新型鹅细小病毒VP2蛋白的原核表达及其多克隆抗体的制备  

Prokaryotic expression of a novel goose parvovirus VP2 protein and preparation of its polyclonal antibodies

作　　者：刘琳敏 田甜[1,2] 郝雪飘 庞洪泽 雷白时 赵款[1,2] 张武超 袁万哲 LIU Lin-min;TIAN Tian;HAO Xue-piao;PANG Hong-ze;LEI Bai-shi;ZHAO Kuan;ZHANG Wu-chao;YUAN Wan-zhe(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医科学》2023年第9期1145-1151,共7页Chinese Veterinary Science

基　　金：河北省自然科学基金面上项目(C2021204028);河北省重点研发计划项目(22326618D);石家庄市科学技术研究与发展计划项目(203500402)。

摘　　要：为探究造成鸭短喙与侏儒综合征的病原即新型鹅细小病毒(NGPV) VP2蛋白的生物学功能,根据NGPV SD株序列,扩增VP2基因克隆至原核表达载体pET-32a,转化到大肠杆菌BL21(DE3)中表达重组蛋白。将纯化的蛋白按照标准程序免疫7周龄BALB/c小鼠制备多克隆抗体。结果显示,NGPV VP2基因全长为2 199 bp;通过大肠杆菌BL21(DE3)成功表达出大小约为83 ku的His-VP2融合蛋白。ELISA结果显示,制备的多克隆抗体效价为1∶204 800。Western-blot结果显示,制备的多克隆抗体能特异性识别VP2蛋白。间接免疫荧光试验表明,制备的多克隆抗体能够特异性识别受感染细胞中的NGPV和鹅细小病毒。综上所述,本研究成功表达并纯化了VP2蛋白,并成功制备了效价高反应性好的多克隆抗体,为进一步探究NGPV VP2蛋白生物学功能奠定了基础。In order to explore the biological function of the VP2 protein of the novel goose parvovirus(NGPV),which is the cause of duck beak atrophy and dwarfism syndrome,the VP 2 gene was amplified and cloned into pET-32a prokaryotic expression vector according to the sequence of NGPV SD strain,and transformed into Escherichia coli BL21(DE3)for recombinant protein expression.Seven-week-old BALB/c mice were immunized with the purified protein according to the standard procedure to prepare polyclonal antibodies.The results showed that the total length of NGPV VP 2 gene was 2199 bp.His-VP2 fusion protein with the size of about 83 ku was successfully expressed by E.coli BL21(DE3).ELISA results showed that the titer of the polyclonal antibody was 1∶204800.Western-blot results showed that the prepared polyclonal antibody could specifically recognize VP2 protein.Indirect immunofluorescence assay showed that the prepared polyclonal antibodies were able to specifically recognize NGPV and goose parvovirus in infected cells.To sum up,this study successfully expressed and purified VP2 protein,and successfully prepared polyclonal antibody with high titer and good reactivity,which laid a foundation for further exploring the biological function of NGPV VP2 protein.

关 键 词：鸭短喙与侏儒综合征 新型鹅细小病毒 多克隆抗体 VP2 

分 类 号：S852.659.2[农业科学—基础兽医学]