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作 者:刘庭 汤慧 唐青海 刘莎 薛姣雄 吴广艳 马洁 赵婷芳 唐斯萍 LIU Ting;TANG Hui;TANG Qing-hai;LIU Sha;XUE Jiao-xiong;WU Guang-yan;MA Jie;ZHAO Ting-fang;TANG Si-ping(Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region,Hengyang Key Laboratory of New Detection Technology and Biological Agents of Animal Microorganism,College of Life Sciences and Environment Hengyang Normal University,Hengyang 421008,PRC.)
机构地区:[1]衡阳师范学院生命科学与环境学院\南岳山区生物资源保护与利用湖南省重点实验室\动物微生物新型检测技术及生物制剂衡阳市重点实验室,湖南衡阳421008
出 处:《湖南畜牧兽医》2022年第2期45-50,共6页Hunan Journal of Animal Science & Veterinary Medicine
基 金:湖南省教育厅科学研究项目重点项目(21A0442);湖南省自然科学基金面上项目(2021JJ30060);湖南省重点研发计划(2021NK2026);国家级大学生创新创业训练计划项目(202110546001)。
摘 要:[目的]:提高重组猪表皮生长因子(pEGF)的表达量和纯化量,为后续研究pEGF对仔猪生长发育的影响奠定基础。[方法]:将人工合成的pEGF基因克隆至pEGF4T-1载体,转化至大肠杆菌Rossetta(DE3)中,得到重组菌株Rossetta(DE3)-pGEX-pEGF,IPTG诱导表达,进行表达条件的优化,用GST-tag亲和层析纯化方法进行纯化。[结果]:pEGF基因大小为159 bp,融合表达蛋白分子量为32k Da,最佳诱导表达条件为IPTG浓度0.01mmol/L、诱导温度15℃和诱导时间24h。流穿液和洗涤液中目的蛋白含量极少,洗脱液得到了高纯度的GST-pEGF融合蛋白,纯化较为成功。[结论]:成功构建了pEGF表达菌株,得到了最优的诱导表达条件和最优的纯化工艺,为pEGF相关产品的开发以及应用奠定了基础。[Objective]:To improve the expression and purification amount of recombinant porcine epidermal growth factor(pEGF),and lay a foundation for the follow-up study on the effects of pEGF on the growth and development of piglets.[Methods]:The recombinant strain Rosseta(DE3)-p GEX-pEGF was cloned into pGEX4T-1 vector and transformed into Escherichia coli Rossetta(DE3).The recombinant strain Rosseta(DE3)-p GEX-p EGF was induced to express by IPTG.The expression conditions were optimized and purified by GST-Tag affinity chromatography.[Results]:pEGF gene size was 159 bp,the molecular weight of the fusion expression protein was 32k Da,and the optimal induction conditions were IPTG concentration of 0.01mmol/L,induction temperature of 15℃and induction time of 24h.GST-pEGFF fusion protein was obtained in the eluent with high purity,and the purification was successful.[Conclusion]:The p EGF expressing strain was successfully constructed,and the optimal expression conditions and purification process were obtained,which laid a foundation for the development and application of pEGF related products.
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