人MASP1 N端片段原核表达载体的构建及其表达

目的:在大肠杆菌中表达人甘露聚糖结合凝集素(MBL)相关丝氨酸蛋白酶1(MASP1)N端片段.方法:采用PCR技术从含人MASP1 cDNA的质粒pGEM-MASP1中扩增MASP1-N端基因片段,将其插入原核表达载体pGEX4T-1,转化BL21(DE3)感受态菌诱导表达MASP1-N端蛋白,通过GSTrap亲合层析柱纯化目的蛋白,以SDS-PAGE和Westernblot进行鉴定,并以ELISA分析了目的蛋白与重组MBL-CLR、重组MBL的结合活性.结果:从pGEM-MASP1中扩增得到约860 bp的基因片段,构建成重组载体经酶切出现约4 900 bp和860 bp片段,测序结果与预期的完全一致.纯化蛋白经SDS-PAGE可见Mr60000蛋白带,该蛋白可与抗GST抗体反应并能与重组人MBL-CLR、重组人MBL蛋白结合.结论:获得了表达人MASP1 N端片段的大肠杆菌菌株和重组人MASP1 N端片段/GST融合蛋白,为MASP1分子的进一步研究提供了条件.

Prokaryotic expression of N terminal fragment of mannan-binding lectin associated serine protease-1

AIM: To express the N-terminal fragment of human mannan-binding lectin (MBL) associated serine proteases-1 (MASP1-N) in E.coli. METHODS: The target sequence in pGEM-MASP1 plasmid that contains human MBL-MASP1 cDNA was amplified by PCR, inserted into prokaryotic expression vector pGEX4T-1 and identified by restriction mapping and sequencing. The recombinant expression vector was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by GSTrap Immobilized Metal Affinity Chromatography(IMAC) and identified by SDS-PAGE and Western blot assay, its binding-activity with the collagen-like region of human MBL(MBL-CLR) and with recombinant human MBL was analyzed by an indirect enzyme-linked immunosorbent assayèELISAé. RESULTS: The DNA fragment of 860 bp, which encode the N-terminal region of human MASP1, was amplified from pGEM-MASP1 plasmid and the recombinant expression vector, pGEX4T-MASP1-N, was constructed, whose restriction maps and sequence were consistent with those expected. The component of M(r) 60 000 in the purified recombinant product was found by SDS-PAGE and could be recognized by anti-GST antibody in Western blot assay. The purified recombinant product could react with human MBL-CLR and human MBL in the indirect ELISA. CONCLUSION: The prokaryotic cell strain that expresses efficiently recombinant human MASP1-N(rhMASP1-N) protein and the purified rhMASP1-N protein were successfully obtained, which provides the basis for further research of MASP1 molecule.