Prokaryotic Expression Of Infectious Bovine Rhinotracheitis Virus Glycoprotein D And Development Of An Indirect ELISA For Detection Antibodies Against Ibrv

Infectious bovine rhinotracheitis virus (IBRV) is main pathogen of infectious bovinerhinotracheitis (IBR)，IBRV often caused severe respiratory and genital tract infection, and it’salso main object of quarantine institute of animals. In the present study, an indirect ELISAdetection method was established to detect antibody against IBRV using purified gD protein,expecting that the method can make use of fast detecting and diagnosing IBR. The methodwill provide some technical support for quarantine and decontamination of IBR.GD protein was located on the surface of virus envelope, which can stimulate organismto generate neutralizing antibody, so it was also used to diagnostic reagent for IBRV. Thepresent study contains two researches:1. A pair of primers was designed according to the IBRV gD sequences in GenBank toamplify the complete open reading frame.The PCR product was then inserted into theprokaryotic expression vector PET-32a (+)，then transform the recombinant plasmids intocompetent cells of E.coli BL21. After induced by IPTG, the recombinant protein wasdissolved using urea with the concentration of8M and was purified by dialysis against ureaconcentration gradient. The result of RFLP and recombinant plasmids sequencing showedthat the prokaryotic expression vector recombinant plasmids were successfully constructed.SDS-PAGE demonstrated that gD fusion protein was effectively expressedas inclusion bodyin Escherichia coli. Dissolved by8M urea solution and dialysis by urea gradient solution, wesuccessful obtained the fusion protein of52ku, consistent with our prediction. Theconcentration of the purified recombinant protein was0.471mg/ml. Westen blot proved thatthe purified gD fusion protein can be identified by the immunized IBR positive sera,demonstrating a good immunogenicity.2. An indirect ELISA detection method was successfully established using the purifiedrecombinant gD protein as antigen, and through optimal reaction condition, antigen coatedwas at9.42μg/ml, coating antigen1.5h at37℃and overnight at4℃,sera dilution was at1:20, serum reaction1h at37℃, blocking solution at3%BSA, dilution at1:5000, andsubstrate reaction15min, the critical value of negative-positive sample was0.224. The method had a high specificity and fine reproducibility and it is more sensitive than virusneutralization test. The method was used to detect422bovine serum samples from Shaanxiprovince, with the positive rate of serum antibody22.9%. The result indicated that ELISAmethod had a fine sensitivity and specificity, showing a satisfactory application prospection.