增强型绿色荧光蛋白与MAGE-n共表达载体的构建及表达  

Construction and expression of enhanced green fluorescent protein and MAGE-n co-expression vector

作　　者：黄亚渝[1] 童卫[2] 马加海[3] 董海龙[3] 叶菁[3] 陈广生[3] 张秀敏[3] 隋延仿[3] 

机构地区：[1]第四军医大学西京医院血液科,陕西西安710032 [2]第四军医大学医教部教务科,陕西西安710032 [3]第四军医大学基础部病理学教研室,陕西西安710033

出　　处：《现代肿瘤医学》2006年第7期783-786,共4页Journal of Modern Oncology

基　　金：国家自然科学基金(30271464);全军医药卫生科研基金(01Z084)

摘　　要：目的构建增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)与人黑色素瘤抗原-n(melanomaantigenn,MAGE-n)真核共表达载体,并在小鼠黑色素瘤B16细胞中进行表达。方法采用酶切法从pLXSN-MAGE-n中得到MAGE-n基因片段,克隆至真核表达载体pIRES2-EGFP中,构建真核共表达质粒pIRES2-EGFP-MAGE-n。用脂质体法转染小鼠黑色素瘤B16细胞,G418筛选阳性克隆,荧光显微镜检测阳性克隆中EGFP的表达,RT-PCR、Westernblot和免疫组化分别检测阳性克隆中MAGE-nmRNA和蛋白的表达水平。结果从pLXSN-MAGE-n重组质粒中酶切获得一条约950bp的片段,成功构建了真核共表达载体pIRES2-EGFP-MAGE-n,转染B16细胞后筛选得到阳性克隆,可见到EGFP的表达,产生明亮的绿色荧光,RT-PCR检测到MAGE-nmRNA的表达,Westernblot和免疫组化检测到MAGE-n蛋白的表达。结论成功构建了共表达质粒pIRES2-EGFP-MAGE-n,获得了稳定共表达EGFP和MAGE-n的小鼠黑色素瘤B16细胞系,为进一步研究MAGE-n在肿瘤免疫治疗中的作用奠定了基础。Objective ：To construct the enhanced green fluorescent protein and MAGE - n co - expression vector and to express the plasmid in mouse melanoma B16 cells. Methods：The MAGE -n gene was obtained from pLXSN - MAGE - n by digestion, and subcloned into the muhiclonal site of pIRES2 - EGFP, then the pIRES2 - EGFP - MAGE - n plasmid was constructed. The plasmid was transfected into the mouse melanoma B16 cells under mediation of lipofectamine. The positive clones were selected by G418. The expression of EGFP was detected by fluoresent microscope, and MAGE - n mRNA and protein in positive clones were detected by RT - PCR , Western blot and immunohistochemistry respectively. Results：The full ORF of MAGE -n, about 950bp fragment, was obtained from pLX- SN - MAGE - n by enzyme digestion. The pIRES2 - EGFP - MAGE - n plasmid was constructed and transfected into B16 cells successfully. Green fluorescence of EGFP expressed in B16 cells was observed under fluoresent microscope and it showed posotive signals of MAGE - n mRNA and protein in the transfected cells. Conclusion：The co - expression plasmid pIRES2 - EGFP - MAGE - n was constructed successfully. The B16 cells that stably transfected MAGE - n was obtained which laid a foundation for the research of MAGE - n in the tumor immunotherapy.

关 键 词：黑色素瘤抗原n 基因转染 绿色荧光蛋白 基因表达 

分 类 号：R730.3[医药卫生—肿瘤]