重组人N端缺失MBL的原核表达及其活性研究  

Prokaryotic expression and biological activities of recombinant human MBL N-terminal deletant(rhMBL△N)

作　　者：雷鸣[1] 左大明[1] 王明永[1] 张雅妮[1] 陈政良[1] 

机构地区：[1]南方医科大学免疫学教研室,广州510515

出　　处：《免疫学杂志》2008年第3期346-349,共4页Immunological Journal

摘　　要：目的原核表达具生物学活性的重组人N端缺失甘露聚糖结合凝集素(rhMBL△N)蛋白。方法应用PCR技术,从含中国人野生型MBL cDNA的质粒pGEM-MBL中扩增rhMBL△N基因片段,插入pET43.1a载体后,转化E.coliBL21(DE3)感受态菌诱导表达rhMBL△N蛋白。应用Ni2+-NTA琼脂糖柱纯化目的蛋白,以SDS-PAGE、Western blot和ELISA进行鉴定。结果从pGEM-MBL中扩增到长约620bp的DNA片段,构建的重组表达载体经BamHⅠ和EcoRⅠ酶切后出现约7270bp和620bp的片段,测序结果与预期的完全一致。表达的重组蛋白纯化后,经SDS-PAGE鉴定为Mr97000的蛋白。ELISA证实,纯化蛋白能与抗重组人MBL抗体结合,具备配体结合活性并能与人甘露聚糖结合凝集素相关丝氨酸蛋白酶1、2的N端片段结合。结论获得了可表达rhMBL△N的菌株和具活性的rhMBL△N蛋白,为进一步研究提供了条件。Objective To express recombinant human mannan-binding lectin N-terminal deletant （rhMBLΔN） protein possessing biological activities in E. coli. Methods The rhMBLΔN gene was amplified by PCR from pGEM-MBL plasmid, and then inserted into prokaryotic expression vector pET43, la. After identification by restriction mapping and sequencing, the recombinant plasmid pET43.1a/His rhMBLΔN was transformed into E. coli BL21 （DE3） cells. The expressed product was purified by immobilized metal affinity chromatography （IMAC）, and then identified by SDS-PAGE, Western blotting, and ELISA. Results The cDNA fragment of about 620 bp was amplified from pGEM-MBL plasmid and the recombinant expression vector pET43.1a/His rhMBLΔN was constructed, whose restriction maps and sequence were consistent with those expected. A components of Mr 97 0130 in the purified recombinant product was detected by SDS-PAGE and could be recognized by anti-6His antibody. The purified recombinant product could react with the antibody against the recombinant human MBL protein and could bind to mannan and MBL associated serine protease 1/2-N proteins specifically. Conclusion The prokaryotic expression strains that express efficiently rhMBLΔN and the rhMBLΔN protein with biological activities are obtained successfully, which will help the further research of MBL molecule.

关 键 词：甘露聚糖结合凝集素 N端缺失 原核表达 活性 

分 类 号：R392.1[医药卫生—免疫学]