Tian Renli, Zhu Jingying, Yin Xiaotao, Wang Wei, Lin Xiaoliang, Xu Yuanji, Yan Jinqi, Zhang Wei, Gao Jiangping, Yu Jiyun
Department of Urology, PLA General Hospital, Beijing 100853, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Nov;29(11):1200-4.
To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity.
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-1-mCD40. The expression vector was transformed into E.coli BL21 (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.
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.
The prokaryotic expression plasmid pGEX-6P-1-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.
构建小鼠CD40（mCD40）胞外区的原核表达质粒，在大肠杆菌中表达mCD40/GST重组蛋白，纯化该重组蛋白并鉴定其抗原性。
从细胞系DC2.4中通过PCR扩增小鼠CD40的胞外区，然后将其克隆到原核表达载体pGEX-6P-1中，构建重组表达载体pGEX-6P-1-mCD40。将该表达载体转化至大肠杆菌BL21（DE3）中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导融合蛋白mCD40/GST表达。通过琼脂糖4B纯化融合蛋白。然后通过蛋白质免疫印迹法（Western blotting）和酶联免疫吸附测定（ELISA）验证纯化后的mCD40/GST蛋白的抗原性。
DNA测序验证PCR产物与GenBank上小鼠CD40的序列一致。通过双酶切成功鉴定重组质粒。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示IPTG诱导的融合蛋白相对分子质量为45 000。蛋白质免疫印迹法和酶联免疫吸附测定表明纯化后的mCD40/GST蛋白具有良好的抗原性。
成功构建了原核表达质粒pGEX-6P-1-mCD40。在转染该质粒的大肠杆菌BL21（DE3）中，通过IPTG诱导表达了mCD40/GST融合蛋白。纯化后的mCD40/GST融合蛋白具有高抗原性，为后续CD40的研究提供了有力支持。
