Yolk Antibody Preparation Against Chinese Sacbrood Virus VP2 Protein And Establishment Of Indirect ELISA Test Method

ObjectiveTo optimize the codon managing VP2 structural protein of Chinese sacbrood virus(CSBV)and conduct prokaryotic expression by bioinformatics software in order to prepare yolk antibody and to establish indirect ELISA test method detecting yolk antibody against CSBV by using purified VP2 protein as a coating antigen.MethodsVP2 gene sequence of CSBV-LN strain as a representative strain(Gen Bank:HM237361.1)was selected as a reference sequence by comparing VP2 gene sequences of 17 strains sacbrood virus(SBV)with those of 7 strains Chinese sacbrood virus(CSBV).Moreover,after taking the place of the amine acid at the corresponding position of VSBV-LN strain VP2 by relatively strong conservative amine acid in all CSBV strains,prokaryotic expression plasmid-p ET-28a-opti VP2 was constructed by optimizing the codon using online software(http://www.jcat.de/,http://genomes.urv.es/OPTIMIZER/).Prokaryotic expression plasmid p ET-28a-opti VP2 was transferred to BL21(DE3)to conduct induction expression.The expression products were identified by SDS-PAGE and Western blotting.For the standards-compliant,expression protein was purified and white Leghorn hen was immunized to it.The antibody level and lymphocyte added index of immunized hen were detected to explore immunogenicity of CSBV VP2 protein.Meanwhile,immunized eggs were collected to prepare yolk antibody.Purified yolk antibody interacted with CSBV for one hour.After that,Apis carana Fabr.larvae in 2-3 days old were inoculated with this antibody and mortality should be recorded to investigate neutralization ability of yolk antibody to CSBV viruses.In order to detect the anti-CSBV VP2 yolk antibody,purified VP2 protein was used as a coating antigen and indirect ELISA test method detecting yolk antibody against CSBV was established by determining coating antigen concentration,yolk antibody dilution,dilution multiple of enzyme labeled secondary antibody,yin-yang critical value and conducting sensitivity test and repeatability test.ResultsHereditary evolutional analysis for 17 SBV separation strains show that SBV comprised of two branches,including SBV infected with Apis mellifera L and SBV infected with Apis cerana F’ containing 7 CSBV strains.The comparison of VP2 amine acid sequences among 7 CSBV strains and analysis suggest that amine acid homology for different strains was 95~99.6%,in which CSBV-LN strain(Gen Bank: HM237361)was relative highly homologized with other strains(94~99.6%).Hence,VP2 gene sequence of CSBV-LN strain was selected as a reference sequence.Meanwhile,relatively strong conservative amine acid in all CSBV strains took the place of the amine acid at the corresponding position of VP2 of CSBV-LN strain.Asparagine(N),histidine(H)and Valine(V)at 49,84 and 148 of VP2 of CSBV-LN strain were replaced by serine(S),threonine(T)and isoleucine(I)respectively to conduct codon optimization and prokaryotic expression.The optimized opti VP2 can highly expressed in colibacillus,the expression product inoculated with white Leghorn hens could induce specific anti-CSBV antibodies,and the antibody level was significantly higher than that of PBS group(P<0.05).The results of the measurement of spleen lymphocyte proliferation index showed There was no significant difference in SI value between VP2 protein immunized group and purified virus group(P> 0.05),but it was significantly different from PBS group(P<0.05).Neutralization test results show the virus inoculation amount is less than or equal to 1x107 copies /larvae,the mortality of the egg yolk antibody group and the non-immunized egg yolk antibody group were significantly different(P<0.05),but there was no significant difference from PBS group.Meanwhile,the most suitable coating concentration of antigen was 800ng/hole and optimized dilution of yolk antibody was 1:100 for ELISA.In this optimized condition,rabbit-anti-chicken enzyme labeled secondary antibody was diluted in multiple proportion to determine its optimized diluting multiple and it was 1∶1000.Based on this,yin-yang critical value judging criteria was 1.2661 for ELISA,which had favorable sensitivity and repeatability.Conclusions1 The efficient expression of CSBV structural protein VP2 in prokaryotic system was achieved by optimizing the codon.2 Chicken immunization test showed that VP2 has good immunogenicity and the yolk antibody prepared by it has the ability to neutralize CSBV,which lays the foundation for further development of biological preparation for prevention and treatment of CSBV.3 CSBV VP2 purified protein is used as a coating antigen to establish indirect ELISA test method for yolk antibody against CSBV.