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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
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王宏俊1,2，张培君2，龚玉梅2，李永清2，陈小玲2 ，杨汉春1
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(1.中国农业大学 动物医学院，北京100094；2.北京市农林科学院 畜牧兽医研究所，北京100097 )
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摘要：
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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（1035bp），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Hpg HA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。 |
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关键词：
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副鸡嗜血杆菌；血凝素基因；原核表达 |
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中图分类号：S 852.612：Q 786 文献标识码：
A 文章编号：1673-4696(2006)10-0773-04 |
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Cloning and expression of HA gene of Haemophilus paragallinarum
serotype A in E.coli and identification of biological
activity of the recombinant protein |
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WANG Hong-jun1,2, ZHANG Pei-jun2 ，GONG
Yu-mei2 ，
LI Yong-qing2，CHEN Xiao-ling2，YANG Han-chun1 |
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(1.College of Veterinary Medicine, China Agricultural University, Beijing 100094, China; 2. Institute of Animal
Science and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, Beijing
100097, China)
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Abstract: 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 1035bp 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. |
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Key words: Haemophilus paragallinarum； hemagglutinin gene；prokaryotic expression |
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