《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (12): 3121-3126.doi: 10.16431/j.cnki.1671-7236.2016.12.006

• 生物技术 • 上一篇    下一篇

大约克猪SLA-1原核表达载体的构建及表达

张秀娟, 杨杰, 孙永强, 高凤山   

  1. 大连大学生命科学与技术学院, 大连 116622
  • 收稿日期:2016-06-16 出版日期:2016-12-20 发布日期:2016-12-22
  • 通讯作者: 高凤山 E-mail:gfsh0626@126.com
  • 作者简介:张秀娟(1993-),女,甘肃平凉人,学士,研究方向:生物工程,E-mail:1270853789@qq.com
  • 基金资助:

    国家自然科学基金项目:猪源病毒CTL多肽表位与SLA-Ⅰ结晶研究(31172304);2015年辽宁省大学生创新创业训练计划项目:育-大约克猪SLA-1原核表达载体的构建及表达研究(20150086);2014年大连大学大学生创新创业训练计划项目重点项目:育-大约克猪SLA-1原核表达载体的构建及表达研究(2014066)

Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig

ZHANG Xiu-juan, YANG Jie, SUN Yong-qiang, GAO Feng-shan   

  1. College of Life Science and Technology, Dalian University, Dalian 116622, China
  • Received:2016-06-16 Online:2016-12-20 Published:2016-12-22

摘要:

为构建大约克猪SLA-1胞外区的原核表达载体及表达目的蛋白,试验设计1对引物,经PCR扩增获得大约克猪SLA-1胞外区基因(命名为SLA-1-DYKe),将此片段克隆至pMD®19-T Simple Vector,转化大肠杆菌TOP10感受态细胞,经Nde Ⅰ和Xho Ⅰ双酶切筛选阳性克隆菌并测序,将目的基因插入到原核表达载体pET-28a(+)中,转化至宿主菌BL21(DE3)进行诱导表达,用SDS-PAGE检测目的蛋白的表达情况,大量诱导提取包涵体并检测。结果显示,PCR成功扩增SLA-1-DYKe的胞外区,得到大小为837 bp的目的基因,目的基因成功克隆至pMD®19-T Simple Vector,并获得序列正确的重组质粒。以得到的重组质粒成功构建了SLA-1-DYKe/pET-28a(+)表达载体,目的蛋白大小约为34 ku。本研究成功构建了大约克猪SLA-1原核表达载体,获得了表达蛋白,为今后研究大约克猪SLA-1的空间结构和基因功能奠定了基础。

关键词: 大约克猪; SLA-1基因; 原核表达载体

Abstract:

In order to construct the prokaryotic expressing vector of SLA-1 derived form Yorkshire pig and express the interest of protein, a pair of primers was designed to amplify the extracellular domain of SLA-1 gene from Yorkshire pig (named SLA-1-DYKe) by PCR. Then the PCR product was cloned into pMD®19-T Simple Vector and transformed into Escherichia coli TOP10. After cleaved by Nde Ⅰ and Xho Ⅰ, the positive clones were selected to be sequenced. Analyzing by biological soft, the fragment from positive clone with correct sequence was inserted into pET-28a (+) and transformed into E.coli BL21(DE3). After induction and expression, the interest of protein was detected by SDS-PAGE. The results showed that the extracellular domain of SLA-1-DYKe was successfully amplified with the fragment length of 837 bp. The interest of SLA-1 gene was successfully cloned into pMD®19-T Simple Vector and the positive recombinant plasmids with correct sequences were obtained. The SLA-1-DYKe from positive recombinant plasmids was further inserted into pET-28a(+). After transformed into E.coli BL21(DE3) and induction, the SLA-1-DYKe was successfully expressed. The molecular weight of the protein was about 34 ku. It was concluded that the prokaryotic expressing vector of SLA-1 was constructed successfully from Yorkshire pigs and then the expressed protein was obtained, which would lay a base for studying on the structure and function of SLA-1 from Yorkshire pig in the future.

Key words: Yorkshire pig; SLA-1 gene; prokaryotic expressing vector

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