G9P[8]型A组轮状病毒VP7蛋白抗体的制备及其免疫印迹检测方法的建立

Preparation of antibodies against G9P[8] group A rotavirus VP7 protein and establishment of its Western blotting detection method

摘要:

A组轮状病毒(group A rotavirus, RVA)是引起婴幼儿患急性胃肠炎的主要病原体之一。本研究旨在建立一种基于RVA VP7蛋白抗体的RVA免疫印迹(Western blotting)检测方法，用于RVA感染的诊断。首先对5株RVA新乡株进行全基因组测序和VP7基因遗传进化分析；构建VP7基因原核表达载体，并在大肠杆菌BL21(DE3)中诱导表达纯化VP7蛋白；随后免疫BALB/c小鼠制备抗VP7多克隆抗体，利用纯化的抗体作为检测抗体建立RVA的Western blotting检测方法，评估其特异性和敏感性；最后采用Western blotting与逆转录PCR (reverse transcription-polymerase chain reaction, RT-PCR)方法对60份临床粪便标本检测，分析其符合率。结果显示，5株RVA新乡株均为G9P[8]型，其VP7基因序列同源性为100%，属于G9-Ⅵ谱系。本研究成功表达和纯化了VP7蛋白并制备了高效价的多克隆抗体，所建立的Western blotting检测方法可检测到G9P[8]型RVA，与人肠道腺病毒和人诺如病毒无交叉反应。敏感性试验结果表明抗体的最高有效稀释度为1:32 000。与RT-PCR方法相比，Western blotting检测方法具有较高的敏感性，阳性符合率为100%，阴性符合率为92.1%，总符合率为95%。综上所述，本研究建立的Western blotting检测方法具有较好的特异性和敏感性，为RVA感染的实验室诊断提供了一种检测手段，具有一定的临床应用前景。

Abstract:

Group A rotavirus (RVA) is one of the leading pathogens responsible for acute gastroenteritis in infants and young children. This study aimed to develop a Western blotting assay using antibodies against the RVA VP7 protein for the diagnosis of RVA infection. First, five strains of RVA from Xinxiang were subjected to whole-genome sequencing and phylogenetic analysis of the VP7 genes. Subsequently, a prokaryotic expression vector for the VP7 gene was constructed, and the VP7 protein was induced for expression in Escherichia coli BL21(DE3) and purified. Then, BALB/c mice were immunized to generate polyclonal antibodies against VP7. With the purified antibodies as detection antibodies, a Western blotting assay for RVA detection was established and its specificity and sensitivity were evaluated. Finally, 60 clinical stool specimens were tested by both Western blotting and RT-PCR, and the concordance rate of results obtained with the two methods was analyzed. The results showed that all five strains of RVA from Xinxiang were identified as genotype G9P[8], with the VP7 gene sequences exhibiting 100% homology, and thus the strains were classified into the G9-VI lineage. The VP7 protein was successfully expressed and purified, and a high-titer polyclonal antibody was subsequently prepared. The established Western blotting assay specifically detected G9P[8] RVA strains and showed no cross-reactivity with human adenovirus or norovirus. Furthermore, sensitivity testing demonstrated that the highest effective dilution of the antibody was 1:32 000. Compared with the RT-PCR method, the Western blotting assay exhibited improved sensitivity, with a positive concordance rate of 100%, a negative concordance rate of 92.1%, and an overall concordance rate of 95%. In summary, the Western blotting assay established in this study demonstrates good specificity and sensitivity, providing an alternative detection method for the laboratory diagnosis of RVA infection and showing the potential for clinical application.