截断型人可溶性CD40L的表达与纯化

Expression and Purification of Soluble Human CD40L Truncate

目的 表达相对分子质量 180 0 0的截断型人可溶性CD4 0L。方法 针对人CD4 0L(Glu10 8 Leu2 6 1)设计引物 ,以全长人CD4 0L为模板 ,扩增目的基因 ,经pGEM T中间载体进一步连接到pQE 4 0载体 ,构建pQE sCD4 0L表达载体。转化E .coliM15后经IPTG诱导表达 ,分离、洗涤包涵体 ,并利用Ni NTA亲和层析纯化目的蛋白 ,经复性后检测对骨髓瘤细胞SP2 0细胞株的抑制作用。结果 克隆的目的基因为 4 80bp ,经测序与文献报道一致 ,所构建的菌株 (M15 pQE sCD4 0L)经诱导表达目的蛋白量为 2 6 7% ,相对分子质量为 180 0 0 ,纯化后蛋白纯度为96 5 % ,对SP2 0细胞有生长抑制作用。结论 原核表达的截断型人可溶性CD4 0L具有抑制肿瘤细胞生长作用。

Objective To express soluble human CD40L truncate with a relative molecular weight of 18 000.Methods Design primers according to the sequence of CD40L (Glu1 08-Leu261) and amplify the goal gene using the full-length of human CD40L as a template.Insert the amplified gene into pGEM-T vector then subculture to plasm id pQE-40 for the construction of expression vector pQE/sCD40L.Transform the re combinant plasmid pQE/sCD40L into E.coli M15 and express under induction of IPTG.Isolate and wash inclusion body and purify goal protein by Ni-NTA affinity chromatography.Re-naturalize the goal protein and detect its inhibiting effect on myeloma cell SP2/0 strain.Results The sequence of cloned goal gene,at a length of 480 b p, was consistent with that reported.The goal protein expressed in the constructed strain M15/pQE/sCD40L contained 26.7% of total somatic protein.The expressed pr otein,with a relative molecular weight of 18 000,reached a purity of 96.5% afte r purification and showed significant inhibiting effect on the growth of SP2/0 cells.Conclusion The soluble human CD40L truncate expressed in prokar yotic cells showed inhibiting effect on the growth of tumor cells.