The Study On Construction Of Genomic Expression Library Of Haemophilus Parasuis And Screening & Immunogenicity Of Its Capsular Polysaccharide Export Protein Gene

Haemophilus parasuis (Hps) is the etiological agent of Glasser's, which has become one of the most important bacterial diseases of pig worldwide. But systemic research on H. parasuis has not been reported, especially in pathogenesis and immune mechanism. The further studies on H. parasuiss pathogenesis and immune mechanism were important for disease prevention, diagnosis and control. In this study, H. parasuis SC-1was used to construct expression library. In vivo induced (IVI) genes were selected and identified by in vivo induced antigen technology. The capsular polysaccharide export protein gene of H. parasuis was selected and identified. The main contents were as follows:1. Construction of Haemophilus parasuis SC-1 genomic expression libraryIn this study, the Haemophilus parasuis SC-1 (serotype 4) strain was used to construct genomic expression library by molecular cloning methods. Genomic DNA of Haemophilus parasuis SC-1 strain was extracted by SDS-proteinase K and possessed high quality and purity.2.5μg total genomic DNA was partially digested with 0.25μL Sau3AⅠ(10 U/μL) at 37℃The digestion to produce 500 bp-2 000 bp fragments was the best choice. Which were recovered by agarose gel electrophoresis. BamHⅠwas used to digest the expression vectors pRSET A, pRSET B, pRSET C respectively. And the expression vectors were dephosphorylated. The genomic DNA fragments were ligated into the dephosphorylated vectors pRSET A/B/C. The recombinants were transformed into E. coli (DH5a) and identified randomly by colony PCR. The results of colony PCR showed that the size of inserted fragments ranged from 500 bp-2 000 bp and the rate of insertion reached 80%. The plasmids in DH5αwere extracted and purified. The plasmids were transformed by electroporation into BL21. The capacity of Haemophilus parasuis genomic expression library reached up to 1.2×105CFU. The colonies were picked out to sequence randomly. The sequencing results indicated that all the foreign inserted fragments shared more than 99% in the homology comparing with Haemophilus parasuis genomic DNA. The Haemophilus parasuis genomic expression library was constructed successfully. It was a basis for screening of Haemophilus parasui.in vivo induced antigen genes.2. Screening and identification of in vivo induced antigen genes during swine infected with Haemophilus parasuis SC-1 In vivo induced antigen genes were screened and identified from the Haemophilus parasuis SC-1 strain genomic expression library with the pig sera infected Hps. In order to remove the non-specific antibody subsets expressed against H. parasuis SC-1 and E.coli BL21 antigens in vitro. The positive sera were absorbed with bacterial whole cell and ultrasonic lysis of both H. parasuis SC-1 and E.coli BL21 in vitro, respectively. The absorbed sera were detected by ELISA, and D450 nm value decreased gradually until it was stable in the absorption progress.The antibodies expressed against antigens in vitro were not found by Western blot with the absorbed sera. The expression library was screened three times. Ultimately five positive colonies were found. The inserted nucleotide sequence was more than 99% in the homology comparing with Haemophilus parasuis genomic DNA in GenBank. Five pairs of primers were designed to detect mRNA of H. parasuis in vitro by RT-PCR. The results indicated that these genes were not transcribed in vitro. It was successful to screen the in vivo induced antigen genes of Haemophilus parasuis SC-1 strain. We finally confirmed five biologically meaningful ORF including tyrosine phosphatase (IVI-1), isoleucyl-tRNA synthetase (IVI-2), capsular polysaccharide export protein (IVI-3) and two hypothetical proteins with unknown functions (IVI-4, IVI-5). The analysis of antigens indicated that these proteins had multiple antigenic sites. The successful screening and identification of capsular polysaccharide export protein gene make it possible to further study on its immunogenicity.3. Cloning, expression and immunogenicity of Haemophilus parasuis SC-1 capsular polysaccharide export protein gene.The capsular polysaccharide export gene was used to study on its immunogenicity. According to the sequence of capsular polysaccharide export gene in NCBI (GenBank accession number:HM063414), a pair of primers was designed to amplify capsular polysaccharide export gene by PCR. The specific product of PCR was subcloned into BamHⅠ/XhoⅠsite of prokaryotic expression vector pET32a (+) to construct recombinant expression plasmid pET32a-CPEP. It was transformed into Escherichia coli BL21 (DE3) and expressed with IPTG inducer. The molecular weight of recombinant was 35 kD by SDS-PAGE analysis. The optimum expression conditions were defined (1mM IPTG and 5 hours). The result of immunoblotting test showed that the recombinant protein had favorable reactogenicity with anti-Hps SC-1 pig sera.The purified recombinant protein (group A) and the inactivated Haemophilus parasuis SC-1 (group B) were used as antigen and emulsified with Freund's adjuvant, respectively. The mice were injected with these antigens in two weeks interval. The PBS with adjuvant (group C) and PBS (group D) were used as the control. Then mice sera were collected at 0 d,14 d and 28 d post immunization. The specific antibodies were detected in group A and B at 14 d post immunization by ELISA. The antibodies of group B and group A were improved at 28 d post immunization. The mice of group C and D did not generate the specific antibodies by ELISA. The results showed that the recombinant protein had immunogenicity.The challenge tests was taken in 1 000 times median lethal dose of H. parasuis two weeks after the last immunization. The mice in group A died in three days after injection. All mice in group C and D died in five days after injection. Six mice in group A died and the protection rate was 40%. The mice in group B did not die and the protection rate was 100%. All the mice died in group C and D. The results indicated that the mice were protected by inoculating with recombinant protein.