| Infectious bronchitis (IB) is an acute highly contagious respiratory disease of poultry caused by infectious bronchitis virus (IBV). As there are many serotypes, and new variants and genotypes are constantly emerging, and there are low cross-protection between different serotypes, immune failure often occurred causing serious economic losses to the poultry industry. Presently, there is no specific treatment of this disease, and the development of a rapid and accurate method to detect the antibody is important for the precaution of IB. The amino acid sequence of S1gene IBV ZY3strain was analyzed by DNAStar software, and four regions rich of epitopes were selected and ligated together by using flexible peptide linker. The tandem epitopes was then subcloned into prokaryotic expressionand a indirect ELISA assay was established by using the expressed recombination protein. The specificity, repeatability and sensitivity of this method were also measured.This study made a foundation for the assembly of Indirect Elisa kit and the development of chimeric multi-epitope vaccine against IBV.1. The amino acid sequence of S1gene IBV ZY3strain was analyzed by DNAStar software, including secondary structure prediction, Antigenic Index, Hydrophilicity Plot, Flexible Regions.And compared with H120, H52,QX, M41,491, SCYA, SCNC, SCDY strain by MegAlign software, then four epitopes from SI protein (26-51AA、165-249AA、295-391AA、46-505AA) were selected.To ensure that all epitopes of the respective natural senior conformation, independent function will not be disturbed each other, do not affect a series of protein biochemical characteristics, and the G4S spacers as Linker into a series of new genes F. And in the5’included a initiation codon ATG,and in the3’included a the termination codon TAA, in order to ensure the effective termination when mRNA translation. The analysis for the tandem gene showed that the coded tandem protein showed favourable flexibility, antigenicity and hydrophilicity. The study provided foundations for the prokaryotic expression of the multi-epitope tandem gene,and to establish an Indirect ELISA method.2. The constructed recombinant plasmid was digested by BamH I and Xhol enzyme,which was inserted into pET-32a(+) vector to obtain the recombinant plasmid pET-32-F.The recombinant plasmid was transformed into E. coil BL21.The bacterium was induced by IPTG.Recombinant protein got a lot of expression. Use of ultrasonic cracking law cracking bacteria, SDS-PAG results showed that the recombinant protein was a42kD protein in the form of inclusion body fusion expression, And Western-blote results showed the expressed recombinant proteinwas recognized specifically by positive serum of IBV. The expression conditions of chimeric F protein were optimized with proper inducing conditions of0.8mmo1/L IPTG for4hours at37℃temperature. Using the puridied tandem recombinant protein as coating antigen, the optimal concentration of F protein for coating of plate was12.5μg/mL;the working concentration of HRP sheep-anti-chicken IgG was1:2000, the dilution of antibody was1:60;the threshold value of ELISA assay was0.363,Sensitive test indicated that the OD45onm value was still0.374when the positive serum of IBV was diluted to1:500,suggesting a high sensitivity,and Specific test indicated that it could detect antibodies to IBV,and Not with AIV (H5), AIV (H9), NDV, IBDV standard positive serum happen cross reaction.The detection results indicated that lenv-ELISA was quick, specific and sensitive for large scale surveys of IBV antibodies.The present experiment was to investigate the flexibility, antigenicity and hydrophilicity of the coded tandem protein, and in its ELISA test the application of the positive and beneficial exploration,for its commercialization, large scale application laid solid foundation. |
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