1型鹅星状病毒ORF2蛋白原核表达及其多克隆抗体的制备与鉴定

[目的] 利用原核表达系统表达1型鹅星状病毒（Goose astrovirus 1，GAstV-1）结构蛋白ORF2，并制备其多克隆抗体。[方法] 根据GAstV-1 TZ03株基因序列，对ORF2基因序列进行大肠杆菌密码子偏爱性优化和合成，将其克隆至原核表达载体pET-30a（+），构建重组质粒pET-ORF2，转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导表达重组蛋白，通过SDS-PAGE和Western blotting鉴定重组蛋白；将鉴定正确的重组蛋白纯化后免疫家兔制备多克隆抗体，以间接免疫荧光试验鉴定多克隆抗体的特异性。[结果] 酶切和测序结果显示，成功获得重组质粒pET-ORF2；SDS-PAGE结果显示，表达的重组蛋白分子质量约为70 ku，主要以包涵体形式存在；Western blotting结果表明，该蛋白能与抗His标签鼠单克隆抗体发生特异性反应。间接ELISA检测制备的兔抗ORF2蛋白多克隆抗体效价达1：128 000；间接免疫荧光试验结果显示，制备的多克隆抗体能与GAstV-1发生特异性反应。[结论] 原核表达的GAstV-1 ORF2重组蛋白具有良好的免疫原性，制备的兔源多克隆抗体具有较高的效价和特异性，为研制GAstV-1相关诊断试剂奠定了基础。

Prokaryotic Expression of ORF2 Protein of Goose Astrovirus 1 and Preparation and Identification of Its Polyclonal Antibody

[Objective] The purpose of this study was to express the structural protein ORF2 of Goose astrovirus 1 (GAstV-1) by prokaryotic expression system and prepare its polyclonal antibody. [Method] According to the gene sequence of GAstV-1 TZ03 strain, the ORF2 gene sequence was optimized and synthesized. It was cloned into prokaryotic expression vector pET-30a(+). The recombinant plasmid pET-ORF2 was constructed and transformed into E. coli BL21(DE3) competent cells. The recombinant protein was induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting. The polyclonal antibody was prepared by immunizing rabbits with the purified recombinant protein, and the specificity of the polyclonal antibody was identified by indirect immunofluorescence assay (IFA). [Result] The results of enzyme digestion and sequencing showed that the recombinant plasmid pET-ORF2 was successfully obtained. SDS-PAGE showed that the molecular weight of the expressed recombinant protein was about 70 ku, mainly in the form of inclusion body. Western blotting showed that the protein could react specifically with mouse monoclonal antibody against His tag. The titer of rabbit polyclonal antibody against ORF2 protein detected by indirect ELISA was 1:128 000. IFA results showed that the polyclonal antibody could react specifically with GAstV-1. [Conclusion] The prokaryotic expression of GAstV-1 ORF2 recombinant protein had good immunogenicity, and the rabbit polyclonal antibody had high titer and specificity, which laid a foundation for the development of GAstV-1 related diagnostic reagents.