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中国人民解放军总医院老年心血管病研究所
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中华老年多器官疾病杂志编辑委员会
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创刊人 王士雯
总编辑 范利
副总编辑 陈韵岱
执行主编 叶大训
编辑部主任 王雪萍
ISSN 1671-5403
CN 11-4786
创刊时间 2002年
出版周期 月刊
邮发代号 82-408
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安冬洁,魏调霞,高春辰,曹秀丽,赵俊龙,秦鸿雁.14-3-3η蛋白的生物信息学分析及原核表达[J].中华老年多器官疾病杂志,2018,17(3):216~221
14-3-3η蛋白的生物信息学分析及原核表达
Bioinformatics analysis and prokaryotic expression of 14-3-3η protein
投稿时间:2017-10-25  修订日期:2017-11-20
DOI:10.11915/j.issn.1671-5403.2018.03.047
中文关键词:  计算生物学;巨噬细胞;原核表达;蛋白纯化;14-3-3η
英文关键词:computational biology; macrophages; prokaryotic expression; protein purification; 14-3-3η
基金项目:国家自然科学基金(81530018;31570878;31371474)
作者单位E-mail
安冬洁 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032  
魏调霞 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032  
高春辰 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032  
曹秀丽 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032  
赵俊龙 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032  
秦鸿雁 空军军医大学基础医学部遗传学与发育生物学教研室,西安 710032 hongyanqinfm@gmail.com 
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中文摘要:
      目的 分析巨噬细胞不同极化状态下14-3-3η mRNA表达及其生物信息,并构建14-3-3η蛋白原核表达载体,为研究其在巨噬细胞活化中的作用提供实验基础。方法 提取C57BL/6小鼠骨髓原代巨噬细胞mRNA,反转录后用实时定量聚合酶链式反应(PCR)检测14-3-3η mRNA水平的改变。运用ProtParam tool、SOPMA等在线软件对14-3-3η氨基酸序列进行分析。以小鼠巨噬细胞cDNA为模板,采用PCR的方法扩增14-3-3η基因,插入pGEX-4T-3载体中,双酶切和测序鉴定正确的重组质粒转化入大肠杆菌BL21菌株,用异丙基硫代半乳糖苷诱导其原核表达并纯化,SDS-PAGE法和Western blotting法对融合蛋白进行鉴定。采用Graphpad prism 5软件进行统计分析。样本比较均采用配对t检验。结果 M1型巨噬细胞14-3-3η表达降低,M2型趋势相反。生物信息学表明14-3-3η蛋白其二级结构中主要为α-螺旋(占62.60%)且疏水性较差(疏水系数为-0.607)。酶切和测序结果提示重组质粒pGEX-4T-3-14-3-3η构建成功。SDS-PAGE和Western blotting结果表明融合蛋白谷胱甘肽-S-转移酶(GST)-14-3-3η成功表达和纯化。结论 本实验对14-3-3η进行了初步的生物信息学分析,成功表达并纯化融合蛋白GST-14-3-3η,为14-3-3η在巨噬细胞极化上的功能研究奠定了实验基础。
英文摘要:
      Objective To detect the expression profiles of 14-3-3η mRNA in different polarized macrophages, analyze the bioinformation, and construct the prokaryotic expression vector of 14-3-3η in order to provide an experimental basis for further study on macrophage polarization. Methods After the mRNA of bone marrow derived macrophages from C57BL/6 mice was extracted, real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of 14-3-3η after reverse transcription. ProtParam tool, SOPMA and other online statistics were used to analyze the amino acid sequence of 14-3-3η. With mouse macrophage cDNA as template, PCR was employed to amplify the target genes, and the products were inserted into pGEX-4T-3 vector. After verification by double-enzymes digestion and sequencing, the correct recombinant plasmids were transformed into the Escherichia coli strain BL21. Then the bacteria were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the expressed proteins were purified and identified by SDS-PAGE and Western blotting. Graphpad prism 5 was used to perform the statistical analysis. Paired t test was employed for the comparison among different samples. Results The mRNA expression level of 14-3-3η was decreased in M1 macrophages, but it was increased in M2 macrophages. Bioinformatic analyses showed that α-helix (62.60%) was the main component of its secondary structure, and it had poor hydrophobicity (hydrophobic coefficient=-0.607). Enzymes digestion and sequencing indicated that the vector pGEX-4T-3-14-3-3η was successfully constructed. GST-14-3-3η protein was induced and identified by SDS-PAGE and Western blotting. Conclusion Primary bioinformatic analyses, successful expression and purification of fusion protein GST-14-3-3η are achieved in this study, which founds the experimental basis for 14-3-3η function in macrophage polarization.
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