中国兽医科学2023，Vol.53Issue(12)：1523-1530,8.DOI:10.16656/j.issn.1673-4696.2023.0209
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
摘要
Abstract
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 con-ducted antigenicity analysis on PAstV5 Cap through DNAStar software,selected the antigen dominant re-gion 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 induc-tion 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 rab-bits to prepare polyclonal antibodies.The titer of polyclonal antibodies determined by indirect ELISA method was 1∶51 200.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 poly-clonal antibody had good reactivity.This study lays the foundation for the establishment of a detec-tion 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Key words
porcine astrovirus type 5/capsid protein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
胡艺欣,李泽辉,张璐,吴兴一,郝晨琳,祖少坡,靳晓慧,胡慧..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[J].中国兽医科学,2023,53(12):1523-1530,8.基金项目
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) （新型非注射型疫苗佐剂设计及诱发免疫机制研究,232300421001）
