Prokaryotic Expression and Immunogenicity of lpxM Protein of Avibacterium paragallinarum
[Objective]This experiment was airxed to investigate the immunogenicity of Avibacterium paragallinarum outer membrane protein lpxM,and provide a basis for the prevention of infectious rhinitis in chickens.[Method]The pET-28a-lpxM prokaryotic expression vector was constructed.The positive plasmid identified by PCR amplification and sequencing was transformed into Escherichia coli BL21(DE3)competent cells.Induced expression was performed with IPTG,and the induction time and concentration were optimized.The recombinant protein lpxM was purified using urea buffer and identified by SDS-PAGE.The specificity of the recombinant protein was detected by Western blotting.After the recombinant protein was combined with MONTANIDEISA 71 VG adjuvant to form the vaccine v-lpxM,and its immunization effect was observed by animal experiments.[Result]Recombinant plasmid pET-28a-lpxM transformed Escherichia coli BL21(DE3)competent cells and was successfully expressed after induction by IPTG,with specific bands of target protein at 37 ku.Protein expression was highest at 37 ℃,700 mmol/L IPTG and 4 h after induction.Recombinant protein lpxM was expressed in both supernatant and precipitation,and the expression level was relatively high in precipitation.Western blotting results showed that the positive chicken serum of Modesto strain of serotype C Avibacterium paragallinarum could react with the purified recombinant protein.The results showed that the protection rate of v-lpxM vaccine against serotype A,B and C Avibacterium para gallinarum was 0,20％and 60％,respectively.[Conclusion]The prokaryotic expression vector pET-28a-lpxM was successfully constructed,achieving the efficient expression of lpxM protein.The prepared lpxM protein showed good immunogenicity.The results laid a foundation for exploring the immunological properties of Avibacterium para gallinarum outer membrane protein lpxM,as well as for the prevention of chicken infectious rhinitis and the development of vaccines.
Avibacterium para gallinarumlpxM proteinprokaryotic expressionimmunogenicity
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