嵌合锚定融合分子在T淋巴细胞的荧光表达及检测  

Construction and Identification of Fluorescent Protein Expression Vector with Chimeric Molecule in T Lymphocytes

作　　者：徐宏勇[1] 徐立[2] 李开宗[3] 窦科峰[3] 

机构地区：[1]解放军第451医院肝胆普外科,西安710054 [2]第四军医大学西京医院消化病研究所,西安710032 [3]第四军医大学西京医院肝胆外科,西安710032

出　　处：《中国普外基础与临床杂志》2010年第10期1046-1049,共4页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金(项目编号:39900068);陕西省自然科学基金(项目编号:2007C259)~~

摘　　要：目的构建含有由抗肿瘤相关糖蛋白72(anti-TAG72)单链抗体片段(single chain variable fragment,scFv)及CD28胞内区及跨膜区的融合基因anti-TAG72 scFv-CD28的重组绿色荧光表达载体pEGFP-C3-anti-TAG72 scFv-CD28,并转染获得嵌合锚定T淋巴细胞。方法将anti-TAG72单链抗体及CD28胞内区及跨膜区基因的嵌合锚定融合分子克隆入绿色荧光蛋白真核表达载体pEGFP-C3,获得pEGFP-C3-anti-TAG72 scFv-CD28表达载体。用卡那霉素筛选阳性克隆,经酶切和测序鉴定。将上述重组质粒pEGFP-C3-anti-TAG72 scFv-CD28以脂质体法瞬时转染外周血单核细胞(PBMC),以获得嵌合锚定T淋巴细胞,用荧光显微镜检测嵌合分子anti-TAG72 scFv-CD28在PBMC中的表达,并检验嵌合锚定融合分子anti-TAG72 scFv-CD28的表达。结果重组绿色荧光表达载体pEGFP-C3-anti-TAG72 scFv-CD28中anti-TAG72 scFv-CD28片段为1 002 bp,与已知的序列相符。酶切、测序结果表明,嵌合锚定融合分子基因片段anti-TAG72 scFv-CD28被正确插入pEGFP-C3载体;转染后anti-TAG72 scFv-CD28片段及绿色荧光物质表达于嵌合锚定T淋巴细胞胞膜表面及胞浆中,为绿色荧光显色。结论完成了绿色荧光表达载体pEGFP-C3-anti-TAG72 scFv-CD28的构建,并成功在PBMC瞬时表达。Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28,containing anti-TAG72 single chain variable fragment（scFv） fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene,and to transfect it into peripheral blood mononuclear cells.Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector.Recombinant clones were selected by Kanamyein,and then identified by PCR,enzyme digestion analysis and DNA sequencing.The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection.The recombinant protein expression was confirmed by immunocytochemistry,laser scanning confocal microscope,PCR and Western blot analysis.Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid,and it was confirmed by enzyme digestion and DNA sequencing.The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells.Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells,which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte,for generating of modified T lymphocytes targeting gastrointestinal tumors.

关 键 词：单链抗体片段 CD28 pEGFP-C3载体 肿瘤相关糖蛋白72 转染 

分 类 号：R392[医药卫生—免疫学]