可溶性mPDL1-hIgGFc表达载体构建、表达及对诱导细胞增殖与凋亡的影响

Vector construction and expression of soluble mPDLI-hIgGFc and its effect on the proliferation and apoptosis of cells in vitro

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hIgGFc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Western blot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MLC，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。

Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apoptosis of cells in vitro. Methods The extracellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phlgGFc vector. The recombinant pmPDLl-hlgGFc was transfected into CHO cells by LipofectAMINE ^TM2000, and the transfected ceils were named as CHOp. The expression of mPDLI-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 Flowmetry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by Hind Ⅲ and Kpn Ⅰindicated the recombinant plasmid pmPDLl-hIgGFc was successfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDLI-hIgG- Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activated T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hlgGFc protein could inhibit lymphocyte proliferation and induce the apoptosis of the activated T cells.