小鼠CD25分子胞外段的原核表达载体的构建及表达鉴定  

Construction of prokaryotic expression vector of mouse CD25 extracellular domain

作　　者：徐林[1] 张凤[2] 周涯[3] 罗军敏[1] 覃明[1] 

机构地区：[1]贵州省遵义医学院免疫学教研室,贵州遵义563000 [2]复旦大学上海医学院免疫学系,上海200032 [3]贵州省遵义医学院医学物理学教研室,贵州遵义563000

出　　处：《第三军医大学学报》2010年第3期278-281,共4页Journal of Third Military Medical University

摘　　要：目的构建小鼠CD25分子胞外段的原核表达载体,并在大肠杆菌中进行表达。方法从BALB/c小鼠脾细胞中提取总RNA,利用RT-PCR方法获得CD25分子胞外段序列;将其亚克隆入原核表达载体PET-32a,构建好PET-32a-CD25胞外段,并进行酶切及测序鉴定;将PET-32a-CD25胞外段转染大肠杆菌BL21进行表达,并对表达产物进行纯化和鉴定。结果RT-PCR扩增出708 bp的CD25胞外段的基因片段;pET32a-CD25胞外段/BL21经IPTG诱导后,SDS-PAGE显示有新生的蛋白表达条带,相对分子质量约为47×103,重组蛋白用镍树脂纯化后经Western blot检测显示具有良好的免疫反应性,并且可以在体外有效刺激自体T细胞疫苗免疫小鼠脾细胞的增殖并高分泌IL-4。结论CD25胞外段蛋白在原核细胞中成功表达,纯化后的蛋白保持良好的免疫反应性。Objective To construct the prokaryotic expression vector of mouse CD25 extracellular domain and to express it in E coll. Methods Total RNA was isolated from splenocytes of Balb/c mice. The CD25 extracellular domain gene was amplified by RT-PCR and cloned into the PET-32a vector. A positive recombinant, PET-32a-CD25e, was identified by enzyme cleaving and sequencing before its expression in E. coli, and transferred into E. coli BI21 （DE3） plysS for its expression. After purification with Ni ＋ resin and renaturation in vitro, a relative molecular mass （Mr） of the interesting protein was detected by SDS-PAGE and Wes-tern blotting. Effect of the purified interesting protein on the proliferation of splenocytes from T cell vaccine-immunized syngeneic mice was detected by MTT assay. Results The cloned CD25 extracellular domain gene was identified to be functional by sequencing and expression. The purified interesting protein could significantly induce the proliferation and IL-4 secretion of splenoeytes from T cell vaccine-immunized mice in. vitro. Gonclusion Mouse CD25 extracellular domain gene can be successfully expressed in prokaryotic cells with biological activity, which lays a foundation for further relative studies.

关 键 词：CD25胞外段 原核表达 T细胞疫苗 

分 类 号：R392.13[医药卫生—免疫学] R394-33[医药卫生—基础医学]