Establishment Of Indirect Enzyme-linked Immunosorbent Assays For IBR And Preparation Of Recombinant Glycoprotein E And Its Monoclonal Antibodies

Infectious bovine rhinotracheitis virus (IBRV) belongs to alpha-herpesvirinae, asubfamily of the herpesviridae. It causes diseases in cattle such as respiratory,abortions, genital diseases and conjunctivitis, sometimes encephalitis of newborncalves. Recently IBR spreads worldwidely now. The genome of IBRV is a lineardouble-strand DNA molecule of approximately 138 kb,with the content of G+C 71-72%.It consists of two unique parts called the long unique (UL) and the short unique(US), and the two reverse repeat units are located at internal and terminus. It contains54 to 59 genes, encoding 11 glycoproteins and other structural and non-structuralproteins.Glycoprotein E (gE)gene is one of the main virulent genes of IBRV. It canexpress on the surface of virion and the infected cell′s at high-level. It is the maintarget for neutralizing antibodies. The gE gene may affect the virus release from theinfected cells by some special ways. And the gE gene deletion may affect the virustransmission from cell to cell. Thus, the gE gene is essential for its virulence invivo.But it is not dispensible for the virus propagation in vitro. So it is regarded asnon-essential glycoprotein.IBR was found in the imported breed cattle in our country in 1970′s. It has beenspreading in most regions now. Which caused great loss and confined beef exporttrade seriously.At present the research on IBRV gene deleted marker vaccine isproceeding in China in order to fill out the blank as soon as possible.The only standardmethod to diagnose is neutralization experiment now.So it is important to explode thediagnose of IBR and preparation of monoclonal antibodies against IBRV and IBRV gEglycoprotein.We develope an indirect ELISA against antibody of IBRV to monitor theIBR's spreading and an indirect ELISA against antibody of IBRV gE envelopeglycoprotein to distinguish immunized cattles from natural ones. The preparedmonoclonal antibodies against IBRV and IBRV gE envelope glycoprotein can beused to detect the corresponding antigen.An Indirect ELISA Against Antibody of Infectious Bovine Rhinotracheitis VirusInfectious bovine rhinotracheitis virus(IBRV) was grown in Madin-Darby bovinekidney(MDBK) and purified by ultracentrifugation. The purified virus was disruptedusing an ultrasonic disintegrator. An indirect enzyme-linked immunosorbentassay(ELISA) was developed to detect specific antibody to infectious bovinerhinotracheitis virus(IBRV) in serum with prepared diagnostic antigen. OD valuehigher than 0.369 was determined as positive standard for the ELISA and lower than0.295 as negative standard. The ELISA was confirmed to be specific and reproducible.96.3% agreement was obtained compared to IBR ELISA kit of France ,and 95.8%compared to virus neutralization experiment. The infection with IBRV wasinvestigated by ELISA in some districts of China. The result showed that the infectionrate with IBRV is 67.10%in these districts.Cloning, expression and identification of IBRV gE envelope glycoproteinAfter infectious bovine rhinotracheitis virus(IBRV) Bartha Nu/67 strain wascultivated in Madin-Darby bovine kidney(MDBK) cell cultures, the glycoprotein E(gE)gene was cloned from the virus DNA by polymerase chain reaction (PCR) , andexpressed with pET32a expression system as six fragments. Two of the six fragmentswere expressed in soluble form and as fusion proteins, and one fragment wasexpressed in inclusion bodies , the other three were not expressed. There was a sixhistidines tag at amino acid terminus of the soluble expressed products. They werepurified by immobilized metal ion affinity chromatography under native conditions.The purified recombinant proteins showed reactivity to IBRV positive serum samplesand no reactivity to normal bovine sera in indirect ELISA and immuoblot assay. Theseassays demonstrates that recombinant gE proteins have very good antigenicity andspecificity.An Indirect ELISA Against Antibody of IBRV gE envelope glycoproteinAn indirect enzyme-linked immunosorbent assay(ELISA) was developed todetect glycoprotein E(gE) specific antibody to infectious bovine rhinotracheitisvirus(IBRV) in serum with recombinant gE protein. The optimum conditions ofiELISA were that the fold of the gE protein coated was 1.075μg. The rabbitanti-bovine IgG conjugated with perosidase is 1:5000, and the sera were 1:40. ODvalue higher than 0.582 was determined as positive standard for the ELISA and lowerthan 0.472 as negative standard. The ELISA was confirmed to be specific andreproducible. 94.35% agreement was obtained compared to imported IBR ELISA kit,97.98% compared to the indirect ELISA of IBRV and 85.71% compared to virusneutralization. The infection with IBRV was investigated by ELISA in some districtsof China. The result showed that the infection rate with IBRV is 68.74% in thesedistricts.Preparation and identification of monoclonal antibodies against IBRV and IBRV gEenvelope glycoproteinWith purified IBRV and recombinant protein of pET32gEE and pET32gEB asimmunogen, and the developed indirect ELISA of IBRV and recombinant gE proteinsfor selection,we obtained three hybridoma cell strains producing monoclonalantibodies against IBRV ,named IBRVM3G4,IBRVM7D10 and IBRVM8E10, twohybridoma cell lines gEEM2G11 and gEEM3G2 producing monoclonal antibodiesagainst gEE and two hybridoma cell lines gEBM4C10 and gEBM5B3 producingmonoclonal antibodies against gEB. The numbers of chromosomes of the hybridomacell lines were between 95 and 108.The specifity of the McAbs were confirmed byindirect immunofluorescence assay.Immunoglobulin subtype experiments showed that only IBRVM8E10 was IgAand others were all IgG1. The ELISA titers of ascites produced by McAbs werebetween 100×25 and 100×27. Antibody sandwich ELISAs which detected antigensdemonstrated that all 7 McAbs were strongly positive with IBRV Bartha Nu/67 andOD490 value could be high over 0.69. At the same time they were negative withcell-cultivated supernatant and the OD490 value were all under 0.092.The McAbs IBRVM3G4, IBRVM7D10 and IBRVM8E10 to IBRV were positivewith IBRV gE-/LacZ+ and the OD490 value could be high over 0.85. The McAbs togEE gEEM2G11 and gEEM3G2 had weak reaction with IBRV gE-/LacZ+ and theOD490 value were all under 0.4. Furthermore, the OD490 value was lower than 1/2 ofits with IBRV Bartha Nu/67.The McAbs to gEB gEBM4C10 and gEBM5B3 were thesame as gEE′s.