藏猪IGF-1成熟肽基因的克隆及原核表达

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p MD19-T-IGF-1,以p MD19-T-IGF-1质粒为模板,克隆IGF-1成熟肽序列并构建成熟肽p ET-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p ET-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 k Da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p ET32α-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。

Cloning of Tibetan Pig IGF-1 Mature Peptide Gene and Its Prokaryotic Expression

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 pMD19TIGF-1 plasmid,which was identified by sequencing.The pMD19-T-IGF-1 plasmid 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-1 fusion 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 SefinoseTM 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.