An Indirect ELISA For Detection Of Antibody Against Infectious Bovine Rhinotracheitis Virus Using Recombinant Truncated Glycoprotein B Expressed In E.coli

The gene, which codes the amid acid residency from 190 th to 524 th of Infectious Bovine Rhinotracheitis Virus(IBRV) glycoprotein B(g B), was amplified by PCR technique with specific primers after analyzing glycoprotein B with biosoftware DNAstar. Then the amplified product was cloned into T-vector. After identifying with double-enzyme cut and sequencing for the accuracy of the product of amplification, the expected band on the agarose gals was recovered and directional inserted into p ET30 a vector. The recombinant vector was transformed into Rosetta SDS-PAGE analysis showed that the target protein was expressed in inclusion body in E. coli with a size of 41.4 ku. Western blotting analysis showed that the target protein had a good antigenic reactivity. The recombinant protein was checked by the indirect enzyme-link assay, which showed that it retains antigenicity of the nature protein. An indirect enzyme linked-assay(antigen concentration: 1.25 ?g/m L; serum dilution: 1:20) was developed with the recombinant protein as a diagnosis antigen, the positive criterion was S/P≥0.294.There was no cross reaction with positive sura of other six diseases, including BVD-MD(Bovine viral diarrhea), FMD(Bovine foot-andmouth disease),BTB(Bovine tuberculosis), BR(bovine brucellosis). the CV was less than 11%. Furthermore, the results of the indirect ELISA developed by this study were compared with the IDEXX g B-ELISA results, the positive coincidence rate was 84.28%, and the negative coincidence rate was 85.71%. The assay was confirmed to be specific and sensitive by the results above, suggesting a good application prospect.The serologtical investigation of IBR covered 7 regions of Heilongjiang such as Harbin,Qiqihar,Acheng,Mishan,Suihua,Raohe,Heihe include 10 farms.Result display:IBR 1377 serum samples, the highest positive rate was 65.52%; the lowest positive was 5.56%; the average positive rate was 18.01%.Between 2009 and 2014, Antibody positive rate as high as 37.22% in 2011, a minimum of 10.23% in 2012, from 2012 to 2014, the antibody positive rate showed a trend of increasing.