重组人IZ-CD40L在大肠杆菌中的高效表达和生物学活性的鉴定

发布时间：2018-06-04 14:04

本文选题：CD40配体 + 异亮氨酸拉链　；参考：《苏州大学》2005年硕士论文

【摘要】：CD40配体(CD40 ligand,CD40L)通过与其受体CD40的相互作用在T细胞依赖的体液和细胞免疫中发挥重要的作用。本实验克隆了CD40L胞外功能区(E107-L261)基因,并在其N端融合了异亮氨酸拉链(isoleucine zipper,IZ)促使其形成三聚体活性形式,同时为了后期纯化的需要,在IZ的N端融合6个His,将所融合得到的IZ-CD40L克隆进入pET30a表达载体,转化大肠杆菌BL21(DE3)表达体系进行诱导表达。结果表明,重组蛋白IZ-CD40L以包涵体形式在大肠杆菌(BL21)中得到较高水平的表达。通过蛋白的变性、复性和HiTrap~(TM)亲和层析获得了纯度达95%以上的IZ-CD40L重组蛋白。经凝胶过滤层析和非还原SDS-PAGE分析,验证了其确系三聚体形式。激光共聚焦实验结果表明,重组蛋白IZ-CD40L可与多发性骨髓瘤细胞株XG2表面CD40分子结合,并促使其在膜表面发生聚集。体外活性实验结果表明,重组蛋白IZ-CD40L可有效促使Dentritic cell(DC)成熟,表现为DC膜表面CD83、CD86、HLA-DR分子上调表达,分泌IL-12增加,以及抗原摄取能力明显下降。
[Abstract]:CD40 ligand CD40 ligand CD40L plays an important role in T cell dependent humoral and cellular immunity by interacting with its receptor CD40. In this study, the CD40L E107-L261) gene was cloned and fused with isoleucine zipperidin IZ at its N-terminal to promote the formation of trimer activity. Six Hiss were fused at the N-terminal of IZ, and the fused IZ-CD40L was cloned into pET30a expression vector and transformed into E. coli BL21DE3) expression system for induction expression. The results showed that the recombinant protein IZ-CD40L was expressed in E. coli BL21 in the form of inclusion body. IZ-CD40L recombinant protein with purity of more than 95% was obtained by denaturation, renaturation and affinity chromatography. The results of gel filtration chromatography and non-reductive SDS-PAGE showed that it was a trimer. The results of confocal laser scanning showed that the recombinant protein IZ-CD40L could bind to the CD40 molecules on the surface of multiple myeloma cell line XG2 and promote its aggregation on the membrane surface. The results of in vitro activity test showed that recombinant protein IZ-CD40L could effectively promote the maturation of Dentritic cells. The expression of HLA-DR on DC membrane was up-regulated, the secretion of IL-12 was increased, and the ability of antigen uptake was obviously decreased.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【共引文献】