›› 2009, Vol. 21 ›› Issue (05): 0-454.

• 论文 •    

鸡γ干扰素在酵母中的分泌表达

魏雪涛,李银,刘宇卓,张敬峰   

  1. 江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-09-25 发布日期:2009-09-25

Secretory expression of the chicken γ interferon gene in yeast Pichia pastoris

WEI Xue-tao;LI Yin;LIU Yu-zhuo;ZHANG Jing-feng   

  1. Institute of Veterinary Medicine ,Jiangsu Academy of Agricultural Sciences ,Key Laboratory of Animal Diseases Diagnostic and Immunology,Ministry of Agriculture ,National Center for Engineering Research of Veterinary Bio-products ,Nanjing 210014 ,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-09-25 Published:2009-09-25

摘要:

根据GenBank已发表的鸡γ干扰素(ChIFN-γ)eDNA基因序列设计一对引物,以ConA刺激8h后的鸡全血淋巴细胞提取的总RNA为模板.通过RT—PCR方法克隆出ChIFN-γ基因,将其连接到pMD18-T载体上获得阳性重组质粒。测序结果表明,该ChIFN-γ与GenBank上发表的ChIFN-γ序列的同源性可达99.6%,其含有1个495bp的开放性阅读框架,编码164个氨基酸。通过KpnⅠ和NotⅠ两个酶切位点将该基因插入酵母表达载体(pPICZa—A),并将该重组酵母ChIFN-γ载体线性化,通过电转化使ChIFN-γ基因整合到酵母(X-33)基因组上,经甲醇诱导后,成功表达出ChIFN-γ。利用Western—blotting分析测定表达的ChIFN-γ蛋白的活性。结果表明,本试验得到的重组酵母ChIFN-γ是具有反应原性的蛋白。

 

关键词: 鸡γ干扰素, 克隆, 分泌表达

Abstract:

A pair of primers specific to ChIFN-γ gene were designed and synthesized according to the known cDNA sequences from GenBank,and ChIFN-γ gene was cloned and amplified by reverse transcripition-polymerase chain reaction (RT-PCR) from the total mRNA in the lymphocyte of chicken blood stimulated with ConA for 8 hours. RT- PCR product was cloned into pMD18-T vector. The recombinant plasmid was identified by digestion with restriction endonucleases and the nucleotide sequence was determined. The ChIFN-γ gene was a 495 bp open reading frame encoding a polypeptide of 164 amino acids. The gene was inserted into the expression vector pPICZa-A,which had been cleaved by Kpn I and Not I. The recombinant vector was transfered into yeast Pichia pastoris strain X-33 ,and the ChIFN-γ gene was successfully expressed after induced by methanol. The activity of the recombinant ChIFN-γ was detected by the Western-blotting. The results showed that the recombinant yeast ChIFN-γ was a protein with reactivity.

Key words: chicken y interferon, gene cloning, secretory expression