Study On Immunity Characteristic Of PRRSV-gp5 Protein Of The LTb As Intramocecular Adjuvant

In the present study, we explored the new vaccine design against PRRSV, We construct LTb -PRRSV- gp5 fusion protein, in animal immune tests, we show the adjuvant LTb clarify role of the new-type PRRS vaccine for exploring new ways. Main contents are as follows:1.Cloning, Expression and Identification of ORF5 of PRRSVThe ORF5 gene of PRRSV was amplified by PCR with a size of 375bp.The amplified DNA fragment was cloned into pMD18-T, and sequenced. The result of sequencing showed that the consistency was 98% compared with that of standard strains. Inserted in pET-28a(+),and transformed in Escherichia coli BL21(DE3), the fragment was expressed after induced with IPTG, The results of SDS-PAGE and Western Blotting showed that the protein was 13.75KD in size and specifically reacted with PRRSV-positive sera from pigs, not negative sera.2.Cloning, Expression and Identification of the fusion gene of E.coli LTb and ORF5 of PRRSVIn this research the E.coli LTb gene and reconstructed ORF5 gene of PRRSV was by overlap extention PCR. A seven-aminoacid,proline-containing linker was included between the LTB and ORF5. The fusion gene LTb-ORF5 was cloned into pMD18-T and sequenced. The result showed that the consistency was 100% compared with that of parent gene. then the fusion gene was inserted in pET-28a(+),and transformed in Escherichia coli BL21(DE3), the fragment was expressed after induced with IPTG, The results of SDS-PAGE and Western Blotting showed that the protein was 26KD and specifically reacted with PRRSV-positive sera from pigs, not negative sera.3.Compared the result of the immunity of gp5 and LTb-gp5 proteinSix to eight weeks'ICR mices were immuned with the expressed gp5 and LTb-gp5 proteins and the control group established by indirect ELISA and MTT assay in mouse serum levels of specific antibody and T,B cell proliferation. Results show that gp5 protein intramuscular way can induce better immune effect; The gp5 of specific antibody achieve higher titiers after three weeks later. The LTb-gp5 IM and the gp5 emulsion group have no significant difference eache other after the third immunination. The two groups and gp5 IM have significant difference from the antibody types. After the third immunination the LTb-gp5 and the gp5 emulsification can produce the same three antibody subtypes. The gp5 IM can still produce only one antibody subtype. LTb-gp5 IM, gp5 emulsion gp5 IM group and the spleen can induce both T and B lymphocyte proliferation; LTb-gp5 IM, gp5 emulsion group were significantly higher than those gp5 IM.4.Preparation and Identification of the Monoclonal Antibodies of Escherichia coli LTb proteinThe Escherichia coli LTb gene was cloned into a prokaryotic expression vector pPIC9K, then transformed the recombinant plasmid in to SMD1168 to express recombinant protein. The recombinant protein was immunized to BALB/C mice. After the third immunization, the splenocytes of the immunized mice were collected and fused with murine myeloma cells. An indirect ELISA based on LTb recombinant protein was used to screen hybridom as which could produce specific antibody. Four hybridoma clones which could stably produce monoclonal antibody (McAb) against LTb recombinant protein was obtained. These MAbs named as 1F2,2C2,3D6,4H6 were characterized to be IgG2a isotype. The supernant of hybridoma clones could be recognised by LTb recombinant protein in Western blot analysis.