猪圆环病毒3型Cap蛋白的表达纯化及单克隆抗体的制备  

Prokaryotic expression of porcine circovirus 3 Cap and preparation of monoclonal antibodies against PCV3

作　　者：陈桂华[1] 孙媛[1] 周玲[1] 李迪 白杨[1] 麦凯杰 高金铭 马静云[1] CHEN Gui-hua;SUN Yuan;ZHOU Ling;LI Di;BAI Yang;MAI Kai-jie;GAO Jin-ming;MA Jing-yun(College of A nimal Science,South China A gricultural Unive rsity ,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院

出　　处：《中国兽医科学》2019年第1期13-19,共7页Chinese Veterinary Science

基　　金：“十三五”国家重点研发项目(2016YFD0501304)

摘　　要：猪圆环病毒3型(porcine circovirus 3,PCV3)是新近被发现的病原,与之相关的疾病包括母猪繁殖障碍、猪皮炎与肾病综合征、猪呼吸道疾病综合征、仔猪先天性震颤等。本研究采用PCR方法从PCV3/CN/GDLC1/2016株中扩增出衣壳蛋白(Cap)基因,对Cap基因进行序列分析并预测其二级结构,将去除信号肽序列的PCV3 Cap基因片段克隆到表达载体pET-32a(+)中,测序验证后转化至大肠杆菌BL21(DE3)感受态细胞中进行原核表达,经镍柱纯化表达蛋白,通过免疫BALB/c小鼠制备单克隆抗体(MAb)。结果表明,重组PCV3 Cap蛋白为包涵体,大小约38 ku,最佳诱导时间为4 h。通过镍柱成功纯化PCV3 Cap蛋白,筛选获得A8F、C6D、C7E、C6E、C9C、C9E、B7D和C11C等8株能稳定分泌抗PCV3 Cap蛋白抗体的杂交瘤细胞株。ELISA结果表明,A8F、C6D、C9E和B7D MAb均属于IgG2b亚类,C7E、C6E和C9C均属于IgM亚类,C11C属于IgG1亚类;A8F、B7D和C11C MAb效价均大于1∶64 000,C9E效价为1∶32 000,C6E效价为1∶8 000,C6D、C7E和C9C效价均为1∶1 000。经Western-blot分析,重组PCV3 Cap蛋白能与抗His标签抗体和抗PCV3 Cap蛋白MAb进行特异性免疫印迹反应,证实表达蛋白具有良好的反应原性。本研究为今后PCV3的防控提供了技术支持和实验基础,具有重要的生产实践意义。Porcine circovirus 3(PCV3) was identified as a newly discovered pathogen in swine with reproductive failure,porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory syndrome(PRS),congenital tremor(CT)and other symptoms.Capsid protein(Cap)gene of PCV3 strain PCV3/CN/GDLC1/2016 originated from porcine was amplified by PCR. After being sequenced,the truncated PCV3 Cap gene was cloned into the p ET32 a(+)expression vector and transformed into Escherichia coli BL21(DE3). A recombinant protein with 38 ku in molecular weight was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography.The optimal induction time of the recombinant was 4 h.BALB/c mice were immunized with purified PCV3 Cap protein to obtain the monoclonal antibodies(MAb).Eight hybridoma cell lines secreting the MAb against PCV3 Cap,namely A8 F,C6 D,C7 E,C6 E,C9 C,C9 E,B7 D and C11 C,have been selected,respectively.The immunoglobulin subtypes of eight MAbs were identified and the result showedthat A8 F,C6 D,C9 E and B7 D were IgG2 b,C7 E,C6 E and C9 C were IgM and C11 C were IgG1.The titers of A8 F,B7 D and C11 C were greater than 1 ∶ 64 000,C9 E was 1 ∶ 32 000,C6 E was 1 ∶ 8 000,and C6 D,C7 E and C9 C were 1 ∶ 1 000.Western-blot analysis confirmed that PCV3 Cap reacted with the MAb specifically and possessed antigenicity. This study provides a good technical support and theoretical basis for future control of PCV3 and has a significant meaning for pig production.

关 键 词：猪圆环病毒3型 CAP蛋白 原核表达 纯化 单克隆抗体 

分 类 号：S852.659.2[农业科学—基础兽医学]