人野生型MBL基因在大肠杆菌中的表达

Cloning and Expression of Human MBL Wild-type Gene inEscherichia coli

为获得人MBL蛋白,并对其功能进行初步研究,用DNA重组法构建了组氨酸标签融合原核表达质粒pET 28(b)-MBL。将重组质粒转入大肠杆菌BI21(DE3),经IPTG在37℃条件下诱导培养,利用SDS-PAGE、Westem-blot检测目的蛋白的表达,用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白,纯化的MBL浓度约为844μg/mL,为制备MBL的基因工程抗体奠定了基础。

The study is aimed at obtaining MBL protein and doing some research about its function. A recom-binant prokaryotic expression vector, pET28-MBL, was constructed by inserted the MBL gene into plasmid pET28 (b) , and after transfected it into E. coli BL21 (DE3) and induced with IPTG, recombinant products were checked by SDS-PAGE , western-blot assay and was purified by IMAC. SDS-PAGE and western-blot assay, showed that the recombinant MBL protein was expressed successfully. The expressed product was 29 kD and could be recognized by and-6His MBL antibody. It was purified by affinity chromatography. The successful expression of MBL fusion protein will provide material for further studies the function of MBL. As this work is a part of the project of mechanisms, diagnosis and therapy of MBL deficiency, it provides the basis for further research of MBL deficiency.