血小板生成素拟肽的原核表达、纯化及生物学活性评估  

Prokaryotic expression,purification and activity analysis of thrombopoietin mimic peptide

作　　者：邹洪胜 宋磊 赵文明 刘洋 李胜 赵腾宇 王伟浩 ZOU Hongsheng;SONG Lei;ZHAO Wenming;LIU Yang;LI Sheng;ZHAO Tengyu;WANG Weihao(Department of Intensive Care Medicine,Rongcheng People's Hospital,Shandong Province,Rongcheng 264300,China;Department of Business,Shandong Quangang Pharmaceutical Co.,Ltd.,Shandong Province,Jinan 250014,China;Department of General Surgery,Second Affiliated Hospital of Shandong First Medical University,Shandong Province,Tai'an271016,China;School of Stomatology,Shandong First Medical University,Shandong Province,Jinan 250117,China)

机构地区：[1]山东省荣成市人民医院重症医学科,山东荣成264300 [2]山东泉港药业有限公司业务部,山东济南250014 [3]山东第一医科大学第二附属医院普外科,山东泰安271016 [4]山东第一医科大学口腔医学院,山东济南250117

出　　处：《中国当代医药》2025年第11期20-26,共7页China Modern Medicine

基　　金：山东省科技型中小企业创新能力提升工程项目(2022TSGC2198);山东省济南市“新高校20条”资助项目(2021GXRC060)。

摘　　要：目的在原核表达系统中实现血小板生成素拟肽(TMP)的表达,并对TMP进行纯度和活性测定。方法通过基因合成得到编码TMP的基因片段,利用基因重组技术构建原核表达质粒pET-22b-TMP,转化至大肠杆菌。工程菌经高密度发酵,收集菌体经超声破碎、包涵体复性后利用Protein A亲和层析和阳离子交换层析对目标蛋白进行纯化。纯化的目的蛋白利用32D(c-mpl)细胞进行活性分析。结果工程菌在发酵过程中TMP的表达水平超过菌体总蛋白的30%;表达产物经两步纯化得到有效分离,蛋白收率为300 mg/L发酵液;纯化的TMP纯度达到了95%以上;生物学活性与国外同类一致。结论TMP纯化工艺和检测方法的建立将为其规模化生产和进一步的临床研究提供良好的基础。Objective To express thrombopoietin mimic peptide(TMP)in a prokaryotic expression system,and to purify and assay the activity of TMP.Methods The gene fragment encoding TMP was synthesized by gene synthesis.The prokaryotic expression plasmid pET-22b-TMP was constructed by gene recombination technology and transformed into Escherichia coli.After high-density fermentation,the collected bacteria were crushed by ultrasonic,and the inclusion bodies were renaturated.Protein A affinity chromatography and cation exchange chromatography were used to purify the target proteins.The purified target protein was analyzed by 32D(c-mpl)cells.Results The expression level of engineering bacteria fermentation accounted for more than 30%of total bacterial protein.The expression product was purified by two steps and effectively separated with a yield of 300 mg/L in fermentation broth.The purity of purified TMP was more than 95%.The biological activity was consistent with that of foreign counterparts.Conclusion The establishment of purification technology and detection method of TMP will provide a good basis for its large-scale production and further clinical research.

关 键 词：血小板生成素模拟肽 原核表达 纯化 生物活性 

分 类 号：R973.3[医药卫生—药品]