摘要
为了研制针对新型鹅星状病毒(GAstV)的血清学检测方法和高效抗体,克隆新型GAstV HNZZ-4毒株的ORF2基因序列,构建pET-32a-Cap重组质粒,进行衣壳蛋白(Cap)原核表达;用重组Cap蛋白免疫兔只制备多克隆抗体,并采用Western blot和免疫过氧化物酶单层试验鉴定制备的多克隆抗体的反应原性.结果表明,重组质粒pET-32a-Cap经限制性内切酶NdeⅠ和XhoⅠ进行酶切后,得到大小约5 000 bp和750 bp的2个与预期一致的条带;阳性重组质粒测序结果显示,成功构建了重组表达载体pET-32a-Cap;pET-32a-Cap原核表达获得了分子质量约为27.5 ku的GAstV重组Cap蛋白;制备的兔抗GAstV Cap多克隆抗体能够与重组Cap蛋白发生特异性反应,且能特异性结合感染LMH细胞的GAstV.综上,制备的兔源抗新型GAstV Cap多克隆抗体保留了抗原性,特异性较好.
Abstract
In order to develop serological detection methods and highly effective antibodies against the novel goose astrovirus(GAstV),the ORF2 gene sequence of the novel GAstV HNZZ-4 strain was cloned and the pET-32a-Cap recombinant plasmid was constructed.Prokaryotic expression of capsid protein(Cap)was performed.Polyclonal antibody was prepared by immunizing rabbits with recombinant Cap.Western blot and immunoperoxidase single-layer assay were used to determine the reactivity of the polyclonal antibody.The results showed that the recombinant plasmid pET-32a-Cap was digested by the restriction enzymes NdeⅠand XhoⅠ,and two bands with the expected size of about 5 000 bp and 750 bp were obtained.Sequencing results of positive recombinant plasmid showed that the recombinant expression vector pET-32a-Cap was successfully constructed.Prokaryotic expression of pET-32a-Cap obtained GAstV recombinant Cap with molecular weight of about 27.5 ku.Rabbit polyclonal antibodies against GAstV Cap could react specifically with the recombinant Cap and bind to GAstV infecting LMH cells.The rabbit polyclonal antibody against novel GAstV Cap prepared in this study retains its antigenicity and has good specificity.
基金项目
河南省科技攻关计划项目(232102110109)
河南牧业经济学院科研创新基金项目(XKYCXJJ2020011)
河南牧业经济学院预防兽医重点学科项目(XJK202202)
国家科技期刊平台
NSTL
万方数据