细胞穿透肽CCL融合蛋白的构建与表达
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吴君

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R3  Q2-33

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湖北省教育厅重点项目支持(D20112101)


Construction and expression of cellpenetrating peptide CCL fusion protein expression vector
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    摘要:

    目的评估细胞穿透肽CCL融合蛋白构建的可能性。方法将CCL6PEP6XHis构建至pABP质粒,然后提取pABPCCL6PEP质粒进行人胚肾HEK293细胞转染表达,以及CCL6PEP6XHis 蛋白层析纯化和检测。结果成功构建并纯化细胞穿透肽CCL融合蛋白。将CCL6PEP6XHis Tag 基因经PCR扩增、接入T 载体、克隆、培养,并提取质粒进行测序鉴定,所得序列与目的基因一致。成功将CCL6PEP6XHis基因构建至哺乳动物细胞表达载体pABP 中,经质粒提取和酶切鉴定,电泳结果显示,HindⅢ + XbaⅠ切出约430 bp的条带,符合预期,酶切鉴定正确。蛋白质印迹法(Western Blot)检测结果阳性,表明纯化得到的目标蛋白带有hisx6标签。结论细胞穿透肽CCL融合蛋白能够人工构建,并通过真核细胞进行表达。

    Abstract:

    ObjectiveTo evaluate the construction of expression vector for fusion protein of cellpenetrating peptide CCL (PEPCCL). MethodsCCL6PEP6XHis was inserted into plasmid pABP, pABPCCL6PEP plasmid was extracted and then transfected into HEK293 cells, CCL6PEP6XHis was expressed and purified by chromatography and detected with Western Blot. ResultsPEPCCL express vector was successfully constructed and purified. PCR product of CCL6PEP6XHis Tag was ligated with T vector, recombinant was transferred into the host cells, then host cells were cultured, plasmid was extracted and sequenced, the sequence was identical to targeted gene. CCL6PEP6XHis was successfully inserted into the eukaryotic expression vector pABP, plasmid was extracted and digested, electrophoresis results revealed that a fragment with 430bp was digested by Hind Ⅲ+XbaⅠ, which was identical to the expected value. Western Blot revealed that CCL6PEP fusion protein could be recognized by His monoclonal antibody. ConclusionPEPCCL express vector can be constructed and expressed in eukaryotic cells.

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刘勇,安艳芳,等.细胞穿透肽CCL融合蛋白的构建与表达[J]. 中国感染控制杂志,2016,15(6):361-366. DOI:10.3969/j. issn.1671-9638.2016.06.001.
LIU Yong, AN Yanfang, et al. Construction and expression of cellpenetrating peptide CCL fusion protein expression vector[J]. Chin J Infect Control, 2016,15(6):361-366. DOI:10.3969/j. issn.1671-9638.2016.06.001.

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  • 收稿日期:2015-08-23
  • 最后修改日期:2015-11-12
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  • 在线发布日期: 2016-06-30
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