Tian Renli, Yin Xiaotao, Wang Wei, Lin Xiaoliang, Zhu Xiaoming, Xu Yuanji, Yan Jinqi, Zhang Wei, Gao Jiangping, Yu Jiyun
Department of Urology, General Hospital of PLA, Beijing 100853; Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 May;30(5):476-9, 484.
To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purity the CT40L fusion protein and verify its antigenicity.
Gene sequences of Coxsackie and adenovirus receptor (CAR), bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a(+) to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 (DE3) and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA.
The recombinant expression vector was verified correct by double digestion with Nco I and EcoR I. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity.
The prokaryotic expression plasmid pET42a-Ct40L was successfully constructed and expressed in E. coli, and the purified fusion protein was proved to have a good antigenicity.
构建CT40L的原核表达质粒，在大肠杆菌中表达目的蛋白，纯化CT40L融合蛋白并验证其抗原性。
在GenBank中查找柯萨奇病毒和腺病毒受体（CAR）、噬菌体T4纤维蛋白和小鼠CD(40)L的基因序列。然后将三个分子的功能域连接形成融合序列，并对其进行原核表达优化。将优化后的序列克隆到原核表达载体pET42a(+)中，构建重组表达载体pET42a-CT40L。将重组载体转化到BL21(DE3)中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导融合蛋白CT40L/GST表达。然后用GST亲和层析法对融合蛋白进行纯化，并用蛋白质免疫印迹法（Western blotting）和酶联免疫吸附测定（ELISA）鉴定其免疫活性。
经Nco I和EcoR I双酶切验证重组表达载体正确。IPTG诱导后，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示融合蛋白的相对分子质量约为78 kDa，纯化后的蛋白纯度达到90%。蛋白质免疫印迹法和酶联免疫吸附测定表明纯化后的融合蛋白具有有效的抗原性。
成功构建了原核表达质粒pET42a-Ct40L并在大肠杆菌中表达，纯化后的融合蛋白具有良好的抗原性。
