Prokaryotic Expression And Immunogenicity Of Truncated VP4 Protein Of Porcine Rotavirus

Porcine rotavirus(PoRV)is one of the main pathogens causing acute diarrhea and dehydration in young piglets.The mortality rate of young piglets infected with PoRV is high.Vaccination is the most effective and economical prevention and control measure.In recent years,the infection rate of porcine rotavirus has increased significantly,second only to porcine epidemic diarrhea virus,which poses a great threat to the healthy development of pig industry.There are many serotypes of porcine rotavirus,and the cross-protection between different types is poor.Epidemiological survey showed that G9P7 and G9P23 were the predominant serotypes in China,but no vaccines were available.Some enterprises have tried to use inactivation technology to develop inactivated whole virus vaccines,and it is expected that after the approval of the combined vaccine,the situation of vaccine availability will be alleviated to a certain extent.VP4 is an important protein with neutralizing activity of porcine rotavirus.Immunization with VP4 alone can induce a high level of immune response and has a good ability to neutralize the virus,which can completely avoid the risk of stray virus of inactivated whole virus vaccine.In this study,we attempted to develop bivalent porcine rotavirus subunit vaccines based on the truncated VP4* of PoRV G9P7(NJ2012)and G9P23(AHFY2022).First,the vp4* gene sequence was designed,optimized and cloned into recombinant plasmids vp4*P7 and vp4*P23.The recombinant immunogen was efficiently expressed in E.coli.Then,the VP4*P7 and VP4*P23 recombinant proteins were emulsified with adjuvants to immunize mice.The immunogenicity of PoRV alone or in combination was evaluated by neutralizing antibodies,ELISA antibodies and cellular immune indicators,which laid a foundation for the development of new combined PoRV genetic engineering vaccines.1.Prokaryotic expression and protein purification of VP4*P7 and VP4*P23.According to the bioinformatics analysis of the dominant immunogenic region of VP4,the gene sequence corresponding to 26-476 aa was selected and sent to the company for preferred codon optimization.The recombinant plasmids p Cold-sumo-vp4*P7 and p Cold-sumo-vp4*P23 were successfully obtained by cloning into the expression vector p Cold-sumo.All plasmids contained a 6×His tag and a sumo tag.VP4*P7(9 mg/L)and VP4*P23(16 mg/L)were expressed in Escherichia coli BL21(DE3)competent cells,and the soluble proteins were collected,which provided a reliable way to reduce production cost and simplify production process.2.Immunogenicity analysis of VP4*P7 and VP4*P23 recombinant proteins.The purified sumo tag protein,VP4*P7 protein,VP4*P23 protein and VP4*P7+VP4*P23mixed protein were used as immunomonins and emulsified with ISA201 adjuvant to immunize mice.Serum samples were collected from immunize mice on days 0,14,28 and 42 to detect neutralizing antibodies against the two viruses.ELISA was used to measure the level of Ig G,CCK8 method was used to measure the proportion of lymphocyte proliferation,flow cytometry was used to detect the proportion of T lymphocyte subtypes,and cytokine detection kit was used to detect the secretion of cytokines in mice.The results showed that the specific Ig G,neutralizing antibody titers and cellular immune indexes in the serum of the immunized mice were significantly increased,and the combination of VP4*P7+VP4*P23 was better than that of VP4*P23alone.In summary,we successfully prepared a bivalent subunit vaccine using porcine rotavirus VP4* protein,which was determined to have good immunogenicity and was able to stimulate the production of high titers of neutralizing antibodies in mice by preliminary tests.