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中国农学通报 ›› 2010, Vol. 26 ›› Issue (22): 12-15.

所属专题: 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

犬传染性肝炎病毒IX基因的克隆及原核表达

杨拓 张洋 孟焕 徐春瑛 袁宝 文力正 陈承祯 任文陟 陈健   

  • 收稿日期:2010-04-21 修回日期:2010-05-21 出版日期:2010-11-20 发布日期:2010-11-20
  • 基金资助:

    吉林省实验动物质量检测中心平台建设项目

Expression of The IX Gene of CAV-I in E.coli BL21

  • Received:2010-04-21 Revised:2010-05-21 Online:2010-11-20 Published:2010-11-20

摘要:

摘 要:本试验通过对犬传染性肝炎病毒(CAV-I)基因组的提取,通过PCR扩增获得了大小为1109bp CAV-I病毒结构IX基因片段, 成功地将IX基因克隆至载体pMD-18T中,构建了pIX原核表达载体pGEX-4T1-IX,将重组质粒pGEX-4T1-IX转化至大肠杆菌BL21(DE3)感受态细胞中,并实现了IX基因在该菌中的高效表达。对表达产物进行了Western blot 检测,分析结果表明表达蛋白能与抗CAV-I阳性血清发生特异性的抗原-抗体反应,为CAV-I的血清学诊断和免疫监测提供大量优质的抗原奠定了基础。

关键词: 横断山北部高山区, 横断山北部高山区, 土壤真菌, 物种多样性

Abstract:

Abstract: we firstly adopted the method of concentration of the virus and extract its genome. Than referring to the sequence which is published in Gene Bank, we designed a pair of primers. product of the PCR amplification , pIX protein ,was analyzed by agarose gel electrophoresis and demonstrated to be a specific band of 1099bp.The fragment was cloned into the pMD18-T vector. After cloning it into the T vector, we sequenced the targeted fragment and the result showed that it was right. Then we ligated it into the expression vector pGEX-4T-1 with DNA T4 ligase, which is digested by the same two enzymes. Furtherly digesting by EcoRI and SalI for identification, we finally got the positive expression vector pGEX-4T1-IX. After transforming the recombinant plasmid pGEX-4T1-IX to the host E.coli BL21(DE3)for 2~3 hours ,we induced it by IPTG with the terminal concentration of 1mmol/L.The whole quantity of expression is up to 21% in the host cell. In the Western Blotting assay , the protein expressed can be detected by CAV-I positive serum, which will contribute greatly to the establishment of CAV-I antibody detecting method.