异亮氨酸拉链结构对可溶性CD40L生物学活性的影响

Effect of Isoleucine Zipper Motif on the Biological Activity of Human Soluble CD40L

CD40配基(CD40L)为TNF超家族中的一员,瞬时表达于激活的CD4+的T淋巴细胞上.自然状态下膜上三聚体的CD40L通过和CD40受体(CD40R)结合,在免疫监视和肿瘤免疫中发挥作用.以PCR方法从pcDNA3 CD40L质粒上扩增得到编码CD40L基因的胞外部分sCD40L,并将该片段插入大肠杆菌表达质粒pET30a上,构建得到大肠杆菌表达质粒pET30a sCD40L;同时,在sCD40L的N端融合了异亮氨酸拉链(IsoleucineZipper,IZ)结构,构建得到表达质粒pET30a IZ sCD40L.表达质粒在大肠杆菌中表达,得到可溶性的重组人CD40L和IZ CD40L.SDS PAGE分析结果表明重组人rhsCD40L和IZ rhsCD40L在相应分子质量位置都有明显的条带产生,并得到Westernblotting的确认.非SDS PAGE电泳结果显示,异亮氨酸拉链结构有利于rhsCD40L蛋白形成聚合体结构.B细胞增殖实验和人骨髓瘤细胞株XG2凋亡实验结果都表明,异亮氨酸拉链结构提高了rhsCD40L的生物学活性.

CD40 Ligand (CD40L), a member of TNF superfamily, is expressed transiently on the surface of CD4+ T cells upon activation. It exists as a membraneanchored trimer. The interaction of CD40L with its receptor, CD40, plays a pivotal role in the immune surveillance and antitumor immunity. In this study, the extracellular domain of human CD40 (sCD40L, Tyr45 to Leu261) was expressed in expression vector pET30a with or without addition of an isoleucine zipper at the Nterminus. The constructs were introduced into E coli BL21 (DE3) strain for expression. The corresponding recombinant proteins, rhsCD40L and IZrhsCD40L, were purified by Ni2+chealating chromatography, and confirmed by SDSPAGE analysis followed by Western blotting. The fusion protein IZrhsCD40L showed a higher percentage of trimeric formation than rhsCD40L as revealed by NonSDSPAGE analysis. The biological activity of IZrhsCD40L was increased by 10fold compared with that of rhsCD40L on proliferation of B cells and apoptosis of XG2 cell line.