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。
The aim of this study was to clone IGF-1 mature peptide gene from Tibetan pig and study the IGF-1prokaryotic 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 p MD19-T vector to construct p MD19TIGF-1 plasmid,which was identified by sequencing. The p MD19-T-IGF-1 plasmid was used as a template to amplify IGF-1 mature peptide fragment which was cloned into vector p ET32α to obtain recombinant plasmid p ET-32α-IGF-1 and transformed into E. coli BL21（ DE3）. The p ET-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 p ET-32α-IGF-1 fusion protein with Ni-NTA SefinoseTMResin. 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 p ET-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 p ET-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.
出处
《华北农学报》
CSCD
北大核心
2017年第1期80-85,共6页
Acta Agriculturae Boreali-Sinica
基金
四川省高等学校科技创新团队资助项目(KM406183.1)
全国大学生创新训练计划项目(201410626028)
