吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (01): 59-63.doi: 10.13481/j.1671-587x.20160112

• 基础研究 • 上一篇    下一篇

hISO基因原核表达载体的构建及其在大肠杆菌中的表达及鉴定

邵敏1, 王新颖1, 刘星1, 王燕1, 周鹤峰1, 葛正龙2   

  1. 1. 遵义医学院珠海校区生物化学教研室, 广东珠海519041;
    2. 遵义医学院生物化学教研室, 贵州遵义563003
  • 收稿日期:2015-06-15 发布日期:2016-01-26
  • 通讯作者: 周鹤峰,教授,硕士研究生导师(Tel:0756-7623310,E-mail:hfztim@163.com) E-mail:hfztim@163.com
  • 作者简介:邵敏(1978-),女,黑龙江省七台河市人,副教授,医学硕士,主要从事分子生物学方面的研究。
  • 基金资助:

    贵州省科技厅社会发展攻关项目资助课题[黔科合SY字(2012)3091)];遵义医学院青年科研启动基金资助课题(F-501)

Construction of prokaryotic expression vector of hISO gene and its expression in E.coli and identification

SHAO Min1, WANG Xinying1, LIU Xing1, WANG Yan1, ZHOU Hefeng1, CE Zhenglong2   

  1. 1. Department of Biochemistry, Zhuhai Campus, Zunyi Medical University, Zhuhai 519041, China;
    2. Department of Biochemistry, Zunyi Medical University, Zunyi 563003, China
  • Received:2015-06-15 Published:2016-01-26

摘要:

目的: 构建原核表达载体pET-28a(+)-hISO,探讨其融合蛋白在大肠杆菌中的稳定表达情况,为后续实验研究奠定基础。 方法: 以Caco-2细胞的总RNA为模版,采用RT-PCR方法扩增出大小为1770bp的人异麦芽糖酶(hISO)基因片段,将其插入到pET-28a(+)中,构建重组载体pET-28a(+)-hISO。经PCR、酶切及测序鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达,亲和层析纯化重组蛋白,利用SDS-PAGE电泳、Western blotting对重组蛋白进行分析和鉴定。 结果: 经PCR、酶切及测序鉴定后,重组质粒pET-28a(+)-hISO构建正确,表达重组蛋白相对分子质量为68860,将融合蛋白进行纯化,经40和60mmol·L-1咪唑缓冲液洗脱后能够得到浓度和纯度相对较高的蛋白。 结论: 成功地构建了hISO基因的原核表达载体,并在大肠杆菌中获得了融合蛋白表达。

关键词: 异麦芽糖酶, 原核表达, &alpha, -葡萄糖苷酶, 大肠杆菌

Abstract:

Objective: To construct the prokaryotic expression vector pET-28a(+)-hISO and to explore the expression of hISO fusion protein in E.coli,and to provide a foundation for follow-up experiment research.Methods: The 1 770 bp fragment of hISO gene was amplified from the total RNA of Caco-2 cells by RT-PCR and inserted into pET-28a(+)to construct the recombinant plasmid pET-28a(+)-hISO.The recombinant plasmids were transformed into E.coli BL21(DE3)to induce the protein expression with IPTG after PCR,digestion and DNA sequencing.The recombinant plasmid was analyzed and identified by SDS-PAGE and Western blotting method.Results: The recombinant plasmid pET-28a(+)-hISO was constructed successfully after identified by PCR,digestion and sequencing. The fusion protein was 68 860 when the soluble fusion protein was purified,and using elution buffer containing 40 mmol·L-1 and 60 mmol·L-1 imidazole could get high concentration and purity protein.Conclusion: The prokaryotic expression vector pET-28a(+)-hISO is constructed successfully and the recombinant protein is expressed in the E.coli BL21(DE3).

Key words: isomaltase, prokaryotic expression, α-glucosidase, Escherichia.coli

中图分类号: 

  • Q786