MORF4L1蛋白抗体制备及生物信息学分析

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中文摘要：[目的]构建MORF4L1原核表达载体和纯化表达产物，制备MORF4L1蛋白抗体并进行生物信息学分析。[方法]利用PCR将MORF4L1基因片段酶切、克隆、转化至大肠杆菌Bl21(DE3)，经IPTG诱导表达蛋白。利用His-Tag技术纯化重组蛋白，用重组蛋白免疫新西兰白兔，采集抗血清后通过硫酸铵沉淀法将多克隆抗体从抗血清中纯化，应用Western Blotting验证多克隆抗体与内外源性MORF4L1蛋白的结合特异性。利用在线软件(GEPIA、UALCAN)进行生物信息学分析。[结果]成功构建了重组蛋白，蛋白纯化后在相对分子质量(Mr)43000位置处有单一条带，重组His-MORF4L1蛋白与His抗体发生特异性结合。制备的抗血清与MORF4L1蛋白特异性结合，纯化后仅在相对分子质量(Mr)55000和25000位置处有条带。纯化的多克隆抗体与内外源性MORF4L1蛋白发生特异性反应。MORF4L1的表达与肝癌不良预后相关。[结论]成功构建MORF4L1蛋白及抗体，MORF4L1在肝癌中高表达预后差，对进一步研究MORF4L1蛋白的结构和生物学功能具有重要意义。

外文标题：Preparation of rabbit polyclonal antibody and bioinformatics analysis of MORF4L1 protein

外文摘要：[Objective]To construct MORF4L1 prokaryotic expression vector and purify the expression product,to prepare an-tibody against MORF4L1 protein and to perform bioinformatics analysis.[Method]The MORF4L1 gene fragment was digested,cloned and transformed into E.coli Bl21(DE3)using PCR,and the protein was induced to express by IPTG.The recombinant protein was purified using His-Tag technology,and New Zealand white rabbits were immunised with the recombinant protein,the polyclonal antibody was purified from the antiserum by ammonium sulphate precipitation after collection of the antiserum.Western Blotting was applied to verify the binding specificity of the polyclonal antibody to endogenous and exogenous MORF4L1 protein.Bioinformatics analysis was performed using online software(GEPIA,UALCAN).[Result]The recombinant protein was successfully constructed,and after protein purification,there was a single band at the relative molecular mass(Mr)43000 position,and the recombinant His-MORF4L1 protein bound specifically to the His antibody.The prepared antiserum binds specifically to the MORF4L1 protein,and after purification has bands only at the relative molecular mass(Mr)55000 and 25000 positions.The purified polyclonal antibody reacts specifically with endogenous and exogenous MORF4L1 protein.[Con-clusion]The successful construction of MORF4L1 protein and antibody,which is highly expressed in hepatocellular carcinoma with poor prognosis,is of great significance for further research on the structure and biological function of MORF4L1 protein.

外文关键词：

mortality factor 4 like 1 proteinvector constructionrecombinant proteinprokaryotic expressionprotein purifi-cationpolyclonal antibodyantibody preparationbioinformatics analysis

作者：

丁圆圆、陈泽锋、邱腾、施雯、任德续、王伟玲、吉敬、刘彬

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作者单位：

江苏海洋大学药学院,江苏省海洋药物活性分子筛选重点实验室,江苏连云港 222005

关键词：

致死因子4样蛋白1(MORF4L1) 载体构建重组蛋白原核表达蛋白纯化多克隆抗体抗体制备生物信息学分析

出版年：

2024

DOI：

10.16519/j.cnki.1004-311x.2024.01.0004

生物技术