Expression Of The S1 And NP Gene Of Infectious Bronchitis Virus And Development Of ELISA Based On The Recombinant Nucleocapsid Protein

IBV has many different serotypes and clinical situations. Weak cross protection appear between different serotypes. Resently, IBV seriously hinder the development of poulty industry with the occurrence of new variant strains and serotypes. In order to affords strong specificity, high sensitivity ,excellent coincidence and can apply to detect the field sera samples in bulk and procvide an effective method for diagnosis and immunal monitoring of IB ,and also can incomplement VN test and HI test to evaluate the effect of the vaccine. we will do the research on NP-ELISA and S1-ELISA.The NP gene was obtained by RT-PCR , which nucleotide sequence is 1614bp in length .Then NP gene was cloned and recombined to the pET28a to contrast the expressed vector pET-NP. pET-NP was proved to be right and was induced by IPTG in bulk and was clearaged by ultrasonic wave to obtained the solubily N protein, The expessed N protein was purified by HisTrap HP Kit. The result demonsrated that the purified NP was suitable to be ELISA antigen. The purified N protein was used to establish an indirect ELISA assay for the detection of antibody againt IBV. The optimal concentration of coating antigen was1.45μg/mL, and the optimal dilution of serum and HRP labeled rabbit anti-chicken was 1:80 and 1:800. The border of negative and positive serum in OD₄₅₀ value was 0.270,when the OD₄₅₀ value was equal or over 0.27,it was positive, or else negative. NP-ELISA had no reaction to the positive serums of AIV H9,H5,H7,ND,IBD and EDS₇₆ ,which directed that the expressed N protein was only reacted to the serum of IBV. The result have no relativity bewent NP-ELISA and HI test. The results above indicated the NP-ELISA not only affords strong specificity, high sensitivity .excellent coincidence and can apply to detect the field sera samples in bulk.Complete S1 gene of Avian infectious bronchitis Virus M41 strain was amplified by RT-PCR, which nucleotide sequence is 1614bp in length. According to analysis of S1 pprotein, two pairs of primers were designed to obtain the 1533bp and 801bp of S1 gene, which one was deletaded the signal peptide and another included important antibody sites. The four fragments were cloned into the pGEM-T easy vector to construct the recombinant clone plasmids. The plasmids were proved to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences. Then the four fragments were recombinated into prokaryotic expression vectors pGEX-6P-1 to construct the expression vectors pGEX-S1-2,pGEX-S1-3, which were identified to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences, and then transformated into E.coli BL21 (DE3). The recombinant expression plasmid was induced by 1mmol/L IPTG at 37℃for 5 hours and analyzed by 12% SDS-PAGE. The results described that the recombined vector (pGEX-S1-2) was lowerly expressed in inclusion body which molecular weight was about 80 KDAa as expected by the analysis of SDS-PAGE. The result of western-blot demensrated the fusion proteins of S1 have partical antigen, which indicats the expression products have clinical value as the diagnosis antigen and gene engineering vaccine.