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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 recombinant green fluorescent protein (EGFP) expression vector containing anti-TAG72 scFv-CD28, which is a fusion gene of anti-TAG72 single chain variable fragment (scFv) and intracellular and transmembrane regions of CD28 Vector pEGFP-C3-anti-TAG72 scFv-CD28 and transfected to obtain chimeric anchored T lymphocytes. Methods The chimeric and anchored fusion protein of anti-TAG72 single chain antibody and CD28 intracellular and transmembrane region genes were cloned into the green fluorescent protein eukaryotic expression vector pEGFP-C3 to obtain the expression of pEGFP-C3-anti-TAG72 scFv-CD28 Vector. The positive clones were screened with kanamycin, identified by restriction enzyme and sequencing. Peripheral blood mononuclear cells (PBMCs) were transiently transfected with the above recombinant plasmid pEGFP-C3-anti-TAG72 scFv-CD28 to obtain chimeric anchored T lymphocytes. Fluorescence microscopy was used to detect the chimeric molecules anti- TAG72 scFv-CD28 expression in PBMCs and the expression of the chimeric-anchored fusion molecule anti-TAG72 scFv-CD28 was examined. Results The fragment of anti-TAG72 scFv-CD28 in the recombinant green fluorescent vector pEGFP-C3-anti-TAG72 scFv-CD28 was 1 002 bp, which was consistent with the known sequence. The results of sequencing showed that anti-TAG72 scFv-CD28 gene was inserted into pEGFP-C3 vector correctly. After transfection, the fragment of anti-TAG72 scFv-CD28 and green fluorescent substance were expressed in chimeric anchored T Lymphocyte membrane surface and cytoplasm, the green fluorescent color. Conclusion The construction of the green fluorescent expression vector pEGFP-C3-anti-TAG72 scFv-CD28 was completed and was transiently expressed in PBMC.