Eukaryotic Expression Of HP-PRRSV (HuN4) ORF3 Gene And Preparation Of Monoclonal Antibodies Against GP3

Porcine reproductive and respiratory syndrome virus (PRRSV) was classified in the family Arteriviridae in the newly created order Nidovirales. Typical clinical symptom of PRRS were fever, abortion, prematurity, stillbirths and respiratory disease in pigs of any age. Although PRRS was first reported in the United States in 1987, it spread rapidly North American and Europe, and was first emerged in china in 1996. A highly pathogenic disease was outbreaked in the south of China in 2006. Most experts had demonstrated that PRRSV mutant was one of the main etiologies. PRRSV genome contained 9 open reading frames (ORFs). GP3 which was highly glycosylated envelope protein and was encoded by ORF3 play an important part in pathogenicity, replication and variability of virus, but the concrete function of GP3 was still unclear.A pair of primer for ORF3 gene of PRRSV was designed according to the published genome sequences of PRRSV HuN4 strains available in GenBank. The ORF3 gene of PRRSV was amplified by RT-PCR from PRRSV HuN4 strains and cloned into the baculovirus donor vector pFastBac HTb. The recombinant plasmid pF-ORF3 DNA was transformed into E.coli competent cells DH10Bac containing baculovirus shuttle vector, and the recombinant bacmid rBac-ORF3 was verified by PCR. The bacmid rBac-ORF3 was transfected into Sf9 insect cells and the recombinant baculovirus rAc-ORF3 was obtained and confirmed by PCR. Western blot and indirect flurescent assay (IFA) with antibody against PRRSV confirmed that the GP3 was expressed in Sf9 cells, the molecular weights of recombinant protein was 27ku.The purified GP3 expressed by recombinant baculovirus was inoculated into BALB/c mice to prepare the monoclonal antibodies and then the specificity of monoclonal antibodies (McAbs) were tested by the ELISA used by the purified GP3 expressed in E.coli. Through limiting dilution and screening and cloning, we obtained three hybridoma strains of secreting stable monoclonal antibody against GP3 of HP-PRRSV HuN4 strain which were named them 1C7,2D1 and 4C6 respectively. The ELISA titers of culture supernatant were1:256,1:3×105.0 and 1:1024, and ascites were 1:12 800, 1:1.6×107.0 and 1:25 600. The light chains of all McAbs are kappa orescent, subtypes of 1C7 and 4C6 ware IgM and subtype of 2D1 was IgG2b. IFA showed that three McAbs could recognized GP3 in PRRSV (HuN4 strain) infected Marc-145 cell.