高茵;李杰峰;张宁;王华健;赵志强;郑丽丽;陆安;田勇;杨威;徐丽娜;
GAO Yin;LI Jie-feng;ZHANG Ning;WANG Hua-jian;ZHAO Zhi-qiang;ZHENG Li-li;LU An;TIAN Yong;YANG Wei;XU Li-na;School of Life Sciences and Food Engineering, Hebei University of Engineering;Institute of Animal Husbandry and Veterinary Medicine of Hebei Province;College of Veterinary Medicine, Hebei Agricultural University;Shijiazhuang Jinyuan Kangmu Pharmaceutical Co., Ltd.;

• 1:河北工程大学生命科学与食品工程学院
• 2:河北省畜牧兽医研究所
• 3:河北农业大学动物医学院
• 4:石家庄市金元康牧药业有限公司

摘要(Abstract)：

为获取猪圆环病毒3型衣壳蛋白(PCV3-Cap蛋白),本试验参照PCV3 HBBD1810毒株(GenBank登录号：MK105924)的基因组序列信息合成cap基因，并将其克隆至原核表达载体pET-32a中，对重组质粒进行测序和双酶切鉴定后，将重组质粒转化Rosetta大肠杆菌，对最佳IPTG诱导浓度和诱导表达时间筛选，并对目的蛋白表达后的存在形式进行检测以及对纯化条件进行筛选，透析复性后利用鼠源抗PCV3-Cap单克隆抗体进行Western blot以验证目的蛋白的反应原性。结果显示，通过构建原核表达系统获得大小为42 kDa的PCV3-Cap蛋白；破菌处理后目的蛋白主要以包涵体形式存在于沉淀中；0.2 mmol/L IPTG 37℃下诱导培养8 h后蛋白表达量较高；在300 mmol/L咪唑洗脱时纯化出清晰条带；经过Western blot验证目的蛋白能够被鼠源抗PCV3-Cap单克隆抗体识别，具有良好的反应原性。结果表明，本试验成功构建了PCV3-Cap蛋白表达载体，成功表达PCV3-Cap蛋白，同时优化了表达和纯化条件，为PCV3亚单位疫苗的研制以及检测试剂盒的开发和研究提供了科学依据。
In order to obtain porcine circovirus type 3 capsid protein(PCV3-Cap protein), the cap gene was synthesized according to the genomic sequence information of PCV3 HBBD1810 strain(GenBank accession number: MK105924)and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid was further verified by sequencing and double digestion. After verification, the plasmid was introduced into Rosetta expression bacteria for prokaryotic expression, and the optimal IPTG concentration and induction time were determined. The presence form of the target protein was then detected and the purification conditions were screened. After dialysis renaturation, Western blot was performed using mouse anti-PCV3-Cap monoclonal antibody to verify the reactivity of the target protein. The results showed that PCV3-Cap protein of 42 kDa in size was successfully expressed in constructed prokaryotic expression system; the target protein mainly existed in inclusion bodies in the precipitate after the treatment of broken bacteria; the protein expression was high after induction with 0.2 mmol/L IPTG for 8 h at 37℃; and the purification effect was good at 300 mmol/L imidazole elution. Western blot showed that the target protein could be recognized by mouse anti-PCV3-Cap monoclonal antibody and had good reactivity. Thus, through construction of PCV3-Cap protein expression vector, expression of PCV3-Cap protein, optimizing of expression and purification conditions, this study laid a sound foundation for the development of PCV3 subunit vaccine and detection kit.

关键词(KeyWords)： 圆环病毒3型;Cap蛋白;原核表达
porcine circovirus 3;Cap protein;prokaryotic expression

Abstract:

Keywords:

基金项目(Foundation): 农业基础性长期性科技工作动物疫病观测监测任务(ZX06S0303);; 河北省青年科学基金项目(c2019313002)

作者(Authors): 高茵;李杰峰;张宁;王华健;赵志强;郑丽丽;陆安;田勇;杨威;徐丽娜;
GAO Yin;LI Jie-feng;ZHANG Ning;WANG Hua-jian;ZHAO Zhi-qiang;ZHENG Li-li;LU An;TIAN Yong;YANG Wei;XU Li-na;School of Life Sciences and Food Engineering, Hebei University of Engineering;Institute of Animal Husbandry and Veterinary Medicine of Hebei Province;College of Veterinary Medicine, Hebei Agricultural University;Shijiazhuang Jinyuan Kangmu Pharmaceutical Co., Ltd.;

参考文献(References)：

• [1] Zhang H H,Wen Q H,Jie Y L,et al.Novel circovirus species identified in farmed pigs designated as porcine circovirus 4,Hunan Province,China[J].Transbound Emerg Dis,2020,67(3):1057- 1061.
• [2] Palinski R,Piňeyro P,Peng C S,et al.A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol,2017,91(1):e01879-16.
• [3] Cheung A K.Comparative analysis of the transcriptional patterns of pathogenic and nonpathogenic porcine circoviruses[J].Virology,2003,310(1):41- 49.
• [4] Nawagitgul P,Morozov I,Bolin S R,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein[J].J Gen Virol,2000,81(9):2281- 2287.
• [5] Phan T G,Giannitti F,Rossow S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2016,13(1):184- 187.
• [6] 王承玉，李杰峰，赵志强，等.猪圆环病毒3型河北株全基因组序列特征和遗传进化分析[J].甘肃农业大学学报，2020,55(1):35- 44.
• [7] Arruda B,Piňeyro P,Derscheid R,et al.PCV3-associated disease in the United States swine herd[J].Emerg Microbes Infect,2019,8(1):684- 689.
• [8] Bera B C,Choudhary M,Anand T,et al.Detection and genetic characterization of porcine circovirus 3 (PCV3) in pigs in India[J].Transbound Emerg Dis,2020,67(3):1062- 1067.
• [9] 苏战强，苏红，尹峥，等.猪圆环病毒3型陕西株Cap基因的克隆、序列分析及原核表达[J].动物医学进展，2020,41(7):7- 10.
• [10] 湛洋，王东亮，王乃东，等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报，2017,48(6):1076- 1084.
• [11] 王忠田，郭鑫，杨汉春，等.猪圆环病毒2型BF株ORF2基因在大肠杆菌中的表达[J].农业生物技术学报，2005(2):171- 174.
• [12] 陈如敬，吴学敏，陈秋勇，等.猪圆环病毒3型去核定位信号的Cap蛋白原核表达[J].动物医学进展，2020,41(11):24- 28.