人甘露聚糖结合凝集素相关丝氨酸蛋白酶2N端片段的原核表达

Prokaryotic expression of the N terminal fragment of mannan-binding lectin associated serine protease-2

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2(MASP2)N端片段。方法应用PCR技术从含人MASP2 cDNA的质粒pGEM-MASP2中扩增MASP2-N端基因片段,将其克隆至pGEX4T-1原核表达载体,转化BL21(DE3)感受态菌诱导表达MASP2-N端蛋白,通过GSTrap亲合层析柱纯化目的蛋白,以SDS-PAGE和Western blot进行鉴定,并以ELISA分析了目的蛋白与MBL-CLR结合的活性。结果从pGEM-MASP2中扩增得到约840 bp的基因片段,构建成重组载体经酶切出现约4900 bp和840 bp片段,测序结果与预期的完全一致。纯化蛋白经SDS-PAGE可见Mr 60000蛋白带,该蛋白可与抗GST抗体反应并能与重组人MBL-CLR蛋白结合。结论获得了表达人MASP2 N端片段的大肠杆菌菌株和重组人MASP2N端片段/GST融合蛋白,为MASP2分子的进一步研究提供了条件。

Objective To express the N-terminal fragment of human mannan-binding lectin （MBL） associated serine proteases-2 （MASP2-N） in E, coli,Methods The target sequence in pGEM-MASP2 plasmid that contains human MBL-MASP2 eDNA was amplified by PCR, and inserted into prokaryotie expression vector pGEX4T-1, The recombinant expression vector was identified by restriction mapping and sequencing, and then transformed into E. coli BL21 （DE3） colls, The expressed product was purified by GSTrap immobilized metal affinity chromatography （IMAC） and identified by SDS-PAGE and Western blot assay, Binding activity of the expression product to the collagen-like region of human MBL （MBL-CLR） was analyzed by an indirect enzyme-linked immunosorbent assay （ELISA）, Results The DNA fragment of 840 bp, which encode the N-terminal region of human MASP2, was amplified from pGEM-MASP2 plasmid and the recombinant expression vector, pGEX4T-MASP2-N, was constructed, whose restriction maps and sequence were consistent with those expected. The component of Mτ 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 in the indirect ELISA. Conclusion The prokaryotie cell strain that expresses efficiently the recombinant human MASP2-N （rhMASP2-N） protein and the purified rhMASP2-N protein are successfully obtained, which provides the basis for further research of MASF2 molecule.