Construction Of GG~-/EGFP~+ Gene-deleted Mutant Of IBRV And Preparation Of Monoclonal Antibodies Against IBRV GG Glycoprotein

Infectious bovine rhinotracheitis(IBR) is caused by infectious bovine rrhinotracheitis viruses(IBRV) and known mainly as an acute,pyrexic and contagious disease of respiratory tract which is charaeterized by rhinotracheitis,conjunctivitis,fever, abortion and infectious pustular vulvovaginitis.IBR is epidemic all over the word,and causes significant economic losess ever year.Vaccination is an effective way to control this disease.However,there are some drawbacks of the traditional vaccines.Recently more and more researchers intend to develop live viral vaccines of IBRV.The objective of this study is to construct a recombinant infectious bovine rrhinotracheitis virus with gG gene deletion,providing a platform to develop live IBRV vaccine and a live viral vector expressing exogenous genes.1.Construction of gG~-/EGFP~+ Gene-deleted Mutant of IBRVTwo pairs of primers of gG gene was synthesized based on the genomic DNA sequence of IBRV(GenBank accession No.AJ004801).The upstream and downstream sequences of gG gene homologous arms were amplified by PCR with the two pairs of primers,and were directed-cloned into pcDNA3.1(+)myc-His B vector one by one by restriction enzyme.The EGFP gene was then inserted between the two homologous arms. The recombinant plasmid with deleted gG gene and intact EGFP gene were linearized and co-transfect MDBK cells together with IBRV genomic DNA and pBICP0.The recombinant mutant of IBRV gG~-/EGFP~+ was screened by observing the green fluorescence and purified with plaque formation.2.Preparation of monoclonal antibodies against IBRV gG glycoproteinBalb/c mice were immunized with purified IBRV.The immunized spleen cells of these mice were fusing with SP2/0 myeloma cells.One hybridoma cell strain producing monoclonal antibody against IBRV was established after screening by indirect ELISA and cloning three times by limited dilution.Indirect immunofluorescence assay showed that this McAbs was specific against IBRV.The ELISA titers of ascites were 1:3200.The McAb would be useful for detection of IBRV.