猪星状病毒5型衣壳蛋白抗原优势区域的原核表达及其多克隆抗体的制备与应用  

Prokaryotic expression in the dominant region of porcine astrovirus 5 capsid protein antigen preparation and application of its polyclonal antibodies

作　　者：胡艺欣 李泽辉 张璐 吴兴一 郝晨琳 祖少坡 靳晓慧[1,2,3] 胡慧[1,2,3] HU Yi-xin;LI Ze-hui;HANG Lu;WU Xing-yi;HAO Chen-lin;ZU Shao-po;JIN Xiao-hui;HU Hui(College of Veterinary Medicine,Henan Agriculture University,Zhengzhou 450002,China;Ministry of Education Key Laboratory for Animal Pathogens and Biosafety,College of Veterinary Medicine,Henan Agriculture University,Zhengzhou 450002,China;Key Laboratory for Animal-derived Food Safety of Henan Province,College of Veterinary Medicine,Henan Agriculture University,Zhengzhou 450002,China;Biological Laboratory,Zhengzhou Customs Technology Center Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院,河南郑州州450002 [2]河南农业大学动物医学院,动物病原与生物安全教育部重点实验室,河南郑州450002 [3]河南农业大学动物医学院,河南省动物食品安全重点实验室,河南郑州450002 [4]郑州海关技术中心生物实验室,河南郑州450002

出　　处：《中国兽医科学》2023年第12期1523-1530,共8页Chinese Veterinary Science

基　　金：河南省自然科学基金项目(新型非注射型疫苗佐剂设计及诱发免疫机制研究,232300421001)。

摘　　要：猪星状病毒(porcine astrovirus,PAstV)属于星状病毒科,是哺乳动物星状病毒属家族成员,临床上主要引起仔猪呕吐、腹泻等症状,且常与其他病原混合感染。PAstV衣壳蛋白(capsid protein,Cap)与宿主细胞受体结合,诱导机体产生中和抗体,是开发PAstV疫苗的靶蛋白。为制备抗猪星状病毒5型衣壳蛋白的兔源多克隆抗体,本研究通过DNA Star软件对PAstV5 Cap进行抗原性分析,选择出抗原优势区域ORF2开放阅读框1~642 bp(642 bp),构建原核表达质粒pET32a-Cap-642,测序结果正确后将其转化至大肠杆菌Rosetta。经IPTG诱导,重组蛋白以包涵体形式表达,大小约为43 ku。重组蛋白经尿素透析法纯化,免疫新西兰大白兔,制备多克隆抗体。间接ELISA方法测定多克隆抗体效价为1∶51 200,间接免疫荧光(IFA)和免疫印迹(Western-blot)结果显示抗PAstV5 Cap多克隆抗体能够特异性地识别PAstV5感染猪肾近曲小管上皮细胞(LLC-PK1)中表达的天然抗原;同时将抗PAstV5 Cap多克隆抗体进行了初步应用,结果表明该多抗具有良好反应性。本研究为PAstV5检测方法的建立以及PAstV5衣壳蛋白功能的研究奠定了基础。Porcine astrovirus(PAstV)belongs to Astrovirus,a member of mammalian astrovirus fam-ily.It mainly causes vomiting,diarrhea and other symptoms of piglets in clinic,and is often mixed with other pathogens.The capsid protein(Cap)of PAstV binds to the host cell receptor to induce the body to produce neutralizing antibody,which is the target protein for developing PAstV vaccine.In order to prepare rabbit polyclonal antibodies against porcine astrovirus type 5 capsid protein,this study conducted antigenicity analysis on PAstV5 Cap through DNAStar software,selected the antigen dominant region ORF2 open reading frame 1 bp-642 bp(642 bp),constructed the prokaryotic expression plasmid pET32a-Cap-642,and transformed it into E.coli Rosetta after the sequencing was correct.After induction by IPTG,the recombinant protein was expressed in the form of inclusion bodies,with a size of about 43 ku.The recombinant protein was purified by urea dialysis and immunized with New Zealand white rabbits to prepare polyclonal antibodies.The titer of polyclonal antibodies determined by indirect ELISA method was 1:51200.The results of indirect immunofluorescence(IFA)and Western-blot showed that the anti-PAstV5 Cap polyclonal antibodies could specifically recognize the natural antigen expressed in porcine renal proximal tubular epithelial cells(LLC-PK1)infected with PAstV5,and at the same time,the anti-PAstV5 Cap polyclonal antibodies was preliminarily applied.The results showed that the polyclonal antibody had good reactivity.This study lays the foundation for the establishment of a detection method for PAstV5 and the study of the function of PAstV5 capsid protein.

关 键 词：猪星状病毒5型 衣壳蛋白 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]