PD-L2/CTLA-4融合蛋白的表达,纯化及初步功能鉴定

Expression,Purification and Activity Analysis of a New Fusion Protein as an Immune Attenuator

细胞毒性T细胞相关抗原(Cytotoxic T Lymphocyte Associated Antigen 4,CTLA-4)是体内重要的免疫负调控因子,它可与CD28竞争跟B7分子的结合,抑制T细胞的活化.程序性死亡蛋白配体2(Programmed Death Ligand-2,PD-L2)可以和其受体程序性死亡因子1(Programmed Death 1,PD-1)结合,产生的信号可以抑制TCR(T Cell Receptor)介导的T细胞增殖和细胞因子产生.为了得到一种新型的高效免疫负调控蛋白,从人基因组中克隆了CTLA-4和PD-L2胞外区,并在大肠杆菌中实现了其融合表达和纯化.ConA转化实验表明,PD-L2/CTLA-4融合蛋白能显著抑制小鼠淋巴细胞增殖,抑制率达69%.相比于CTLA-4和PD-L2分子单独使用,抑制率分别提高了23%和10%,混合淋巴实验抑制率达71%,提示其具有免疫负调控功能.

CTLA-4（Cytotoxic T Lymphocyte Associated Antigen 4）plays an essential role in negative control of immune response in vivo,it competitively inhibits the binding of B7 and CD28,as a consequence,down-regulate pathogenic T cell proliferation.PD-L2（Programmed Death Ligand-2）is a second ligand for PD-1,Engagement of PD-1by PD-L2 inhibits TCR-mediated proliferation and cytokine production by T cells.Here the extracellular domain of CTLA-4and PD-L2 were subcloned and merged into a new high-efficiency negative regulator of pathogenic immune response.PD-L2/CTLA-4reconbinant protein were optimization expressed and successfully purified in E.coli.During mouse allogeneic ConA specific lymphocyte transformation tests,when PD-L2/CTLA-4was added the inhibitory rate reaches upto 69%,and was 23% and 10% higher than CTLA-4or PD-L2 alone respectively,which shows the fusion protein can supress cellular immune responses significantly in vitro,also,mouse allogeneic mixed lymphocyte reaction（MLR）shows the inhibitory rate can be reaches to 71%.