Development Of An Indirect ELISA For Detection Of IgA Antibodies Against Procine Pseudorabies Virus And Preliminary Evaluation Of Mucosal Adjuvants

Swine Aujeszky's disease is one of the most serious swine diseases in the world,the pseudorabies virus(PRV)is the causative agent of it.Since 2011,PRV mutant strains has appeared in China,and the classical strain vaccine against PRV could not provide complete protection.The development of a new effective vaccine is the main task for prevention of PRV in pigs.Live vaccines could produced protective response after mucosal immunization,but mucosal antibodies were difficult to evaluate.Mucosal immunity mainly produces secretory immunoglobulin A(IgA),which has a wide range of immune protection.IgA antibody is the main index to evaluate the mucosal immune response of vaccines.There is no ELISA kit for the detection of IgA antibody against PRV currently.In this study,gB protein of PRV was used as coating antigen to establish an indirect ELISA method for detection of IgA against PRV,which provides a basic method for evaluation of mucosal immunity effect of vaccine.Inactivated vaccine has high safety and was less likely to cause spread of virus than live vaccine.However,inactivated vaccine is not easy to produce nasal mucosal immune response,it needs to be prepared with mucosal adjuvant for nasal immunity.In this study,the antigen were mixed with two mucosal adjuvants,several inactivated vaccines were prepared and tested in mice,which laid a foundation for the development of new vaccines.In this study,an indirect ELISA for detection of IgA antibody against PRV was established.The truncated gB gene fragment of PRV was amplified by PCR and cloned into the prokaryotic expression plasmid pGEX-6P-1.The recombinant plasmid was transformed into E.coli Transetta(DE3)strain,IPTG was used for induced expression.After identification of SDS-PAGE and Western blot,the soluble recombinant protein gB was expressed in E.coli.The recombinant protein gB was purified by glutathione affinity chromatography column and used as the coating antigen for ELISA.The bronchoalveolar lavage fluid(BALF)with or without IgA against PRV was used as positive or negative standard,and the HRP-conjugated goat anti-pig IgA was used as the secondary antibody.After the main reaction conditions of ELISA were optimized,an indirect ELISA method for the detection of IgA antibody against PRV was established.The ELISA was used to detect IgA antibodies against PRV from piglets immunized with live vaccine by mucosal immunity.The results showed that specific IgA antibody against PRV could be detected in nasal mucus.This method provides a preliminary detection method for the mucosal immune evaluation of PRV vaccine.Previous studies showed that mucosal adjuvant Gel 01 and immunopotentiator VA5 could significantly improve the mucosal immunity of swine and poultry vaccines.In this study,Gel 01,VA5 and inactivated antigen of PRV were used to prepare the vaccine.Two mucosal adjuvants were evaluated the effect on promoting mucosal immunity of inactivated antigen in mice.After inactivation of PRV gE/gI gene deletion strain,different vaccines were prepared by mixing them with VA5 and Gel 01.The mice were randomly divided into five groups:inactivated virus mixed Gel 01 group(V+Gel 01 group),inactivated virus mixed VA5 group(V+VA5 group),V+Gel 01+VA5 group,inactivated virus group(V group)and blank control group(PBS group).Serum and BALF were collected from mice at different times after immunization,and the specific antibody levels were detected.The mice were challenged with PRV wild strain at 28 days after immunization,and recorded the death of the mice.The results showed that the specific IgG and IgA against PRV in BALF of mice from V+Gel 01+VA5 group were significantly higher than those in other groups,and the specific antibody levels in serum were also significantly increased.The survival rate of mice immunized with V+Gel 01+VA5 vaccine was 100%.The experiments showed that the combination of VA5 immunopotentiator and Gel 01 mucosal adjuvant can significantly enhance the ability of PRV inactivated antigen to stimulate in mice to produce local and systemic humoral immune responses after immunized by mucosal immunization,and it also has a good anti-virus protection effect.Therefore,it can be initially used as a candidate vaccine for pseudorabies.