Preparation Of Monoclonal Antibodies To Pseudorabies Virus And Prokaryotic Expression Of Major Domain Of Glycoprotein GB

Porcine pseudorabies (PR) is caused by pseudorabies virus (PRV). It is extremely contagious and acute infectious disease. PRV is a member of Alphaherpesvirinae, herpesviridae. The first PR case of cat was reported in our country in 1948, and with the rapid development of large-scale pig farming industry,more than 31 provinces and cities,including Hongkong and Taiwan,have reported the occurrence of the PR. It results in heavy economic losses in our pig industry. There is significant value to establish its diagnosis methods for the prophylaxis and eradication of the pseudorabies. In this study, the hybridoma cell lines secreting monoclonal antibody against PRV were screened; a gene hagment encoding the main antigtic domain(264bp)of PRV gBwithout signal peptide expressed in Ecoli expression system. 1. Development of hybridoma cell strains secreting monoclonal antibodies against PRV:Baby hamster kidney cells (BHK-21) were infected with the live vaccine strain Batha of pseudorabies virus(PRV). The harvested Cell cultures which repeated freezed and thawed three times were purified by differential centrifugation and sucrose density gradient ultracentrifugation. The BALB/c mice were immunized subcutaneously with purified antigen of PRV. Spleen cells from immunized mice were fused with NS0 cells.Indirect enzyme linked immunosorbent assay(ELISA) and immunohistochemical method were used to screen hybridoma cells, and limiting dilution method was performed to subclone positive hybridoma cells.Eight positive clones were obtained. The McAbs were named 2B2-C1 and 3F1-A1. The specific experiments showed that 2B2-C1and 3F1-A1 had no cross-reactivity with PCV,CSFV,PRRSV,JEV and BHK-21 cells. This research provides a basis for setting up a PRV-testing which has high specificity and sensibility.2.Clone and prokaryotic expression of a gene hagment encoding the main antigtic domain of the PRV gB genes:According to the nucleotide sequence of the PRV gB genes published in Genbank, a pairs of primers were designed to amplify the gB gene fragment by PCR. The fragment which encoding the main antigetic domains of PRV gB was 264bp. The PCR products were cloned into the expression plasmid pET-28a and the recombinant plasmid was named as pET-28a-gB. The recombinant plasmid was transformed into Escherichia coli BL21. After induction by IPTG, the fusion protein was expressed in Escherichia coli BL21. SDS-PAGE and Western blotting analyses revealed the molecular weight of the expressed recombinant pET-28a-gB protein was about 16 kDa, could react with pig serum containing antibody against PRV. It provided the material for further study of the gB function and diagnostic reagents for detecting PRV-gB antibody.