Preparation Of The Monoclonal Antibody Against The VP7Protein Of Group A Porcine Rotavirus(PoRV) And Its Antigenic Epitope Identification

Rotavirus(PoRV), which belongs to the Reovirus Branch, can cause diarrhea in a variety of the young animal. PoRV A serotypes mixed co-infect the piglets, Group A porcine rotavirus usually infects the piglets with mixed serotypes, which can cause anorexia, severe diarrhea, dehydration and even death. The rotavirus has spread throughout the world,and has brought the huge economic losses to the swine industry.Rotavirus contains11dsRNA,which encode12proteins. VP7is the structural protein of the RV, which constitutes the RV virion surface with VP4.VP7not only plays a key role in the uncoating, but also has the antigenic epitope, which can stimulate the host secreting neutralizing antibody。Therefore, identification of the antigenic epitopes were carried out in the study, which laid the foundation for the research on the PoRV detection technology and designing antiviral immunization strategy.To obtain G5-VP7gene and G11-VP7gene, reverse transcription-polymerase chain reaction(RT-PCR) were used. Then the amplified genes were inserted into the vector pMD-18T for sequencing. The truncated G5-VP7gene(G5-tVP7) and the truncated G11-VP7gene(Gll-tVP7) were amplified by polymerase chain reaction(PCR) using the correct plasmid as the template.The truncated genes were inserted into the prokaryotic expression plasmid pGEX-6p-1and the recombinant plasmids were named as G5-6p-tVP7、G11-6p-tVP7. Then the plasmids were transformed into the E.coli (DH5a) for expression. The recombinant protein G5-tVP7-GST、 G11-tVP7-GST were expressed inducted by IPTG. The results of the SDS-PAGE showed that the molecular weights of the recombinant protein G5-tVP7-GST and G11-tVP7-GST were both56Ku. The recombinant protein could react with anti-RV serum in Western blot assay.To prepare MAbs against the VP7of the PoRV, the BALB/c mice were immunized with the purified tVP7protein which was purified by the method of rubber-cutting. Three strains of the hybridomas secreting antibodies against G5-VP7protein and four strains of the hybridomas secreting antibodies against G11-VP7protein were obtained, which named as A6、 G8(IgG2b),H4(IgG1);4B4、4E6(IgG2b),4C5(IgG1),5G2(IgG2a) respectively. The specific immunoflurescence was detected in PoRV infected MA104cells by indirect immunoflurescence assay.In the Western blot assay, MAbs could react with the natural VP7of RV virion.Through a series of expression of the truncated G11-VP7, the antigenic epitope against MAb4C5was preliminarily identified by the western blot, which was "Y175QQTDEANKWISMG188".To exactly determine the antigenic epitope, a series of polypeptides were synthesized, every polypeptide consists of15amino acids. The antigenic epitope against MAb4C5was precisly identified with the polypeptides by the ELISA assay, which was "Q175TDEANKWIS186".Through the homologous analysis indicated that" Q175TDEANKWIS186" was conserved among the different sources of the group A rotavirus and also the different serotypes in swine.In the study, seven strains of hybridomas secreting antibodies against VP7protein was obtained. The antigentic epitope on the VP7of the G11PoRV was identified, which provided the valuable information and research materials for the detection of the group A porcine rotavirus and designing antiviral immunization strategy.