The Establishment Of An Indirect ELISA Assay For Detection Of Anti-CAV Antibody Based On Screening Multi-Epitope Peptides

Chicken infectious anemia disease is caused by chicken anemia virus(CAV)and mainly characterized by anemia and lymphoid organ atrophy,and so on.The disease can cause serious economic loss.At present,high CAV positive rates in chicken flocks have been reported in several countries.Therefore,it is of great significance to control the disease by detecting CAV infection.Here,based on our previous works,an indirect ELISA method for detection of CAV serum antibody was established by varied form of epitope-peptide combinations from different viral proteins(VP1,VP2 and VP3)of CAV.The results will provide evidence for monitoring and controlling CAV infection.Firstly,potential linear epitopes of VP1,VP2 and VP3 proteins coded by CAV genome were analyzed by DNAStar software.Eight of epitope peptides were selected,including three epitopes in VP1,three epitopes in VP2 and two epitopes in VP3 epitope,and connected with a flexible peptide(AAY).The combination modes were determined as VP1+VP2+VP3(CAV123),VP1+VP2(CAV12),VP2+VP3(CAV23),VP1+VP3(CAV13)and Ii-CAV123(an invariant chain key peptide with CAV123),respectively.Secondly,gene amplification of each epitope-peptide chimer and construction of relative prokaryotic expression plasmids.(1)The gene sequence of Ii-CAV123 chimeric-peptide was synthesized by the biotechnology company.Then,the CAV13 chimeric-peptide gene was amplified by the designed specific primers using the SOE-PCR method.Other combinations were obtained directly by PCR method.Restriction enzyme cutting sites(Sal I and Eco R I)were added ends of each gene fragment,and connected to the p ET-32 a prokaryotic expression vector,respectively.Recombinant plasmids were identified by PCR method and gene sequencing.The results showed that five recombinant plasmids including p ET-32a/Ii-CAV123,/CAV123,/CAV12,/CAV23 and /CAV13 had been successfully constructed.(2)The expression of each fusion protein was induced with IPTG and purified by cutting gel slices detected by SDS-PAGE.The results showed that the fusion proteins,including r Ii-CAV123(36.8 k Da),r CAV123(36.3 k Da),r CAV23(30.4 k Da),r CAV12(32.6 k Da)and r CAV13(30.8 k Da),were mainly expressed in solubleanily.The fusion proteins had good reactivity with anti-VP1 and VP2 polyclonal antibody by Western blotting.Finally,the establishment of the indirect ELISA(i ELISA)was used for detection of CAV antibody.(1)Each fusion protein as coated antigen was used to detecting the reactive specificity of i ELISA method through the cross reaction test.The results showed that the r CAV13 protein had no obvious cross-reaction with specific serum sampleslike as ALV-J.Therefore,r CAV13 was selected as the coated antigen of i ELISA method.(2)The r CAV13 protein as antigen was coated at 4 ?overnight and optimized reaction conditions.The results showed optimal reaction conditions: 30 ?g/m L r CAV13 antigen,5% non-fat milk closed for 1 hour at 37?,the dilution ration of 1:100 of samples incubatedat 37 for 45 ?minutes,enzyme-labelled antibody combined for 40 minutes,TMB chromogenic reaction carried out for 8 minutes.i ELISA method with(named CAV13-i ELISA)was established.The Cut Off value is 0.645,and the variation coefficient of OD450 is 2.0% to 11.7%,which indicated that the method has excellent repeatability.(3)CAV13-i ELISA method was used to detect 88 serum samples in this study,and commercial kit was used as the reference.The results showed that the coincidence rate of them was 76.10%,and the positive rate of CAV13-i ELISA was 9.09% higher than that of the kit detection.The different serum samples were randomly selected and reacted with r CAV13 transferred to the PVDF film after SDS-PAGE for the Western blotting test.The results showed consistent with the testing of i ELISA.In conclusion,five modes of CAV protein epitopes compound were designed.r CAV13 was screened to be an effective coated antigen.CAV13-i ELISA method was established based on the optimized reaction conditions.This method has a higher positive rate than the commercial kit and has good specificity for detection of the clinical serum antibodies.The results provide a method for serological investigation and control of CAV infection.