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目的:构建pRetro-On/LTβR载体,获得可表达淋巴毒素β受体((lymphotoxin beta receptor,LTβR)的Jurkat细胞,探讨LTβR在T细胞凋亡中的作用。方法:应用PCR技术扩增LTβR cDNA片段,将PCR产物纯化后定向连接入pRetro-On载体;重组质粒转入大肠杆菌,LB-Amp培养基筛选阳性克隆,经质粒抽提、纯化、NotⅠ和BamHⅠ双酶切、测序,验证pRetro-On/LTβR载体的正确构建;通过脂质体转染T细胞传代系Jurkat细胞;强力霉素(doxycycline,DOX)诱导后,Western blot鉴定转染细胞中LTβR蛋白的表达,流式细胞术检测细胞表面LTβR表达;以配体LIGHT刺激表达LTβR的Jurkat细胞后以annexin-V-PI双染色流式细胞法检测细胞的凋亡。结果:双酶切及测序结果证实LTβR cDNA正确插入pRetro-On质粒;Western blot和流式细胞术结果证实了LTβR在Jurkat细胞内经DOX诱导得到了表达;表达LTβR的Jurkat细胞经LIGHT刺激后凋亡增加。结论:成功构建pRetro-On/LTβR质粒,pRetro-On/LTβR在Jurkat细胞中可以表达;表达LTβR的Jurkat细胞可以通过LIGHT-LTβR途径导致细胞凋亡。
OBJECTIVE: To construct pRetro-On / LTβR vector and obtain Jurkat cells expressing lymphotoxin beta receptor (LTβR) and to explore the role of LTβR in T cell apoptosis.Methods: The LTβR cDNA was amplified by PCR The PCR products were purified and ligated into pRetro-On vector. The recombinant plasmids were transformed into Escherichia coli and positive clones were screened by LB-Amp medium. The plasmids were extracted, purified, double digested with NotⅠ and BamHⅠ, On / LTβR vector was constructed. The T lymphocyte line Jurkat cells were transfected by lipofectamine. After induced by doxycycline (DOX), the expression of LTβR protein was detected by Western blot. The expression of LTβR protein was detected by flow cytometry And the expression of LTβR was detected.LTβR-expressing Jurkat cells were stimulated with ligand LIGHT.Apoptosis was detected by annexin-V-PI double stained flow cytometry.Results: Double enzyme digestion and sequencing confirmed the correct insertion of LTβR cDNA into pRetro-On plasmid ; Western blot and flow cytometry confirmed that LTβR was expressed in Jurkat cells induced by DOX; apoptosis of Jurkat cells expressing LTβR increased after LIGHT stimulation.Conclusion: pRetro-On / LTβR plasmid was successfully constructed and p Retro-On / LTβR can be expressed in Jurkat cells; Jurkat cells expressing LTβR can induce apoptosis through the LIGHT-LTβR pathway.