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人11β-羟基类固醇脱氢酶基因克隆与表达的实验研究 被引量:1

Cloning and expression human 11β-hydroxysteroid dehydrogenase type
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摘要 目的:构建人Ⅰ型11β羟基类固醇脘氢酶(11β-HSD1)原核表达载体并表达目的蛋白,为代谢综合征的研究奠定基础。方法:从Hep G2细胞系中提取细胞总RNA,经反转录合成c DNA第一链后再以其为模版,扩增11β-HSD1基因。p ET32a原核表达载体和PCR扩增出的产物经过Bam HI和Xho I双酶切后,在16℃过夜的条件下用T4 DNA连接酶连接上述酶切产物,然后用该产物转化Top10大肠杆菌感受态细胞,挑克隆后进行测序。将克隆成功的11β-HSD1重组质粒转化大肠杆菌菌株BL21(DE3),分别用不同浓度、不同时间的IPTG(异丙基-β-D-硫代半乳糖)诱导其表达,并优化诱导条件。表达的蛋白采用聚丙烯酰胺凝胶电泳结合考马斯亮蓝染色的方法观察目的基因的表达情况。结果:挑选的克隆经测序证实p ET32a-11β-HSD1重组质粒构建成功,11β-HSD1基因与携带有6×His-tag标签和肠激酶识别位点的硫氧还蛋白trx A基因融合,在IPTG浓度为10μM,诱导时间为5小时时,上清中有明显的分子量约为52 k Da的重组蛋白产生,且目的蛋白分子量的大小与预期一致。结论:本实验成功构建了人p ET32a-11β-HSD1蛋白原核表达载体,转化大肠杆菌BL21(DE3)后成功诱导了其融合表达。目的蛋白主要以可溶性蛋白的形式存在于上清中,为进一步研究其结构、功能、制备抗体等奠定了基础。 Abjective:To construct recombinant plasmid pET32a-11β-HSD1and express the recombinant protein.Methods:The total RNA was extracted from the HepG2cell line.To obtained11β-HSD1gene fragment,the first strand of cDNA was synthesized by reverse transcription-polymerase chain reaction.Then the11β-HSD1gene was amplified by PCR using the first strand of cDNA as template.pET32a plasmid and PCR production were digested by restriction enzymes Bam HI and Xho I,and then we use T4DNA ligase to ligate them in16℃overnight.E.coli Top10was transformed into the above recombinants.The successfully cloned recombinant plasmid11beta-HSD1was transformed into E.coli strain BL21(DE3).The11β-HSD1gene was induced by isopropy-β-D-thiogalactoside with different concentration and time,and the induction conditions were optimized.The expression of11β-HSD1was measured by SDS-PAGE and Coomassie brilliant blue staining.Results:The selected clones were sequenced to confirm that the recombinant plasmid of pET32a-11β-HSD1was successfully constructed.The11β-HSD1gene was fused with the thioredoxin gene carrying6xHis-tag and enterokinase identification site.When treated with10μM IPTG for5hours,the recombinant protein(about52kDa)was successfully produced in the supernatant,consistent with the expectation.Conclusion:Recombinant plasmid pET32a-11β-HSD1was successfully constructed and after the transformation of E.coli BL21(DE3),the fusion protein was expressed in the form of soluble protein in the supernatant,which laid down the foundation for further investigation on its structure,function and antibody preparation.
作者 胡蒙亮 徐杰 任航江 韩亭亭 满永 唐蔚青 黎健 李国平 HU Mengliang;XU Jie;REN Hangjiang;HAN Tingting;MAN Yong;TANG Weiqing;LI Jian;LI Guoping(The MOH key Laboratory of Geriatrics,Beijing Hospital,National Center of Gerontology,Beijing100730,China)
出处 《心肺血管病杂志》 2018年第11期1019-1022,共4页 Journal of Cardiovascular and Pulmonary Diseases
基金 国家自然科学基金(81471071 81270948) 教育部留学回国人员科研启动基金资助项目(第46批 教外司[2013]693号)
关键词 I型11β羟基类固醇脱氢酶 原核表达 硫氧还蛋白 融合表达 11beta-hydroxysteroid dehydrogenase type1 Prokaryotic Expression Thioredoxin Fusion expression
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