藏猪IGF-1成熟肽真核表达载体的构建及其在毕赤酵母GS115中的表达

发布时间：2018-04-16 03:24

本文选题：藏猪 + 胰岛素样生长因子　；参考：《中国兽医科学》2017年05期

【摘要】：构建藏猪IGF-1成熟肽基因的真核表达载体,通过毕赤酵母表达系统获得具有生物活性的IGF-1成熟蛋白。根据GenBank中猪IGF-1基因设计1对引物,以构建的藏猪pMD19-T-IGF-1重组质粒为模板,克隆IGF-1成熟肽基因,并将其克隆于表达载体pPIC9k,获得重组穿梭质粒pPIC9k-IGF-1,电转毕赤酵母GS115感受态,对重组酵母转化子经MM、MD平板筛选和PCR鉴定后,经G418抗性梯度筛选,获得多拷贝重组菌株,甲醇诱导表达后进行Tricne-SDS-PAGE和Dot blot分析。Tricne-SDS-PAGE和Dot blot分析显示,表达的目的蛋白分子质量约为13 ku,获得成功表达;CCK8法检测显示,纯化后的IGF-1重组蛋白能够促进细胞增殖。表明,本研究成功构建了藏猪IGF-1成熟肽真核表达质粒pPIC9k-IGF-1,并在毕赤酵母GS115中得到了有效表达,表达产物具有促进细胞增殖的生物活性。
[Abstract]:The eukaryotic expression vector of Tibetan pig IGF-1 mature peptide gene was constructed and the bioactive IGF-1 mature protein was obtained by Pichia pastoris expression system.According to the porcine IGF-1 gene in GenBank, a pair of primers were designed. Using the recombinant plasmid of Tibetan pig pMD19-T-IGF-1 as a template, the mature peptide gene of IGF-1 was cloned and cloned into the expression vector pPIC9k. the recombinant shuttle plasmid pPIC9k-IGF-1 was obtained, and the recombinant shuttle plasmid pPIC9k-IGF-1 was transformed into the GS115 receptive state of Pichia pastoris.The recombinant yeast transformants were screened by MMM-MD plate and PCR, and then screened by G418 resistance gradient. After methanol induced expression, the recombinant strains were analyzed by Tricne-SDS-PAGE, Dot blot, Tricne-SDS-PAGE and Dot blot.The molecular weight of the expressed target protein was about 13ku. the successful expression of CCK8 showed that the purified IGF-1 recombinant protein could promote cell proliferation.The results showed that the eukaryotic expression plasmid pPIC9k-IGF-1 of Tibetan pig IGF-1 mature peptide was successfully constructed and expressed in Pichia pastoris GS115. The expressed product has the biological activity of promoting cell proliferation.
【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;绵阳泰科生物技术有限公司;重庆畜牧科学院;
【基金】：“教育部长江学者和创新团队发展计划”创新团队资助项目(IRT0848) 四川省高等学校科技创新团队资助项目(KM406183.1)
【分类号】：Q78;S828

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