副鸡禽杆菌lpxM蛋白的原核表达及免疫原性研究

Prokaryotic Expression and Immunogenicity of lpxM Protein of Avibacterium paragallinarum

[目的]探究副鸡禽杆菌(Avibacterium paragallinarum)外膜蛋白lpxM的免疫原性,为预防鸡传染性鼻炎提供理论参考。[方法]构建pET-28a-lpxM原核表达载体,经PCR扩增和双酶切鉴定后将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞,用IPTG进行诱导表达,并对诱导时间和诱导浓度进行优化,用尿素缓冲液纯化重组蛋白lpxM并进行SDS-PAGE分析。利用Western blotting鉴定重组蛋白的特异性。将重组蛋白与MONTANIDEISA 71 VG佐剂制备成疫苗v-lpxM,通过动物试验观察重组蛋白的免疫效果。[结果]重组质粒pET-28a-lpxM转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后成功表达,在37 ku处有目的蛋白特异性条带,在37℃、700 mmol/L IPTG、诱导4 h时蛋白表达量最高;重组蛋白lpxM在上清和沉淀中均有表达,且在沉淀中表达量相对较高;Western blotting结果显示,C型副鸡禽杆菌Modesto株的阳性鸡血清与纯化后的重组蛋白可发生免疫反应。免疫保护试验结果显示,v-lpxM疫苗对A、B和C型副鸡禽杆菌的保护率分别为0、20%和60%。[结论]本研究成功构建了原核表达载体pET-28a-lpxM,实现了lpxM重组蛋白的高效表达,且表现出良好的免疫原性。研究结果为探究副鸡禽杆菌外膜蛋白lpxM的免疫学特性以及为鸡传染性鼻炎的预防和疫苗的研发奠定了基础。

[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 paragallinarum 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 paragallinarum outer membrane protein lpxM,as well as for the prevention of chicken infectious rhinitis and the development of vaccines.