Study On Pedv Subunit Vaccine With LTB As Carrier And Its Adjuvant

Porcine epidemic diarrhea virus (PEDV) a members of coronavirus family, cause highly contagious swine intestinal diseases world wide especially in Asian countries with repeatedly outbreak and high mortality rate, causing serious economic losses to the pig industry, which is characterized with vomiting, diarrhea, dehydration and high mortality rate of piglets. However, the antigencity for PEDV has been changed in recent years, the corresponding inactivated vaccine or attenuated vaccine in market can not provide efficient protection which although is widely used; Moreover, it is very difficult for PEDV adapting cell line from fetal to vero cell. So it is valuable and urgent to develope a safe, efficient and inexpensive subunit vaccine against PEDV popular strain.The glycoprotein S is the most important structural protein for PEDV, according which virus particles can infect the host cell receptor, then penetration into the cells by membrane fusion, stimulating host to induce the production of antibodies, playing a critical role in developing PEDV vaccine. The COE is the core region of PEDV S protein, containing the most important eutralizing epitope of PEDV considered to be an important target antigen in developing PEDV subunit vaccines. Heat-labile enterotoxin B subnit (LTB) is the binding part for heat labile enterotoxin secreted by E.coli. LTB similar with LT, has mucosal immunogenicity and mucosal immune adjuvant activity, but with no toxicity, is an important target protein for enchancing mucosal vaccine research.The COE part of PEDV TX about 140 Aa of the S protein (502-641Aa) was selected as the target antigen in this study. The Escherichia coli heat-labile enterotoxin B subunit was fused to COE gene spacing with a linker, then the fused gene was cloned into pQZ which is a prokaryotic expression vector, the positive plasmid was named pQZ-COE-LTB. The recombinant protein was expressed when induced with IPTG following pQZ-COE-LTB was transformed into BL21 compent cell. Solubility for recombinant protein was identified with sonication and result proved that the recombinant protein is completely soluble. Western-blot analysis indicated the recombinant protein can react with the positive serum of PEDV.Unpurified recombinant protein COE-LTB supernatant was emulsified with white oil adjuvant. ICR mice received the first immunization at 4-week-old and the second vaccination after 14d, the serum samples were collected at 14d,21d,28d and 55d following the first immunization. Antibody against PEDV was detected with commercial PEDV ELISA antibody test kit. Results showed that mice recived COE-LTB immunization group produced obvious IgG antibody significantly higher than PEDV CV777 positive control group at 14 days after vaccination, which indicated that the recombinant protein is immunogenic, however, IgG antibody descend at 21d,28d and 55d, possiblly due to the antigen used in this experiment was unpurified.In order to enhance the immunogenicity of the recombinant protein, two immune enhancers named CVC1302 and CVC1303 were tested with unpurified recombinant proteins COE-LTB supernatant. ICR mice received the first immunization at 4-week-old and the second vaccination after 14d, the serum samples were collected at 14d,21d,28d and 55d following the first immunization. Agar diffusion test and ELISA kits of detection of serum IgG detection, also ELISA kits for intestinal tissue sIgA levels were performed. Results showed that recombinant proteins combined with CVC1303 immune enhancer group produce significantly both higher levels of IgG and sIgA antibodies in intestine compared with PEDV CV777 inactivated vaccine positive control group, indicated that CVC1303 immune enhancer can effectively enhance the immunogenicity of the recombinant protein.In summary, this study successfully constructed pQZ-COE-LTB/BL21 prokaryotic expression vector, and product was completely soluble when expressed in E.coli. Animal experiments showed that the soluble recombinant protein has good immunogenicity, and CVC1303 immune enhancer combined with recombinant protein can significantly enhance the immunogenicity of recombinant protein.