摘要
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.
出处
《生物技术通讯》
CAS
2008年第2期232-235,共4页
Letters in Biotechnology
基金
国家高技术研究发展计划项目(2005AA246051)
北京市科技新星项目(2005B35)
关键词
副鸡禽杆菌
血凝素
原核表达
Avibacterium paragallinarum
haemagglutinin
prokaryotic expression