首页|缺失内质网驻留信号肽的小鼠钙网蛋白在B16-F1细胞中的表达

缺失内质网驻留信号肽的小鼠钙网蛋白在B16-F1细胞中的表达

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目的:克隆无内质网驻留信号肽的钙网蛋白(CRT),构建表达分泌性钙网蛋白的B16-F1肿瘤细胞株.方法:以前期构建的小鼠钙网蛋白真核表达质粒为模板,采用突变PCR技术获得无内质网驻留信号肽的钙网蛋白cDNA并克隆入真核表达载体中.以脂质体转染法将该质粒转染B16.F1细胞后,用G418筛选单克隆细胞株.分别以PCR,WesternBlot法鉴定成功转染和表达分泌型CRT的B16-F1细胞株.流式细胞术分析该细胞株的细胞周期.结果:成功获得无内质网驻留信号肽编码序列的小鼠CRT真核表达质粒.稳定转染并筛选出表达分泌性钙网蛋白的B16-F1细胞株,CRT在此细胞株中表达并分泌至培养基质中.稳定转染前后的B16-F1细胞株细胞周期没有明显变化.结论:删除CRT编码序列中内质网驻留信号肽(KDEL)后,CRT失去了驻留在细胞内质网中的能力从而以分泌蛋白的形式出现在细胞外培养基质中.本研究为CRT的细胞外转移提供了一条新的途径,同时为以CRT为基础的抗肿瘤免疫研究提供了新的研究工具.
Expression of mouse calreticulin missing endoplasmic reticulum retain signal peptide in B16-F1 cell line
AIM: To clone mouse calreticulin (CRT) in which the endoplasmic reticulum retain signal peptide of CRT (KDEL) is removed and to construct a B16-F1 cell line which can express secretory CRT. METHODS: Mouse CRT cDNA without KDEL-encoding sequence was obtained by PCR mutagenetic techniques and cloned into the eukaryotic expression vector.The vector was then transfected into B16-F1 cells by using liposome and the single clones were selected with G418. The cell line that is successful in transfection andsecretory expression of CRT were identified by PCR and Western Blotting assay. RESULTS: Eukaryotic expression plasmid of secretory CRT was constructed successfully. After transfected into B16-F1 cells, secretory CRT could be expressed and released into the cell medium. CONCLUSION:By removing the KDEL sequence, CRT losted its ability of retaining in endoplasmic reticulumand was released into cell medium as a secretory protein. This study explored a new way for CRT releasing into extracellular environment and offered a new research tool for anti-tumor immunotherapy.

calreticulinsecretory expressionanti-tumor immune

曹春雨、韩钰、任玉珊、王艳林

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三峡大学分子生物学研究所,湖北宜昌443002

三峡大学医学院,湖北宜昌443002

钙网蛋白 分泌型表达 抗肿瘤免疫

国家自然科学基金三峡大学留学回国人员专项基金

30973445200603

2009

第四军医大学学报
第四军医大学

第四军医大学学报

CSTPCDCSCD北大核心
影响因子:0.599
ISSN:1000-2790
年,卷(期):2009.30(23)
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