| 人PSCA真核表达质粒的构建及稳定转染B16细胞系的建立 |
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| 引用本文： | 任杰,高江平,阎瑾琦,王伟,肖毅,江乐,于继云.人PSCA真核表达质粒的构建及稳定转染B16细胞系的建立[J].中国免疫学杂志,2010,26(10). |
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| 作者姓名： | 任杰 高江平 阎瑾琦 王伟 肖毅 江乐 于继云 |
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| 作者单位： | 1. 军事医学科学院基础医学研究所,北京,100850
2. 解放军总医院泌尿外科,北京,100853 |
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| 基金项目： | 国家863基金项目，国家自然科学基金项目 |
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| 摘 要： | 目的:构建真核表达质粒pcDNA3.1-PSCA,通过稳定转染建立高效表达人前列腺干细胞抗原(PSCA)的小鼠黑色素瘤B16细胞系。方法:利用PCR扩增出人PSCA全长基因的cDNA编码区序列,使用基因重组技术将其克隆到真核表达载体pcDNA3.1中,得到重组表达质粒pcDNA3.1-PSCA。双酶切及测序鉴定后,利用脂质体法将其转染入小鼠黑色素瘤B16细胞,G418加压筛选后得到阳性克隆。通过流式细胞术、免疫荧光及Western blot检测PSCA在细胞系中的表达情况。结果:经测序及双酶切鉴定,pcDNA3.1-PSCA重组质粒构建正确;稳定转染人PSCA的B16细胞系表达率接近100%。结论:建立的稳定转染人PSCA的B16细胞系能够高效表达PSCA基因,为抗前列腺癌疫苗的功能研究奠定了基础。
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| 关 键 词： | 前列腺干细胞抗原 稳定转染 肿瘤免疫治疗 |
Construction of eukaryotic expressing plasmid of human PSCA and establishment of stably transfected B16 cell line |
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| REN Jie,GAO Jiang-Ping,YAN Jin-Qi,WANG Wei,XIAO Yi,JIANG Le,YU Ji-Yun.Construction of eukaryotic expressing plasmid of human PSCA and establishment of stably transfected B16 cell line[J].Chinese Journal of Immunology,2010,26(10). |
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| Authors: | REN Jie GAO Jiang-Ping YAN Jin-Qi WANG Wei XIAO Yi JIANG Le YU Ji-Yun |
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| Institution: | REN Jie,GAO Jiang-Ping,YAN Jin-Qi,WANG Wei,XIAO Yi,JIANG Le,YU Ji-Yun.Institute of Basic Medical Sciences,Academy of Military Medical Sciences,Beijing 100850,China |
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| Abstract: | Objective:To construct the eukaryotic expressing plasmid pcDNA3.1-PSCA and establish a stably transfected B16 cell line which expresses human prostate stem cell antigen(PSCA) with high efficacy.Methods:The full length of PSCA cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pcDNA3.1.The recombinant plasmid pcDNA3.1-PSCA was identified by double restriction enzyme digestion and DNA sequencing.Then the plasmid pcDNA3.1-PSCA was transfected into B16 cells by lipofection.After s... |
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| Keywords: | Prostate stem cell antigen Stably transfection Tumor immunotherapy |
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