Construction And Expression Of Multi-epitope Antigenic Gene Of Avian Infectious Bronchitis Virus And The Establishment Of ELISA Method

Infectious bronchitis virus (IBV) is one of the primary causes of respiratory disease in domestic fowl. With the development of large-scale farming, the damage of IB was more serious.Given its economic effects on the commercial poultry industry, the prevention of IB has been continually pursued. Avian infectious bronchitis virus has many serological types and was difficult to prevent. The main immunogen of IBV the structural protein:spike protein(S1), membrane protein(M) and nucleocapsid(N), The ELISA method established by using the single structural protein S1, M or N as coated antigen can only detect the antibody levels of the DNA vaccine which constructed with-gene S1,M or N. Given that default/the subject designed the Multi-epitope diagnostic antigen of IBV which included the S1,M, N protein gene epitopes, and established an ELISA method which can detect the antibody levels of chickens which were immunized traditional vaccine of various strains or DNA vaccine constructed with S1,M or N.1.The dominant epitopes of S1,M, and N protein of IBV M41 strain were analyzed on base of computer bioinformatics softwares and reported references, and then four epitopes from S, M, and N protein (S:221-356AA, M:165-200AA,N:118-270,913-1200AA) were selected. The four minigenes were paiallelled as a single multi-epitope gene separated from each other with flexible peptide.The analysis for the multi-epitope gene indicated that the coded multi-epitope protein showed favourable hydrophilicity, flexibility and antigenicity. The study provided foundations for the prokaryotic expression, ELISA method, eukaryotic expression, and research for DNA vaccine of the multi-epitope gene.2.The constructed multi-epitope gene of IBV was digested by BamHI and XhoI to generate multi-epitope gene D, which was subcloned into the prokaryotic expression vector pGEX-4T-1.The recombinant plasmid was transformed into E.coli BL21.The recombinant bacterium was induced by IPTG. Most of the multi-epitope D protein was expressed fusedly in in supernatant, while minority of the protein was expressed in the form of cytorrhyctes.The expression conditions of multi-epitope D protein were optimized with proper inducing conditions of 1 mmol/L IPTG for 5 hours at 37℃temperature. Wesiem-bloting results showed the expressed recombinant protein occured specific reaction with positive serum of IBV.3.Using the multi-epitope recombinant protein as coating antigen, the optimal concentration of D protein for coating of plate was 20μg/mL; the optimal dilution of antibody was 1:40;the optimal working concentration of HRP rabbit-anti-chicken IgG was 1:2000; the threshold value of ELISA assay was 0.084. According to the determination of condition of enzyme-linked immunosorbent assay (ELISA), an indirect ELISA based on the multi-epitope D protein was developed. The study provided new approachs the detection of ELISA antibodies IBV.