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乳腺癌细胞高亲和融合多肽蛋白的表达以及靶向转运

Expression of fusion peptide with high affinity to human breast cancer and its targeting transportation effect

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【作者】 刘为青董坚赵德萍洪敏杨军邢海霞

【Author】 LIU Wei-qing,DONG Jian,ZHAO De-ping,HONG Min,YANG Jun,XING Hai-xia(Biotherapy Center,the 1st Affiliated Hospital of Kunming Medical College,Kunming 650032,China)

【机构】 昆明医学院第一附属医院生物治疗科

【摘要】 目的探究乳腺癌特异性多肽携带外源生物分子进入特定肿瘤细胞的功能。方法编码乳腺癌细胞特异性多肽(PⅠ)的序列插入质粒pGEX-2T,构建原核表达载体pGEX-2T-PⅠ,表达融合蛋白GST-PⅠ,亲和层析纯化,Western-blot鉴定。GST-PⅠ与人乳腺癌细胞MBA-MB-231体外共培养,免疫荧光检测PⅠ多肽携带GST蛋白的跨膜功能。结果成功构建pGEX-2T-PⅠ原核表达载体,并在大肠杆菌中表达。融合蛋白占细菌总蛋白量的30%左右,纯化后蛋白纯度约为85%。免疫荧光检测显示,GST-PⅠ能特异性进入MDA-MB-231细胞。结论乳腺癌特异性多肽PⅠ能够介导融合蛋白GST-PⅠ穿过细胞膜进入MDA-MB-231细胞内。

【Abstract】 Objective To investigate targeting delivery of a peptide with high affinity to human breast cancer.Methods Gene of the peptide was cloned into pGEX-2T vector.The fusion proteins of glutathione-S-transferase(GST) and peptide sequence CASPSGALRSC(PⅠ),named GST-PⅠ,were expressed in BL21(DE3) and purified by GST affinity column.The purified fusion protein was detected by SDS-PAGE and Western-blotting.Immunostaining analysis was used to determine the transduction of GST-PⅠ fusion protein into the MDA-MB-231 cells and other three human breast cancer cell lines were selected as control.Results A series of pGEX-2T-PⅠ vectors were constructed.The result of Western-blotting showed that the fusion protein was well immunoreacted with murine anti Schistosom a japonica GST monoclonal antibody.Immunostaining analysis showed that GST-PⅠ could specifically permeate into MDA-MB-231 cells.Conclusion GST-PⅠ,a fusion protein with expected molecular mass,was expressed.PⅠ can mediate the fusion proteins across the cell membrane.It can be used as a targeting vector to delivery bimolecular agent in gene therapy of breast cancer.

【关键词】 乳腺癌基因治疗载体
【Key words】 breast cancergene therapyvector
【基金】 国家自然科学基金(30460142);云南省自然科学基金(2003C0006R)
  • 【文献出处】 基础医学与临床 ,Basic & Clinical Medicine , 编辑部邮箱 ,2009年03期
  • 【分类号】R737.9
  • 【被引频次】7
  • 【下载频次】124
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