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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (11): 1-6.

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 •    下一篇

猪α1干扰素基因的克隆与原核表达

杨永胜1,陈春花2,宋勤叶1,左玉柱1   

  • 收稿日期:2009-12-25 修回日期:2010-02-11 出版日期:2010-06-05 发布日期:2010-06-05
  • 基金资助:

    猪圆环病毒2型与猪细小病毒协同感染对猪免疫功能影响的细胞与分子机制

Clone and Prokaryotic Expression of Porcine Interferon-α1 Gene

Yang Yongsheng1, Chen Chunhua2, Song Qinye1, Zuo Yuzhu1   

  • Received:2009-12-25 Revised:2010-02-11 Online:2010-06-05 Published:2010-06-05

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摘要：

摘要：根据猪α1干扰素（pocine interferon-alpha1, poIFN-α1）的全基因序列（EU364896）设计合成一对特异引物，通过RT-PCR从三元杂交仔猪外周血淋巴细胞扩增出poIFN-α1全基因，将该基因克隆到pGEM-T载体，构建重组质粒pGEM-T-poIFN-α1，进行测序。以pGEM-T- poIFN-α1为模板，经PCR扩增带有酶切位点的poIFN-α1基因，将其定向克隆到原核表达载体pGEX-6P-1中，构建重组表达载体pGEX-6P-poIFNα1，转化大肠杆菌BL21(DE3) 进行蛋白诱导表达和鉴定，并确定蛋白最佳表达条件。序列分析表明，克隆的poIFN-α1基因全长546 bp，编码181 aa，前23 aa组成信号肽，无糖基化位点。SDS-PAGE和Western blotting证实重组表达菌经IPTG诱导后，可表达相对分子质量约46 ku的融合蛋白（GST-poIFN-α1）。当以1.0 mmol/L的IPTG于37℃下诱导表达9 h时，蛋白表达量最高。poIFN-α1基因的克隆与表达为进一步研究其抗病毒活性，开发病毒治疗和疫苗免疫增强用生物制剂奠定了基础。

关键词: 美国白蛾, 美国白蛾, 适生区, GARP, 风险评估

Abstract:

Abstract：A pair of specific primer for porcine interferon-alpha1 (poIFN-α1) was designed and synthesized according to gene sequence (EU364896), and poIFN-α1 gene was amplified by RT-PCR from peripherals blood lymphocytes (PBLC) of ternary hybrid piglet.Then poIFN-α1 complete gene was cloned into pGEM-T vector and sequenced. Moreover, poIFN-α1 gene with enzyme sites was cloned into prokaryotic expression vector pGEX-6P-1 to construct recombinant expression plasmid pGEX-6P-poIFN-α1 and transform it into E.coli BL21 (DE3). Recombinat poIFNα1 was expressed and identified with expression conditions confirmed. The analysis of sequence indicated that the cloned poIFN-α1gene consisted of 546 bp encoding 181 aa with a signal peptide of 23 aa but without glycosylation site. The selected recombinant expressed approximately a 46 ku fusion protein with glutathione S-transferase (GST) and poIFN-α1 which was demonstrated by SDS-PAGE and Western blotting. The amount of fusion protein reached the highest when the recombinants were induced for 9 hours on 37℃ with 1.0 mmol/L IPTG. Clone and expression of poIFN-α1 gene laid a foundation for the study of antivirual activity and utilization of poIFN-α1.