Development Of A Indirect ELISA For Detection Of IBRV Serum Antibody By Recombinant Truncated Glycoprotein B

Infectious bovine rhinotracheitis is an acute, febrile contagion caused by IBRV (Infectious Bovine Rhinotracheitis Virus). IBRV is typical pantropic virus, which can invade a variety of organs and tissues. Invasion of cattle's bodies by IBRV can lead to persistent infection. And the infected cattle can carry the virus for a long time or even all their lives. What is more serious is that when the resistance is weak, the body can eliminate virus. IBR is one of the important reasons that cause the cattle industry economic losses, which has a obvious influence on milk yield and birth rate of cow, fertility of bull, growth and fattening of feeder cattle and factitive cattle. Whereas, now there are some reports on the occurrence of infectious bovine rhinotracheitis(IBR) in all the continents of the world. And the cattle of almost all the countries have different degrees of infectious bovine rhinotracheitis. Cattle and their by-products play an important role in agricultural production and economic growth in China. It is significant to establish a sensitive, special and accurate diagnostic method for the prevention and treatment of IBR in China and the detection status quo in port.IBRV glycoprotein B (gB) was chosen as study object. Firstly, the gene sequences of strain Nu/67 of Bovine herpesvirus-1 published in Genbank was analyzed by the biological software DNAstar-Protean. Then the fragments that have better antigenicity, hydrophilicity, and surface display probability was chosen as the target region and the PCR primers were designed according to the target gene sequences of Nu/67. Subsequently the three gene fragments gB1,gB2 and gB3 , respectively with the sequences of 396bp,516bp and 426bp, were amplified by the means of PCR using genomic DNA of IBRV Nu/67 as template. Then the three gene fragments were respectively cloned to the pMD18-T Simple Vector, so as to construct the recombinant plasmids pMD 18T-gB1,pMD 18T-gB2 and pMD 18T-gB3. The result of sequence analysis demonstrated that the concordance of the sequences of the three gene fragments were respectively consistent with those from GenBank.After the enzyme digestion, the three gene fragments of gB were respectively subcloned into the expression vector pET-28(+). Then the three recombinant expression vector 28b-gB1,28b-gB2and 28b-gB3 were constructed followed by transforming into E. coli BL21 (DE3) for expression. The SDS-PAGE electrophoresis result indicated the molecular weight of the three recombinant protein were respectively 18.5kDa,21.4kDa and 17.1kDa in concordance with prediction. Furthermore the conditions of expression were optimized. After 1mmol/L IPTG induction at 37℃for 4 hours, the expressed effectively of three proteins in BL21 with the highest expression amount of 28.09%,30.54% and 35.17% through thin layer scanning analysis. The expressed protein was mainly present in cytorrhyctes observed by SDS-PAGE. Highly purified recombinant were purified by the electroeluting.After reactionogenicity identification of the recombinant protein by the preliminary indirect ELISA and Western-blot, 28b-gB2 was determined as antigen and the indirect ELISA diagnostic method for IBR was established. Accordingly the optimum working conditions were determined: the working concentration of antigen was 50μg/mL, antigen was coated at 4℃overnight; the conditions of blocking was 2% BSA blocking for 120 minutes; antibody was 1?50 diluted,and reacted with antigen at 37℃for 60 minutes; the enzyme-labeled antibody was 1?5000 diluted,and reacted with antibody at 37℃for 60 minutes; substrate reacted for 10 minutes. The result of the cross reaction and the comparative trial shows that there was not any cross reaction, when the positive serum of Bovine Viral Diarrhea (BVD), Bovine Akabane Disease (BAD), Bovine Ephemeral Fever (BEV), Ibaraki Disease Virus(IDV) and Bovine Tuberculosis (BTB) were detected by this indirect ELISA diagnostic method; the sensitivity, specificity and coincidence rate are respectively 93.75%,92.30% and 93.33% compared with IDEXX detection method.The indirect ELISA diagnostic method for IBR is sensitive, specific and accurate. And it is positive and significant for the prevention and treatment of IBR and the detection in port. Moreover, it lays foundations of preparing a rapid detection kit for detecting IBRV.