| The structural protein gene VP2 was amplified from the genome of infectious bursal disease virus (IBDV) by RT-PCR. The recombinant VP2 protein was expressed effectively in E.coli BL21 (DE3) plys S. Using the purified VP2 protein as coating antigen, an indirect ELISA method was successfully developed for detection of anti-IBDV antibody within serum. The establishment of VP2-ELISA provided us the base of rapid diagnostic method.A proteinase K digestion and phenol-chloroform extraction method was used to extract IBDV dsRNA from bursae of infected chicken. The VP2 gene was amplified by RT-PCR, and then cloned into pMD18-T vector for sequence analysis. The results of sequence analysis showed that VP2 gene has 1356 base pair, and the homology of nucleotide sequence to vvIBDV was 97%. The VP2 gene was subcloned into the expression vector pGEX-KG to construct pKG-VP2, and then transformed into E.coli BL21 (DE3) plys S. VP2 protein expression was induced by IPTG. The results of SDS-PAGE and Western blot indicated that the yield of VP2 protein was about 10% of total soluble protein under the optimum expression condition. The molecular weight of VP2 protein was about 40 KDa, and it had strong immunological activity.Using the purified VP2 protein expressed in the E.coli as coating antigen, an indirect ELISA method was developed. Optimized conditions were as following. The concentration of expressed protein for plate coating was 105 ng per well. P/N ratios reached maximum under the condition of serum dilution at 1:80, and demarcation of negative and positive was most obvious at the same dilution. The threshold value of ELISA was 0.32 with OD450. It was found that the positive ratio was 92.8% by detection of 890 serum samples from chicken.It indicated that the study had developed an indirect-ELISA assay with high specificity, good sensitivity and easy manipulation, which provided an available technique for immune detection and serological survey of IBD. |
