The construction of prokaryotic expression system for human Api6 fusion protein

FANG Yan1; LIAN Xue-mei2

doi:10.13406/j.cnki.cyxb.2011.09.006

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The construction of prokaryotic expression system for human Api6 fusion protein

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- The construction of prokaryotic expression system for human Api6 fusion protein
- Journal of Chongqing Medical University Vol. 36, Issue 9, Pages: 1069-1072(2011)
- 作者机构：
  
  1. 重庆医科大学感染性疾病分子生物学教育部重点实验室脂质研究中心
  2. 重庆医科大学公共卫生学院营养与食品卫生学教研室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2011.09.006
  CLC： R363
- Published：2011，
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[1]方严,练雪梅.人Api6融合蛋白原核表达系统的构建[J].重庆医科大学学报,2011,36(09):1069-1072.

FANG Yan1, LIAN Xue-mei2. The construction of prokaryotic expression system for human Api6 fusion protein. [J]. Journal of Chongqing Medical University 36(9):1069-1072(2011)
[1]方严,练雪梅.人Api6融合蛋白原核表达系统的构建[J].重庆医科大学学报,2011,36(09):1069-1072. DOI： 10.13406/j.cnki.cyxb.2011.09.006.

FANG Yan1, LIAN Xue-mei2. The construction of prokaryotic expression system for human Api6 fusion protein. [J]. Journal of Chongqing Medical University 36(9):1069-1072(2011) DOI： 10.13406/j.cnki.cyxb.2011.09.006.

摘要

目的:本研究拟采用蛋白原核表达的方法

诱导表达人Api6（Apoptosis inhibitor 6）融合蛋白

为进一步研究Api6在凋亡抑制、脂质代谢和肿瘤发生中的作用奠定基础。方法:利用PCR技术扩增人Api6基因

将其克隆至原核表达载体ppSUMO

构建重组质粒ppSUMO-Api6;重组质粒转化原核表达宿主菌E.coli Rosetta感受态;采用蛋白原核表达的方法

不同诱导前菌液浓度

不同温度、不同异丙基硫化半乳糖苷（Isopropy1-β-D-thiogalaotopyrano-side

IPTG）浓度、不同诱导时间

筛选诱导表达Api6重组蛋白的最优条件;Western blot鉴定其表达情况。结果:成功构建重组质粒ppSUMO-Api6;成功诱导Api6重组蛋白的原核表达

表达的最优条件为:OD600=0.5

.8的重组菌

16℃、0.5 mmol/L IPTG诱导16 h。结论:含有人Api6 cDNA全长序列的重组质粒ppSUMO-Api6

经转化原核表达宿主菌E.coli Rosetta感受态细胞

在诱导前菌液OD600=0.5

.8、16℃、0.5 mmol/LIPTG、诱导16 h这一优化的诱导条件下

重组菌ppSUMO-Api6-Rosetta可诱导重组蛋白ppSUMO-Api6的原核表达。

Abstract

Objective:To further study the pathophysiological function of human Api6 fusion protein expression using a prokaryotic expression system.Methods:Api6 cDNA was inserted into ppSUMO vector to form recombinant plasmid ppSUMO-Api6;recombinant plasmid ppSUMO-Api6 was transformed into prokaryotic expression host E.coli Rosetta;prokaryotic expression method was optimized to induce expression of recombinant protein Api6 in different conditions of E.coli density

temperature

IPTG concentration and induction time;recombinant protein Api6 was identified by Western blot.Results:The recombinant plasmid ppSUMO-Api6 was successfully constructed;the recombinant protein Api6 was successfully induced

and the expression of recombinant protein ppSUMO-Api6 from the prokaryotic expression system was optimized in the condition of OD600=0.5~0.8

16℃

0.5 mmol/L IPTG and 16 h induction.Conclusion:Recombinant plasmid ppSUMO-Api6 was converted into the prokaryotic expression host E.coli Rosetta to get the recombinant strain ppSUMO-Api6-Rosetta.Recombinant protein ppSUMO-Api6 was expressed using a prokaryotic expression system at the optimal expression conditions.

关键词

凋亡抑制因子6原核表达融合蛋白

Keywords

Api6prokaryotic expressionfusion protein

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