The Establishment And Application Of Indirect ELISA For Detecting Antibodies To Infectious Bovine Rhinotracheitis Virus

Infectious bovine rhinotracheitis(IBR)is an acute,feverish and contagious infectious disease of cattle,caused by infectious bovine rhinotracheitis virus(IBRV).IBR is B species contagious disease lined up by World Organisation for Animal Health(OIE),and also focal point quarantine object for country to enter border animals and international animal trades had rated. An indirect ELISA was developed to detect antibody against IBRV using recombinant gD protein and IBRV virus in my essay.According to the IBRV gene sequence, one pair of primers was designed to clone gD gene.The gene of gD protein was amplified by PCR from the DNA, then cloned into pMD18-T vector. Then the gene of gD protein was subcloned into prokaryotic expression vector pET-30a(+).After identification with restriction endonuclease digestion, the recombinant vector genes were transformed into E.coli competent cell BL21(DE3) pLysS and expressions of proteins were induced by IPTG.The SDS-PAGE result showed that the gene of gD protein was expressed at high level in prokaryotic expression system. And the western blot assay proved the recombinant gD protein of IBRV DQ strain having good reactionogenicity and could be used as an antigen for detection of antibodies against IBRV.The DQ strain of IBRV was propagated in Madin-Darby bovine kidney (MDBK) cells cultured in DMEM and concentrate the virus using differential centrifugation. The concentration of virus is 3.10mg/mL.An indirect ELISA was developed to detect antibody against IBRV using concentrate virus of infectious bovine rhinotracheitis virus.Through the matrix experiments, the optimal coating antigen concentration was 1.25μg/ml, the optimal serum dilution was 1:100, the optimal dilution of HRP-labeled rabbit anti-bovine IgG was 1:5000, the best serum dilution buffer was 5% skim milk.The assay have favourable specific and sensitive. Comparison to the VN Test, the agreement rate is 96.25%.An indirect ELISA was developed to detect antibody against IBRV using recombinant gD protein of infectious bovine rhinotracheitis virus. Through the matrix experiments, the optimal coating antigen concentration was 2.5μg/ml, the optimal serum dilution was 1:200, the optimal dilution of HRP-labeled rabbit anti-bovine IgG was 1:5000, the best serum dilution buffer was 5% skim milk. There is no any cross reaction between the antigen and the positive serum of BCV,BPIV,BRSV.Comparison to the VN Test, the agreement rate of gD-ELISA is 92.5%. The 863 serum samples from Heilongjiang province were detected by this assay. The positive rate is 82.27 %.The positive rate of serum sample in Mudanjiang is 94.79 %, in Heihe is 82.30%, in Harbin is 70.2 %, in Yichun is 74.14 %, in Dqing is 87.17 %, in Qiqihar is 88.82 %.The result is that this ELISA could be used for epidemiology detection for Infectious Bovine Rhinotracheitis.