The ORF129 inclusion body protein was obtained when the initial D_{600 nm} value was 0.4 to 0.8,37℃,220 r/min,the final concentration of IPTG was 1 mmol/L,and the induction time was 3 h.And the protein size was 58 ku.In the arginine and glycerol refolding solution,the protein refolding rate was higher,and the protein was refolded after binding to the Ni column.When the concentration of imidazole was 500 mmol/L,the protein could be eluted.The purified protein was identified by Western blotting and proved to be successfully expressed.In this study,the prokaryotic expression recombinant plasmid of ORF129 gene was successfully constructed,and the ORF129 protein was successfully expressed and purified,which laid a foundation for the establishment of the detection method for orf virus.

Key words: orf virus; ORF129 gene; prokaryotic expression

中图分类号:

S852.65

杜国玉, 刘永杰, 吴锦艳, 王光祥, 尚佑军, 张勇. 羊口疮病毒ORF129基因的原核表达、纯化及鉴定[J]. 《中国畜牧兽医》, 2018, 45(7): 1798-1803.

DU Guoyu, LIU Yongjie, WU Jinyan, WANG Guangxiang, SHANG Youjun, ZHANG Yong. Prokaryotic Expression, Purification and Identification of ORF129 Gene of Orf Virus[J]. , 2018, 45(7): 1798-1803.

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羊口疮病毒ORF129基因的原核表达、纯化及鉴定

Prokaryotic Expression, Purification and Identification of ORF129 Gene of Orf Virus

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