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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 vector containing the extracellular domain of mouse CD40 (mCD40) molecule and express in E. coli. The mCD40 / GST fusion protein was purified and the activity of the antigen was identified. Methods The mCD40 gene was cloned from mouse DC2.4 cell line and cloned into prokaryotic expression vector pGEX-6P-1. The recombinant plasmid pGEX-6P-1-mCD40 was constructed and transfected into E. coli BL21 (DE3) The mCD40 / GST fusion protein was induced by IPTG induction and the recombinant protein was purified using GST agarose gel. The purified recombinant protein was identified by Western blot and indirect ELISA for its antigenic activity. Results The PCR products were identical to those of the extracellular domain of mouse CD40 published by GenBank. The recombinant plasmid was verified by restriction enzyme digestion. SDS-PAGE analysis showed that the molecular weight (M r) was 45 000 recombinant protein; Western blot and ELISA confirmed that the purified mCD40 / GST protein can react with specific antibodies. Conclusion The prokaryotic expression vector pGEX-6P-1-mCD40 was successfully constructed and the soluble expression of mCD40 / GST fusion protein was achieved by prokaryotic expression system and proved to be highly antigenic.