细胞穿透肽CCL融合蛋白的构建与表达

Construction and expression of cell-penetrating peptide CCL fusion protein expression vector

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。

Objective To evaluate the construction of expression vector for fusion protein of cell-penetrating pep-tide CCL （PEP-CCL）.Methods CCL6-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 chromatog-raphy and detected with Western Blot.Results PEP-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.Conclusion PEP-CCL express vector can be constructed and expressed in eukaryotic cells.