人源性RPL22基因的原核表达与纯化及功能分析  

Prokaryotic expression,purification and functional analysis of human RPL22 gene

作　　者：田甜[1] 张星娟 杨明夏[1] Tian Tian;Zhang Xingjuan;Yang Mingxia(Dept of Pulmonary and Critical Care Medicine,The Third Affiliated Hospital of Nanjing Medical University(Changzhou Second People′s Hospital),Changzhou 213003)

机构地区：[1]南京医科大学第三附属医院(常州市第二人民医院)呼吸科,常州213003

出　　处：《安徽医科大学学报》2024年第10期1785-1793,共9页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:81472199)。

摘　　要：目的了解人类核糖体蛋白L22(RPL22)基因的生物信息学,原核表达与纯化人类RPL22重组蛋白,在体外合成人类RPL22(标记为试剂M),研究试剂M对NSCLC A549细胞增殖、周期、凋亡的影响,分析试剂M和顺铂(DDP)联合进行化疗的效果。方法分析RPL22生物信息学。PCR克隆RPL22基因,进行单链寡核苷酸的设计及合成,获得目的基因后连接pET-28α载体,构建pET-28α-RPL22重组质粒,转化大肠埃希菌感受态细胞DH 5α,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot分析重组蛋白RPL22的表达情况。CCK-8检测试剂M和DDP在A549细胞中的半抑制率(IC_(50))以及试剂M对A549细胞增殖的影响;流式细胞术检测试剂M对A549细胞凋亡、周期的影响;qPCR、Western blot检测试剂M和DDP分别对磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)、腺苷5′-单磷酸激活蛋白激酶/哺乳动物雷帕霉素靶蛋白(AMPK/mTOR)信号通路的影响。结果RPL22蛋白为不稳定亲水性蛋白质。诱导表达的重组蛋白经溶解纯化后获得了高浓度重组蛋白。在A549细胞中,试剂M的IC_(50)为400μg/L,DDP的IC_(50)为10μmol/L,试剂M抑制细胞增殖,其浓度与细胞活性成反比;试剂M(200μg/L)与DDP(2.5μmol/L)联合运用时,效果与单独使用10μmol/L DDP抑制增殖比相似;试剂M能诱导G 2细胞周期停止、促进细胞凋亡,且可能参与PI3K/AKT、AMPK/mTOR通路的调节。结论该研究首次克隆得到人类RPL22重组蛋白,使其成功纯化表达。Objective To understand the bioinformatics of human ribosomal protein L22(RPL22)gene,prokaryotic expression and purification of human RPL22 recombinant protein,synthesis of human RPL22(labeled as agent M)in vitro,study the effects of agent M on proliferation,cycle and apoptosis of NSCLC A549 cells,and to analyze the effect of combination chemotherapy with M and cisplatin(DDP).Methods RPL22 bioinformatics was analyzed.RPL22 gene was cloned by PCR,the single chain oligonucleotide was designed and synthesized to obtain the target gene,and connect the pET-28αvector.The pET-28α-RPL22 recombinant plasmid was constructed,and the receptive cell DH 5αof Escherichia coli was transformed.The expression of recombinant protein RPL22 was analyzed by SDS-PAGE and Western blot.The semi-inhibitory rate(IC_(50))of M and DDP in A549 cells and the effect of M on the proliferation of A549 cells were detected by CCK-8.The effect of agent M on apoptosis and cycle of A549 cells was detected by flow cytometry.qPCR and Western blot assays were used to detect M and DDP on phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)and adenosine 5′-monophosphate activation of protein kinase/mammalian target of rapamycin(AMPK/mTOR)signaling pathway,respectively.Results RPL22 protein was an unstable and hydrophilic protein.The recombinant protein was dissolved and purified to obtain high concentration of recombinant protein.In A549 cells,the IC_(50) of reagent M was 400μg/L,and the IC_(50) of DDP was 10μmol/L,and the concentration of reagent M was inversely proportional to the cell activity.The inhibitory effect of reagent M(200μg/L)combined with DDP(2.5μmol/L)was similar to that of DDP(10μmol/L)alone.Reagent M could induce G 2 cell cycle arrest and promote apoptosis,and might be involved in the regulation of PI3K/AKT and AMPK/mTOR pathways.Conclusion The recombinant RPL22 protein is cloned and expressed successfully.

关 键 词：核糖体蛋白L22 非小细胞肺癌 生物信息学分析 原核表达与纯化 功能分析 

分 类 号：R56[医药卫生—呼吸系统]