Modesto株副鸡禽杆菌血凝素的原核表达及其生物活性鉴定

Prokaryotic Expression of Recombinant Haemagglutinin of Modesto Strain of Avibacterium paragallinarum and its Biological Activity Research

目的:克隆并原核表达Modesto株C型副鸡禽杆菌(Apg)的血凝素(HA)基因,鉴定该重组HA的生物学活性。方法:根据GenBank上已发表的Modesto株Apg的HA基因序列,设计合成了1对特异性引物,克隆Apg HA基因;以Modesto株Apg中提取的细菌DNA为模板,利用PCR扩增ApgHA全长基因(1026bp),将其克隆到pET-32a(+)载体上,构建原核表达载体pET-HA,在大肠杆菌BL21(DE3)中表达并纯化重组HA;通过Western印迹及血凝和血凝抑制试验鉴定该重组蛋白的生物学活性。结果:表达并纯化了Apg重组HA,该蛋白可以和C型Apg抗血清特异性结合,并且可以凝集鸡红细胞。结论:构建了Modesto株Apg HA基因的原核表达载体,并表达纯化了Apg HA融合蛋白,该重组HA具有凝集鸡红细胞的活性,为进一步研究Apg HA的免疫功能奠定了基础。

Objective： To clone and express the haemagglutinin（HA） gene of Modesto strain of Avibacterium paragalli-narum（Apg） in E.coli and to study the biological activity of the recombinant protein. Methods： According to the published HA gene sequence of Modesto strain of Apg, a pair of primers was designed and synthesized, which can amplify Apg＇s major protective antigen HA gene. The whole HA gene fragment about 1 026 bp was amplified through PCR from Apg Modesto strain. The HA gene was cloned into the expression vector pET-32a（＋） and over-expressed in E.coli strain BL21（DE3）. The purified recombinant protein was confirmed as Apg haemagglutinin by Western-blotting, hemagglutination test and hemagglutination inhibition （HI） test. Results： The Apg HA fusion protein was expressed and purified, which could react with both the chicken red blood cells and the chicken antibody against Apg of serotype C. Conclusion： The expression vector of recombinant pET-HA was constructed and the Apg HA fusion protein was expressed, which paves the way for studying the immunological function of Apg HA.