一株猪Ａ群轮状病毒的分离及 VP6 多克隆抗体的制备

Isolation and identification of porcine group A rotavirus and preparation of VP6 polyclonal antibody

摘要

旨在分离鉴定某猪场猪Ａ群轮状病毒（PoRVA）的流行毒株及制备VP6蛋白的多克隆抗体。选择 PoRVA 呈阳性的腹泻仔猪粪便样品，处理后接种 Marc145 细胞进行分离培养，通过 RT-PCR、间接免疫荧光（IFA）检测进行鉴定；扩增 VP6 全长片段，克隆至原核表达载体pET-28a，将测序正确的重组表达质粒转化至大肠杆菌 BL21（DE3）感受态细胞，对表达的重组蛋白进行可溶性分析与镍柱纯化，通过 SDS-PAGE 和 Western blot 鉴定蛋白的表达与纯化效果；将纯化后的 VP6 蛋白免疫 BALB/c 小鼠，收集血清后进行 Western blot 和 IFA 鉴定。结果：成功分离到1株可在体外培养的PoRVA毒株 GDYT2024，VP7 基因型为 G12；诱导表达的重组蛋白主要以包涵体形式存在，纯化后条带单一（43 kDa），与预期结果一致；间接 ELISA 方法测定制备的鼠源多抗效价均在1∶64000以上，Western blot（稀释比1∶500至1∶3000）和 IFA（稀释比1∶500至1∶1500）检测 PoRVA 感染的样品均具有明显的阳性信号，表明制备的多抗可与病毒蛋白产生良好的免疫反应性。本研究为后续 PoRVA 的分离鉴定及其感染与致病的分子机制研究奠定了基础。

Abstract

The aim of this study was to isolate and identify the prevalent strains of porcine group A rotavirus (PoRVA) in a pig farm and to prepare polyclonal antibodies for the VP6 protein. The positive fecal samples of PoRVA from diarrheic piglets were collected, selected, processed, and inoculated into Marc145 cells for virus isolation. The strains were identified through RT-PCR and indirect immunofluorescence (IFA) assays. The full-length sequence of VP6 was amplified and cloned into the prokaryotic expression vector pET-28a. The correctly recombinant expression plasmid was transformed into the Escherichia coli BL21(DE3) competent cells. The soluble analysis and nickel column purification of the recombinant protein induced by IPTG were performed, which were identified by SDS-PAGE and Western blot. Finally, BALB/c mice were immunized with the purified VP6 protein, and the serum was collected from the mice for Western blot and IFA identification. The results showed that a PoRVA strain GDYT2024 was successfully isolated, which belonged to G12 genotype (VP7). The recombinant proteins were expressed mainly in the form of inclusion bodies. Purification determined the band to be of 43 kDa, consistent with the expected result. The titer of the prepared polyclonal antibody measured by indirect ELISA was more than 1∶64000, and the Western blot (the dilution ratio of polyclonal antibody ranged from 1∶500 to 1∶3000) and IFA (the dilution ratio of polyclonal antibody ranged from 1∶500 to 1∶1500) test results of the samples infected with PoRVA all presented positive signals, indicating that the prepared polyclonal antibody produced a good antigen-antibody reaction with the viral protein. This study laid a foundation for subsequent research on isolation and identification of PoRVA, and on the infection and pathogenic molecular mechanisms of the strain.

关键词

猪Ａ群轮状病毒 / 分离鉴定 / VP6 / VP7 / 原核表达 / 多克隆抗体

Key words

porcine group A rotavirus / isolation and identification / VP6 / VP7 / prokaryotic expression / polyclonal antibody

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基金资助

扬州大学人才引进科研启动项目