医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2014年
5期
252-256
,共5页
茹晓荣%刘永兰%向安
茹曉榮%劉永蘭%嚮安
여효영%류영란%향안
T 淋巴细胞相关抗原-4%293T 细胞%基因克隆%真核表达
T 淋巴細胞相關抗原-4%293T 細胞%基因剋隆%真覈錶達
T 림파세포상관항원-4%293T 세포%기인극륭%진핵표체
cytotoxic T lymphocyte antigen-4 (CTLA-4)%human embryonic kidney 293T cell (293T)%gene cloning%eukaryotic expression
目的：构建稳定表达细胞毒性 T 淋巴细胞相关抗原-4（cytotoxic T lymphocyte antigen-4， CTLA-4）的293T 细胞株。方法首先从人外周血获取 PBMC 加以 T 细胞活化； PCR 扩增 CTLA-4转录本，连接pUCm-T 质粒引入 HindⅢ和 EcoRⅠ双酶切位点后转入真核细胞表达质粒 pcDNA3．1；重组质粒 pcDNA3．1-CTLA-4经脂质体转染293T 细胞，以抗生素 G418筛选表达 CTLA-4的293T 细胞；定量 PCR、免疫荧光分别检测293T 细胞 CTLA-4表达与细胞定位。结果血样来源 T 细胞经活化后表达 CTLA-4转录本。 HindⅢ和EcoRⅠ双酶切证实重组质粒内 CTLA-4插入序列正确； RT-PCR 及免疫荧光检测证实293T 细胞能够稳定表达目的蛋白 CTLA-4，并定位于细胞膜表面。结论成功构建的 pcDNA3．1-CTLA-4重组真核表达载体，在293T 细胞膜表面实现了稳定表达，为进行相关生物学理论与应用研究打下基础。
目的：構建穩定錶達細胞毒性 T 淋巴細胞相關抗原-4（cytotoxic T lymphocyte antigen-4， CTLA-4）的293T 細胞株。方法首先從人外週血穫取 PBMC 加以 T 細胞活化； PCR 擴增 CTLA-4轉錄本，連接pUCm-T 質粒引入 HindⅢ和 EcoRⅠ雙酶切位點後轉入真覈細胞錶達質粒 pcDNA3．1；重組質粒 pcDNA3．1-CTLA-4經脂質體轉染293T 細胞，以抗生素 G418篩選錶達 CTLA-4的293T 細胞；定量 PCR、免疫熒光分彆檢測293T 細胞 CTLA-4錶達與細胞定位。結果血樣來源 T 細胞經活化後錶達 CTLA-4轉錄本。 HindⅢ和EcoRⅠ雙酶切證實重組質粒內 CTLA-4插入序列正確； RT-PCR 及免疫熒光檢測證實293T 細胞能夠穩定錶達目的蛋白 CTLA-4，併定位于細胞膜錶麵。結論成功構建的 pcDNA3．1-CTLA-4重組真覈錶達載體，在293T 細胞膜錶麵實現瞭穩定錶達，為進行相關生物學理論與應用研究打下基礎。
목적：구건은정표체세포독성 T 림파세포상관항원-4（cytotoxic T lymphocyte antigen-4， CTLA-4）적293T 세포주。방법수선종인외주혈획취 PBMC 가이 T 세포활화； PCR 확증 CTLA-4전록본，련접pUCm-T 질립인입 HindⅢ화 EcoRⅠ쌍매절위점후전입진핵세포표체질립 pcDNA3．1；중조질립 pcDNA3．1-CTLA-4경지질체전염293T 세포，이항생소 G418사선표체 CTLA-4적293T 세포；정량 PCR、면역형광분별검측293T 세포 CTLA-4표체여세포정위。결과혈양래원 T 세포경활화후표체 CTLA-4전록본。 HindⅢ화EcoRⅠ쌍매절증실중조질립내 CTLA-4삽입서렬정학； RT-PCR 급면역형광검측증실293T 세포능구은정표체목적단백 CTLA-4，병정위우세포막표면。결론성공구건적 pcDNA3．1-CTLA-4중조진핵표체재체，재293T 세포막표면실현료은정표체，위진행상관생물학이론여응용연구타하기출。
Objective To construct human embryonic kidney 293T cell line that can stably ex-press cytotoxic T lymphocyte antigen 4 (CTLA-4) .Methods Peripheral blood mononuclear cells (PBMCs) were harvested for T cell activation.CTLA-4 transcripts were amplificated by PCR and then connected with pUCm-T plasmid, which was transduced into pcDNA3.1 after introduction of HindⅢ and EcoRⅠ sites.The recombinant plasmid pcDNA3.1-CTLA-4 was transfected into 293T cells by using liposome.Antibiotic G418 was used to select the 293T cells that stably expressed CT-LA-4.The expression of CTLA-4 and its location were determined by quantitative PCR and immun -ofluoresence assay.Results Once activated, the blood-derived T cells expressed CTLA-4 tran-scripts.Double enzyme digestions with HindⅢ and EcoR-I demonstrated that the sequence of CTLA-4 in the recombinant plasmid was correct.RT-PCR and immuno-fluoresence assay revealed that 293T cells could stably express CTLA-4 protein on the cell membrane.Conclusions The recombinant eukaryotic expression vector pcDNA3.1-CTLA-4 was successfully constructed.CTLA-4 was stably expressed on the cell membrane of 293T cells.The present study lays a foundation for CTLA -4-relat-ed studies.
