《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (3): 628-634.doi: 10.16431/j.cnki.1671-7236.2018.03.009

• 生物技术 • 上一篇    下一篇

巴什拜羊SPLUNC1基因的克隆与真核表达

王继雪, 沈文, 孙延鸣   

  1. 石河子大学动物科技学院, 石河子 832003
  • 收稿日期:2017-09-18 出版日期:2018-03-20 发布日期:2018-03-22
  • 通讯作者: 孙延鸣 E-mail:sym@shzu.edu.cn
  • 作者简介:王继雪(1991-),男,山东阳谷人,硕士,研究方向:临床兽医学,E-mail:2502323936@qq.com
  • 基金资助:

    国家自然科学基金项目(31460686)

Cloning and Eukaryotic Expression of SPLUNC1 Gene of Bashibay Sheep

WANG Jixue, SHEN Wen, SUN Yanming   

  1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
  • Received:2017-09-18 Online:2018-03-20 Published:2018-03-22

摘要:

为了克隆巴什拜羊SPLUNC1基因并表达该蛋白,本试验从巴什拜羊肺脏组织中提取总RNA,利用RT-PCR方法扩增出SPLUNC1基因开放阅读框序列,将该基因片段插入到真核表达质粒pPIC9K中构建pPIC9K-SPLUNC1重组质粒,然后将pPIC9K-SPLUNC1重组质粒线性化并电击转化入毕赤酵母GS115中,经甲醇诱导蛋白表达后用SDS-PAGE和Western blotting分析鉴定目的蛋白。结果表明,经RT-PCR扩增成功获得大小为748 bp的SPLUNC1基因;构建的pPIC9K-SPLUNC1重组质粒经PCR、酶切及测序鉴定与预期结果一致;SDS-PAGE及Western blotting检测鉴定结果表明获得大小为25.53 ku的SPLUNC1蛋白。本试验结果为进一步研究巴什拜羊SPLUNC1蛋白的生物学活性奠定基础。

关键词: 巴什拜羊; SPLUNC1基因; 克隆; 真核表达

Abstract:

In order to clone Bashibay sheep SPLUNC1 gene and express its protein,total RNA was extracted from lung of Bashibay sheep,RT-PCR was used to amplify SPLUNC1 gene open reading frame sequence,the gene fragment was inserted into eukaryotic expression plasmid pPIC9K to construct plasmid pPIC9K-SPLUNC1,then the plasmid pPIC9K-SPLUNC1 was linearized and electroporated into Pichia pastoris GS115,the protein was induced by methanol,and the target protein was identified by SDS-PAGE and Western blotting.The results showed that the 748 bp SPLUNC1 gene was successfully amplified by RT-PCR;The plasmid pPIC9K-SPLUNC1 was consistent with expected results after PCR,enzyme digestion and sequencing identification;Detection results of SDS-PAGE and Western blotting showed that the SPLUNC1 protein was successfully expressed with 25.53 ku.This study laid a foundation for further research of SPLUNC1 protein biological activity of Bashibay sheep.

Key words: Bashibay sheep; SPLUNC1 gene; cloning; eukaryotic expression

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