摘要
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 membraneanchored trimer. The interaction of CD40L with its receptor, CD40, plays a pivotal role in the immune surveillance and antitumor immunity. In this study, the extracellular domain of human CD40 (sCD40L, Tyr45 to Leu261) was expressed in expression vector pET30a with or without addition of an isoleucine zipper at the Nterminus. The constructs were introduced into E coli BL21 (DE3) strain for expression. The corresponding recombinant proteins, rhsCD40L and IZrhsCD40L, were purified by Ni2+chealating chromatography, and confirmed by SDSPAGE analysis followed by Western blotting. The fusion protein IZrhsCD40L showed a higher percentage of trimeric formation than rhsCD40L as revealed by NonSDSPAGE analysis. The biological activity of IZrhsCD40L was increased by 10fold compared with that of rhsCD40L on proliferation of B cells and apoptosis of XG2 cell line.
出处
《复旦学报(自然科学版)》
CAS
CSCD
北大核心
2003年第4期564-569,575,共7页
Journal of Fudan University:Natural Science