Indirect ELISA For The Detection Of Specific Antibody In Chickens Immunized With DNA Vaccine And Expression Of IBV M Gene In E.Coli

The antigen of IBV was purified by difference speed centrifugation and discontinuous centrifugation on sucrose gradients in this thesis. Then, an indirect enzyme-linked immunosorbent assay (Indirect ELISA) was developed to detect the specific IgG of infectious bronchitis virus (IBV) in sera from chickens immunized with DNA Vaccine. It suggestted the optimum concentration of coating antigen was 5μg/ml, and the sera dilution 1:320 and the conjugate dilution 1:10000, respectively. Moreover, the discrimination level between positive and negative sera was set an S/N of 1.27.The results showed that IBV specific antibody in chicken was induced by individual recombinant plasmid pc-S1, pc-M and pc-N, or their admixture. The antibodies were significant difference between DNA vaccine groups and the control group and blank plasmid group(P<0.01). The results of challenging chicken with IBV showed that the chickens was inoculated IBV DNA group could obtaine the ability against IBV infection. The protection rate of S1, M and N is 40%(8/20), 70%(14/20)and 70%(10/14) respectively, and the protection rate of three gene DNA mixture group reached 90% (18/20). According to the results ,we can concluded that the recombinant plasmid admixture group shows the higher protection rate than others, and among the DNA vaccine group alone, M DNA vaccine and N DNA vaccine was superior to S1 DNA vaccine.According to the IBV M gene sequences (AY302742—AY302749) downloaded from GenBank, a pair of primers were designed to amplify M gene from IBV by RT-PCR. The PCR product was cloned and sequenced. The M gene was inserted into the multi-clone site of E. coli expression vector pET30-b(+), and the fusion expression vector pET30 -M was recombined and detected. Then, the positive recombinant was transformed into E.coli BL21 (DE3), and cultured with the inducement of IPTG The analysis of SDS-PAGE and Western blotting showed the M protein of IBV was expressed. The result showed that its molecular weight was 26KD and the yield reach 10% of the total soluble protein under the optimum expression condition that cultured in TE medium at 30 ℃, with Kanamycin 100μg/ml and IPTG 0.4mmol/L. The M protein expressed can be used as the basic material of diagnostic Antigens of IB and Genetically Engineered Vaccine.