CAJ | 학술논문

目的可溶性mPDL1-hIgGFc(mouse PDL1-human IgG Fc)表达载体的构建与表达,及其对诱导细胞增殖与凋亡的研究.方法以含有mPDL1基因的载体pmPDL1为模板,采用PCR的方法获得mPDL1胞外段基因,定向连接于含有hlgGFc基因片段的载体phIgGFc,构建表达载体pmPDL1-hIgGFc;采用脂质体将重组子转染CHO细胞,转染后的CHO命名为cHOp;ELISA和Western blot法检测目的蛋白的表达,流式细胞术检测CHOp培养上清对混合淋巴细胞培养(MLC)的影响.结果测序结果和酶切鉴定显示构建的表达载体pmPDL1-higGFc完全正确;ELISA和Westernblot法检测CHOp培养上清中有目的蛋白表达;流式细胞术检测表明CHOp培养上清可体外抑制小鼠MLG,并诱导活化T细胞凋亡,其效应呈剂量依赖性.结论成功构建mPDL1-hIgGFc表达载体;mPDL1-hIgGFc在体外可抑制MLC和诱导活化T细胞凋亡,具有负性调节T细胞的功能.
목적 가용성mPDL1-hIgGFc(mouse PDL1-human IgG Fc)표체재체적구건여표체,급기대유도세포증식여조망적연구.방법 이함유mPDL1기인적재체pmPDL1위모판,채용PCR적방법획득mPDL1포외단기인,정향련접우함유hlgGFc기인편단적재체phIgGFc,구건표체재체pmPDL1-hIgGFc;채용지질체장중조자전염CHO세포,전염후적CHO명명위cHOp;ELISA화Western blot법검측목적 단백적표체,류식세포술검측CHOp배양상청대혼합림파세포배양(MLC)적영향.결과 측서결과화매절감정현시구건적표체재체pmPDL1-higGFc완전정학;ELISA화Westernblot법검측CHOp배양상청중유목적 단백표체;류식세포술검측표명CHOp배양상청가체외억제소서MLG,병유도활화T세포조망,기효응정제량의뢰성.결론 성공구건mPDL1-hIgGFc표체재체;mPDL1-hIgGFc재체외가억제MLC화유도활화T세포조망,구유부성조절T세포적공능.
Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.