| Many fish pathogens had been reported and one pathogen often showed different subspecies or serotypes. Generally, subunit vaccines protect fish from one pathogen infecting, but it is difficult to be used in aquaculture due to low rate of protection and limited protection to pathogens. In this study, template DNA was extracted from pathogenic Aeromonas and Edwardsiella isolated from diseased eels in recent 10 years. Designing primers according to gene sequences coding the outer membrane protein in GenBank database, the whole gene sequences of porin II and ompS2 were amplified from the genus Aeromonas and Edwardsiella tarda respectively. After sequence analysis, two gene fragments coding the outer part of the outer membrane protein of two pathogens(B11 and B79) were combined by fusion PCR in vitro. The expressed vector(pGEX-2T-His-Aero-Edwa) contained two above-mentioned gene fragments was constructed and highly expressed. After the expressed protein was purified, it was used to immunize eel to study the immunogenicity of the expressed product by detecting the titers of serum antibody to the expressed protein in eels. The results indicated that the constructed expression vector could highly express the outer membrane protein encoded by two combined gene fragments, and the purified product could stimulate the eel to produce high titers of antibodies. Results of the study provided foundation for developing bivalent vaccine of gene engineering of outer membrane proteins of eel’s pathogens. The main research contents and results as following:(1) According to the highly conserved sequences of outer membrane protein gene and whole genomic sequences of the genus Aeromonas and Edwardsilla in the GenBank database, we fished out the whole gene length of the outer membrane protein and designed primers according to the two ends near the gene. Ten whole sequences of porin Ⅱ genes from 30 pathogens belonging to the genus Aeromonas and 5 whole sequences of outer membrane protein gene(ompS2) from 5 Edwardsiella tarda were amplified by PCR. After sequence analysis, a porin Ⅱ gene of Aeromonas hydrophila(B11), locating in the center of the genetic distance of 10 porinⅡ, and a ompS2 gene of Edwardsiella tarda(B79) were used for protein structure, hydrophilicity and immunogenicity analysis by some relevant softwares. Two gene fragments, encoding outer membrane region of the porin Ⅱ and ompS2 respectively, with good hydrophilicity and immunogenicity were combined by the “two-steps” fusion PCR. According to the cut sites of BamHⅠand EcoRⅠ of the expression vector(pGEX-2T-His), two restriction enzymes were added in the combined sequence contained two fragments of porin Ⅱ and ompS2 and the recombinant expression vector(pGEX-2T-His-Aero-Edwa)contained two fragments of outer membrane protein was constructed.(2) Optimum expression conditions of the recombinant expression vector were studied on the different IPTG concentration, temperature, time and bacterial concentration. The results indicated different IPTG concentration, temperature, time and bacterial concentration had no distinct influence on expression of the recombinant protein. Available expression conditions were OD600nm=0.8 of the bacterial concentration, 0.25 mM IPTG induced over night at 16 ℃. The expressed protein with the molecular weight of 87.1 kD was purified by Nickel Chelating Affinity Chromatography with ?KTApurifier-100 protein purification system. After the purified protein was renaturated by dialysis, it was used to immunize American eels(Anguilla rostrata) to study its immunogenicity. The results indicated that compare the group of PBS injection, groups of different doses of the recombinant protein injection(i.m) significantly(P <0.05) enhanced the titers of serum antibody in eels at 28 d after immunization, indicating good immunogenicity of the recombinant protein to eels. |
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