大约克猪SLA-1原核表达载体的构建及表达

doi:10.16431/j.cnki.1671-7236.2016.12.006

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

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

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

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/Abstract

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

中图分类号:

Q786

张秀娟, 杨杰, 孙永强, 高凤山. 大约克猪SLA-1原核表达载体的构建及表达[J]. 《中国畜牧兽医》, 2016, 43(12): 3121-3126.

ZHANG Xiu-juan, YANG Jie, SUN Yong-qiang, GAO Feng-shan. Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig[J]. , 2016, 43(12): 3121-3126.

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大约克猪SLA-1原核表达载体的构建及表达

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

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