Molecular Characteristics And Applications Of VP6 Protein In Group A Bovine Rotavirus In Some Areas Of China

Bovine Rotavirus A?BRVA?is an important diarrhea-causing pathogen.The VP6sequence is common target for molecular detecting BRVA.VP6 protein is also common target protein for serological detecting BRVA and candidate antigen for subunit vaccine.Although it is the most conserved structural gene of BRVA,genetic variations are also found in VP6 sequence.Therefore,monitoring variations in VP6sequence is of great significance for the detection and prevention of BRVA.The purpose of this study was to analyze the molecular characteristics of BRVA VP6,to express VP6 protein and to establish an indirect immunoassay and preliminary investigate the effect of subunit vaccine.The results obtained were as follow:1.Molecular characteristics of BRVA VP6 geneA pair of primers for amplifying the complete gene of BRVA VP6 were used to amplify 30 BRVA-positive MA104 cell cultures covering 6 provinces.17 complete VP6 genes covering 4 provinces were successfully obtained,which were all 1356 bp in length and belong to the I2 genotype.The nucleotide similarity of the coding region was 92.3%¹00%,and the mutation frequency was 15.37%?183/1191?.The amino acid similarity was 98.0%¹00%,and the mutation frequency was 5.29%?21/397?.Phylogenetic analysis showed that the 17 nucleotide sequences were clustered into 4small branches,12 sequences were closely related to the rhesus monkey RV strain,3sequences were closely related to the Indian bovine strain,and 1 sequence was closely related to DQ-75 strain from cattle in China and 1 sequence was closely related to the Japanese cat-derived strain.Further analysis showed that although there were certain amino acid variations in the VP6 proteins of individual strains,there were no significant differences in the predicted secondary and tertiary structures of the 17 VP6proteins,and the predicted length and region of the epitopes were also consistent.The results indicated that there was a certain variation in the nucleotide of BRVA VP6genes,but it had no significant differences on the antigenicity of VP6 proteins.It provided an important reference for the molecular detection method targeting VP6sequence.Since gene reassortment was an important evolutionary characteristic in rotavirus,that 13/17 VP6 sequences cloned in this experiment had the closest relationship with non-bovine rotavirus provided a reference for further studying the genetic evolution of domestic BRVA.2.Expression and purification of BRVA VP6 proteinAfter optimizing the codon bias of VP6 ORF of BRVA YG strain,the VP6 ORF sequence of this strain was synthesized with Bam I and Xho I restriction sites at both ends and a histidine tag at the N-terminus.After connecting with the pET-28H vector,the VP6 protein was highly expressed in E.coli Rosetta?DE3?.And the concentration was about 9 mg/mL after purification.The result of SDS-PAGE showed that the recombinant protein had a molecular size of about 44 kD,and the purification effect was good.Western Blot analysis showed that the protein had biological activity,laying a foundation for establishment of BRV antibody detection method and subunit vaccine research subsequently.3.Development and application of indirect ELISA kit based on recombinant VP6proteinThe purified recombinant BRVA VP6 protein was used as the coating antigen.After the reaction conditions such as the antigen coating concentration were optimized,an indirect ELISA assay for detecting BRVA antibody in bovine serum was successfully established.The method had no cross-reaction with antibodys of bovine coronavirus,bovine viral diarrhea virus and bovine enterovirus.And the intra-and inter-assay variation coefficients were less than 10%.Then an indirect ELISA test kit was successfully assembled based on the results in this study.The coincidence rate of the kit with a commercial BRVA antibody detection kit?Ingenasa of Spain?was 100%,but the sensitivity of the kit was higher.Therefore,the ELISA kit for detecting BRVA antibodies developed in this experiment had the characteristics of good specificity and reproducibility as well as high sensitivity which could be used as a substitute of commercial kits.Out of 755 yak serum from northwest Sichuan,99.04%samples were detected as BRVA positive,indicating that the BRVA infection was very common in yak in northwestern Sichuan.4.Preliminary evaluation of immune efficacy of recombinant VP6 protein subunit vaccineIn order to evaluate the protective effect of a subunit vaccine based on recombinant VP6 protein formulated with a mucosal adjuvant in mice,the subunit vaccine formulated with 20?g VP6 protein/dose was used to immunize the female mice intranasally after optimizing the dose of VP6 protein.Mating after twice immunizations,the 8 suckling mice were selected respectively from the immunized and the non-immunized mice,and each of them was intragastrically administered with200?L of BRVA virus(10⁷.32.32 TCID₅₀/mL).The results showed that 8 suckling mice from immunized mothers did not develop diarrhea within 7 days after challenge,while the diarrhea rate of the control mice was 100%?8/8?within 3 days after challenge.Although BRVA nucleic acid were detected in feces in both groups of suckling mice by fluorescent RT-PCR,histopathological examination showed that the intestinal pathological changes of the suckling mice from the immunized mother were lighter than that of control mice.These results indicated that the subunit vaccine based on VP6 protein in this experiment could provide a protection for the offspring mice by passive immunization,which laid a foundation for further investigation on the immunological efficacy of VP6 protein subunit vaccine in cattle.