DR5胞外段基因的表达及功能初步鉴定  

Gene expression and primary functional characterization of human DR5 extracellular fragment

作　　者：陈居杲[1] 王玉刚[2] 赵昆朋[1] 谷欣[2] 黎燕[1] 马远方[1] 沈倍奋[1] 

机构地区：[1]河南大学细胞与分子免疫学实验室,河南开封475001 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《细胞与分子免疫学杂志》2007年第9期791-793,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30571697)

摘　　要：目的:获得具有生物活性的人DR5胞外段蛋白。方法:通过RT-PCR从Jurkat细胞中钓取DR5的胞外段基因(55-183位氨基酸),克隆到pGEM(-T Easy载体中,经核酸序列测定正确后,将其再次克隆入原核表达载体pET30 a中,在E.coliBL21(DE3)实现蛋白表达。通过SDS-PAGE和Western blot鉴定表达产物。ELISA法分析其与抗DR5单克隆抗体(mAb)结合能力。结果:克隆到人DR5基因的胞外段基因,测序结果和报道的一致;构建了人DR5胞外段基因的原核表达载体并实现在E.coliBL21(DE3)中大量表达;SDS-PAGE表明所表达蛋白与预期蛋白的相对分子质量(Mr)一致;Western blot及ELISA的结果表明该蛋白能与抗DR5抗体反应,竞争阻断试验表明DR5胞外段可阻断TRAIL对Jurkat细胞生长的抑制。结论:成功地制备了具有生物活性的DR5胞外区,为后续研究打下了基础。AIM： To acquire human DP,5 extracellular fragment with bioactivity. METHODS： Total RNA was prepared from Jurkat cells by Trizol. Human DP,5 extracellular fragment gene was amplified by RT-PCR, cloned into pGEM-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pET30a with a His-tag at the amino terminus and expressed in E. coli BL21 （ DE3 ）. The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. ELISA method was used to detect its binding activity to anti-DR5 monoclonal antibody （mAb） mDRA-6. RESULTS： Human DP,5 extracellular fragment gene was successfully amplified and high level expression was obtained in E. coli BL21 （ DE3 ） induced by 0. 1mmol/L IPTG. The DR5 extracellular fragment protein was identified by SDS-PAGE and Western blot analysis. ELISA results showed that the purified DP,5 could be recognized by mDRA-6. CONCLUSION： The extracellular region of DP,5 with bioactivity has been successfully expressed and purifled, which lay the foundation for further study.

关 键 词：DIL5 RT-PCR 原核表达 

分 类 号：R730.51[医药卫生—肿瘤]