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

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.