鲤疱疹病毒2型ORF144原核表达和多克隆抗体制备  

The prokaryotic expression and polyclonal antibody preparation of Cyprinid herpesvirus 2 ORF144

作　　者：吴红军 潘李佳 李节 何艳格 杜宏瑶 龚保荣 吴宜靖 鲍传和[1] 朱若林[1] WU Hongjun;PAN Lijia;LI Jie;HE Yange;DU Hongyao;GONG Baorong;WU Yijing;BAO Chuanhe;ZHU Ruolin(School of Animal Science and Technology,Anhui Agricultural University,Hefei 230036)

机构地区：[1]安徽农业大学动物科技学院,合肥230036

出　　处：《安徽农业大学学报》2023年第3期457-462,共6页Journal of Anhui Agricultural University

基　　金：安徽省高校自然科学基金(KJ2019A0164)资助。

摘　　要：鲤疱疹病毒2型(Cyprinid herpesvirus 2,CyHV-2)引起的鲫造血器官坏死病是近年来对鲫(Carassius carassius)养殖业危害最大的疾病之一。通过PCR扩增得到ORF144基因全长,将其与pET-32a原核表达载体连接构建重组载体pET-ORF144,转化到大肠杆菌中,经IPTG诱导表达得到大小约46 kDa的重组蛋白,该蛋白主要以不可溶的包涵体形式存在。将重组融合蛋白纯化后免疫BALB/c小鼠获得多克隆抗体,间接ELISA检测显示抗体效价大于1∶51200。经Western Blot验证该抗体可特异性识别感染CyHV-2的细胞中的蛋白质和纯化的融合蛋白。间接免疫荧光结果显示该蛋白定位于细胞质,呈点状分布。结果表明构建的ORF144多克隆抗体可为病毒的基因功能和致病机制研究提供基础。Haematopoietic necrosis in Carassius carassius is one of the most damaging diseases in recent years caused by Cyprinid herpesvirus 2(CyHV-2).CyHV-2 belongs to the family Alloherpesviridae and the genus Cy-prinivirus.The genus also includes Cyprinid herpesvirus 1(CyHV-1),Cyprinid herpesvirus 3(CyHV-3),and An-guillid herpesvirus 1(AngHV).This virus is a double-stranded DNA virus with a total gene length of about 290 kb and contains 156 open reading frames(ORFs).Analysis of the CyHV-2 genome revealed that it contains four TRIM family genes,a family of E3 ubiquitinylated ligases that play an important role in the host immune system,while the role of TRIM protein analogs present in the virus has less been studied.The function of ORF144,the TRIM analogue of CyHV-2,has not been reported.In this study,the full length of ORF144 gene was obtained by PCR amplification.The recombinant prokaryotic expression vector pET-ORF144,constructed by ligating ORF144 gene with pET-32a,was transformed into competent E.coli cells,and the recombinant protein of about 46 kDa in size was obtained by IPTG-induced expression,which existed mainly as an insoluble inclusion body.The recombinant fusion protein was purified and immunized to BALB/c mice to obtain polyclonal antibodies.The potency of the antibody was greater than 1∶51200 by indirect ELISA,and the antibody was verified by Western Blot to specifically recognize the pro-tein in CyHV-2 infected cells and the purified fusion protein.Indirect immunofluorescence results showed that the protein was localized in the cytoplasm with a punctate distribution.In conclusion,the ORF144 polyclonal antibody constructed in this experiment can lay the foundation for gene function and pathogenesis study of the virus.

关 键 词：鲤疱疹病毒2型 ORF144 原核表达 多克隆抗体 

分 类 号：S941[农业科学—水产养殖]