| 摘要: |
| 【目的】克隆人生长分化因子-15（Human growth differentiation factor-15，hGDF-15）成熟肽基因，构建其原核表达载体，并进行诱导表达，为hGDF-15药理活性和生物学功能研究奠定基础。【方法】利用PCR技术克隆人hGDF-15成熟肽基因，将其连接到pET-28a载体上构建pET-28a-hGDF-15原核表达载体，并将此载体转入Rosetta（DE3）大肠杆菌感受态细胞，获得重组大肠杆菌，对目的蛋白分别采用不同温度（16，25，37 ℃）、IPTG浓度（0.10，0.25，0.50，0.75，1.00 mmol/L）和时间（12，24，36，48 h）进行诱导表达，对表达产物进行SDS-PAGE电泳和Western blot检测，利用Gel Quant Express软件对菌体破碎离心的上清组分和沉淀组分中的蛋白含量进行测定，通过正交试验，分析上清组分和沉淀组分的最适诱导表达条件。【结果】成功获得hGDF-15成熟肽基因，其长度为339 bp；构建了pET-28a-hGDF-15原核表达载体，并诱导表达了hGDF-15蛋白；优化的上清组分最适诱导条件为IPTG浓度0.25 mmol/L、温度16 ℃、时间24 h，沉淀组分最适诱导条件为温度37 ℃、时间36 h、IPTG浓度1.00 mmol/L。【结论】成功克隆了hGDF-15基因，在大肠杆菌中诱导表达出其编码蛋白，并优化出了最适诱导表达条件。 |
| 关键词: 生长分化因子-15 原核表达 IPTG浓度 诱导时间 诱导温度 |
| DOI： |
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| 基金项目:吉林省科技发展计划项目（20130101105JC，20140204018YY）；国家级大学生创新创业计划项目；吉林农业大学大学生科技创新基金项目 |
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| Cloning and prokaryotic expression of hGDF-15 |
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CONG Fanhao,ZHAO Jinqiao,LIU Yi,et al
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| Abstract: |
| 【Objective】This study cloned the mature peptide gene of human growth differentiation factor-15 (hGDF-15),constructed its prokaryotic expression vector and induced its expression to provide foundation for research on pharmacological activity and biological function of hGDF-15.【Method】PCR technology was used to clone hGDF-15 mature peptide gene,and the prokaryotic expression vector pET-28a-hGDF-15 was constructed and transformed into E.coli Rosetta（DE3）for expression.After being induced by different temperatures (16,25,and 37 ℃),IPTG concentrations (0.10,0.25,0.50,0.75,and 1.00 mmol/L) and durations (12,24,36,and 48 h),the expression products were analyzed by SDS-PAGE and Western blot.Using Gel Quant Express software,the content in the crushed bacteria centrifugal supernatant and precipitate was determined.The optimal expression conditions of supernatant and precipitate were then analyzed by orthogonal experiment.【Result】The hGDF-15 mature peptide gene (339 bp) was successfully cloned into pET28a prokaryotic expression vector and proteins were confirmed by Western blot.The optimal expression conditions for supernatant were 0.25 mmol/L IPTG,24 h at 16 ℃ and those for precipitate were 1.00 mmol/L IPTG,36 h at 37 ℃.【Conclusion】The hGDF-15 mature peptide gene was successfully cloned,the hGDF-15 proteins were expressed,and the optimal expression conditions were obtained. |
| Key words: GDF-15 prokaryotic expression IPTG concentration inductive duration inductive temperature |
