摘要： 旨在克隆藏猪胰岛素样生长因子1（IGF-1）的成熟肽基因，并进行原核表达的研究。提取藏猪肝脏组织RNA，通过RT-PCR扩增出藏猪IGF-1全长基因，构建重组质粒pMD19-T-IGF-1，以pMD19-T-IGF-1质粒为模板，克隆IGF-1成熟肽序列并构建成熟肽pET-32α-IGF-1表达质粒，转入大肠杆菌BL21（DE3），对IPTG诱导剂浓度和诱导时间进行优化，Ni-NTA琼脂纯化融合蛋白后采用Western Blot对其鉴定。结果显示，IGF-1成熟肽基因（315 bp），成功构建了成熟肽pET-32α-IGF-1原核表达质粒；重组大肠杆菌BL21-IGF-1以包涵体形式表达出分子量约31 kDa融合蛋白，最优IPTG浓度为0.5 mmol/L，最佳IPTG诱导时间为10 h；纯化获得了高纯度的融合蛋白，经鉴定IPTG诱导表达和纯化的蛋白为IGF-1融合蛋白。结果表明，成功构建了表达质粒pET32α-IGF-1及获得重组大肠杆菌BL21-IGF-1，表达了藏猪IGF-1成熟肽融合蛋白及该蛋白具有抗原抗体反应活性。

关键词: 藏猪, 胰岛素样生长因子1, 原核表达, 蛋白纯化, 蛋白质印迹法

Abstract: The aim of this study was to clone IGF-1 mature peptide gene from Tibetan pig and study the IGF-1 prokaryotic expression.The total RNA was extracted by using TRIzol from the Tibetan pig liver and used as template to amplify IGF-1 gene by RT-PCR, the amplified fragment was cloned into pMD19-T vector to construct pMD19T- IGF-1 plasmid, which was identified by sequencing. The pMD19-T-IGF-1plasmid was used as a template to amplify IGF-1 mature peptide fragment which was cloned into vector pET32α to obtain recombinant plasmid pET-32α- IGF-1 and transformed into E. coli BL21(DE3).The pET-32α-IGF-1fusion protein was induced to express with different IPTG concentration and induction time. Then the expression culture was analyzed for it's solubility and was prepared to purify pET-32α- IGF-1 fusion protein with Ni-NTA Sefinose^TM Resin.Finally,the expressing culture and purified protein was identified with SDS-PACE analysis and Western Blot. The Tibetan pig IGF-1 mature peptide gene was 315 bp,restriction enzyme mapping and sequencing showed that expression vector was constructed successfully.The pET-32α- IGF-1 fusion protein induced in E. coli BL21(DE3) and could be expressed by IPTG induction with 0.5 mmol/L IPTG induction 10 hours for well expression; The fusion protein expressed in an insoluble form of inclusion bodies and a high-purity fused protein was obtained with Ni-NTA agarose purification. The expressing culture and purified protein were proved to be the pET-32α- IGF-1 fusion protein with SDS-PACE and Western Blot analysis. IGF-1 mature peptide gene was cloned and expressed, the fusion protein has the antigen antibody reactivity.

Key words: Tibetan pig, IGF-1, Prokaryotic expression, Protein purification, Western Blot

中图分类号: 

• Q78

• S828