Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells

LAN Lizhen; et al

doi:10.13406/j.cnki.cyxb.2004.05.002

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Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells

更新时间：2024-01-17

- Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells
- Journal of Chongqing Medical University Issue 5, Pages: 563-566(2004)
- 作者机构：
  
  1. 重庆医科大学临床学院内分泌科
  2. 山西医科大学第一医院
  3. 重庆医科大学临床学院内分泌科,重庆,400016
  4. ,太原,030001
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2004.05.002
  CLC： R587.1
- Published：2004，
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[1]兰丽珍,邓华聪,孙辽,郑丹,弓军胜,刘东方,郑宏庭.带信号肽人胰岛素原突变体的构建及在COS-7细胞中的表达[J].重庆医科大学学报,2004(05):563-566+575.

LAN Lizhen, et al. Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells. [J]. Journal of Chongqing Medical University (5):563-566(2004)
[1]兰丽珍,邓华聪,孙辽,郑丹,弓军胜,刘东方,郑宏庭.带信号肽人胰岛素原突变体的构建及在COS-7细胞中的表达[J].重庆医科大学学报,2004(05):563-566+575. DOI： 10.13406/j.cnki.cyxb.2004.05.002.

LAN Lizhen, et al. Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells. [J]. Journal of Chongqing Medical University (5):563-566(2004) DOI： 10.13406/j.cnki.cyxb.2004.05.002.

摘要

目的 :构建带信号肽人胰岛素原突变体的真核表达载体并在COS - 7细胞表达出成熟的人胰岛素。方法 :采用PCR体外定点突变技术（PCR -SDM）

将普通细胞内furin蛋白酶的识别和切割位点Arg -X -Lys -Arg -样序列

引入人胰岛素原C肽两端

同时加上信号肽、Kozak序列和限制性内切酶的酶切位点并克隆至载体pcDNA3.1（+）中

构建成人胰岛素原突变体的真核表达载体

经测序验证后转染COS - 7细胞

RT -PCR、Western印迹、放射免疫法检测其在COS - 7细胞中的表达。结果 :DNA测序结果表明在预期位点突变符合要求

经RT -PCR、Western印迹、放射免疫鉴定

构建的人胰岛素原突变体的真核表达载体转染的COS - 7细胞可表达、分泌人成熟胰岛素。结论 :构建了人胰岛素原突变体的真核表达载体

并成功地在COS - 7细胞中获得人胰岛素的分泌表达

Abstract

Objective:To construct a mutant of human proinsulin with signal peptide code

so that it may be expressed in eukaryotic system to produce mature human insulin in COS-7 cells.Methods:Human proinsulin gene was mutated by the polymerase chain reaction (PCR) for the site-directed mutagenesis method.Two furin recognized sites of Arg-X-Lys-Arg-motifs were introduced into human proinsulin.Signal peptide

Kozak sequence and restriction enzyme cutting site were added and cloned into pcDNA3.1(+) vector and verified by sequencing analysis.Eukaryotic expression vector was constructed and transfected into COS-7 cells.Insulin were evaluated respectively with RT-PCR

radioimmunoassay and Western blot analysis.Results:The sequencing analysis showed that the mutation sites were correct

and the mutant of human proinsulin was constructed successfully.Human insulin mRNA was highly expressed in transfected COS-7 cells

and secretive recombinant human insulin was detected in the culture supernatant of transfected COS-7 cells by Western blotting.Conclusion:A mutant human proinsulin eukaryotic expression vector is obtained and secretive recombinant human insulin is successfully expressed in COS-7 cells.

关键词

胰岛素原定点突变克隆基因表达COS细胞

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