摘要
目的构建含小鼠CD40(mCD40)分子胞外段序列的原核表达载体,在大肠杆菌中表达,并对mCD40/GST融合蛋白进行纯化和抗原活性鉴定。方法从小鼠DC2.4细胞系中扩增目的基因mCD40并克隆至原核表达载体pGEX-6P-1,构建重组表达载体pGEX-6P-1-mCD40,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达mCD40/GST融合蛋白,并采用GST琼脂糖凝胶纯化重组蛋白。纯化后的重组蛋白经Western blot法、间接ELISA鉴定其抗原活性。结果 PCR扩增产物经测序分析与GenBank公布的小鼠CD40胞外段序列一致;重组表达载体经酶切鉴定正确;IPTG诱导后经SDS-PAGE分析证实得到了相对分子质量(M r)为45 000的重组蛋白;Western blot法和ELISA检测证实纯化的mCD40/GST蛋白能够与特异性抗体发生反应。结论成功构建了原核表达载体pGEX-6P-1-mCD40,利用原核表达系统实现了mCD40/GST融合蛋白的可溶性表达并证明其具有较高的抗原活性。
Objective To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 （ mCD40）, express the mCD40/GST recombinant protein in E. coi, purify the mCD40/GST recombinant protein and characterize its antigenicity. Methods Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-I-mCD40. The expression vector was transformed into E. coli B1_21 （DE3） and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA. Results The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity. Conclusion The prokaryotic expression plasmid pGEX-6P-I-mCD40 was constructed successfully. In E. coli BL21 （DE3） transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第11期1200-1204,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30840094)
国家高技术研究发展计划(863)(2007AA02Z451)
关键词
CD40
原核表达
蛋白纯化
活性检测
CD40
prokaryotic expression
protein purification
antigenicity identification
