A型副鸡嗜血杆菌血凝素基因的克隆表达及其产物的生物学活性鉴定

Cloning and expression of HA gene of Haemophilus paragallinarum serotype A in E.coli and identification of biological activity of the recombinant protein

根据GenBank中登录的A型Hpg Hp8株血凝素基因序列,设计合成了1对特异性引物,以Hpg Hp8株中提取的细菌DNA为模板,利用PCR扩增了Hpg血凝素全长基因(1 035 bp),将其克隆到pET-32a(+)载体上,构建了原核表达载体pET-HA,表达并纯化了重组蛋白,通过免疫印迹及血凝和血凝抑制试验鉴定了该重组蛋白的生物学活性。结果显示,表达并纯化的HpgHA重组血凝素蛋白可以和A型Hpg抗血清特异性结合,并且可以凝集鸡红细胞。表明,成功地构建了Hpg血凝素基因的原核表达载体,并表达、纯化了具有凝集鸡红细胞活性的Hpg-HA融合蛋白。

According to the published hemagglutinin（HA） gene sequence of Hpg Hp8 strain, a pair of primers was designed and synthesized to amplify Hpg＇s major protective antigen HA gene 1 035 bp in size from Hpg Hp8 strain. The PCR amplified 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 Hpg HA by the hemagglutination test, Hemagglutination inhibition（HI） test and Western-blotting. Western- blotting analysis showed that the expressed Hpg-HA fusion protein could react with both the chicken red blood cells and the chicken antibody against Hpg serotype A. It was concluded from the results that the expression vector of recombinant pET-HA expressing the Hpg-HA gene was constructed.