藏猪IGF-1成熟肽基因的克隆及原核表达

发布时间：2018-03-17 08:39

本文选题：藏猪　切入点：胰岛素样生长因子　出处：《华北农学报》2017年01期 　论文类型：期刊论文

【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。
[Abstract]:In order to clone porcine insulin-like growth factor 1 (IGF-1) of the mature peptide gene, and study the prokaryotic expression. The extraction of Tibet pig liver tissue RNA, RT-PCR amplified with IGF-1 gene of Tibet pig, the recombinant plasmid P MD19-T-IGF-1 to P MD19-T-IGF-1 plasmid as template to clone IGF-1 mature peptide sequence and construct mature peptide p ET-32 alpha -IGF-1 expression plasmid was transformed into Escherichia coli BL21 (DE3), the IPTG concentration of inducer and induction time were optimized. The fusion protein was purified by Ni-NTA agar Western Blot for its identification. The results showed that the mature peptide of IGF-1 gene (315 BP), the successful construction of P mature peptide ET-32 alpha -IGF-1 prokaryotic expression plasmid BL21-IGF-1; recombinant Escherichia coli expressed in form of inclusion bodies with molecular weight of approximately 31 K Da fusion protein, the optimal concentration of IPTG was 0.5 mmol/L, the best induction time of IPTG was 10 h; the fusion protein was purified by high purity, identification The IPTG induced and expressed protein was IGF-1 fusion protein. The results showed that the expression plasmid P ET32 -IGF-1 and the recombinant Escherichia coli BL21-IGF-1 were successfully constructed, and the fusion protein of IGF-1 mature peptide was expressed, and the protein had antigen antibody reactivity.

【作者单位】：四川农业大学动物医学院动物微生态研究中心;成都农业科技职业学院;
【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)
【分类号】：Q78;S828

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