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目的获取具有生物学活性的Balb/C小鼠血清型甘露聚糖结合凝集素(MBL-A)糖识别域(CRD)蛋白。方法应用PCR从含Balb/C小鼠MBL-A基因的质粒pmMBL-A中扩增目的基因片段,将其克隆至原核表达载体pET41a(+),在大肠杆菌BL21(DE3)中诱导表达目的蛋白。表达产物(包涵体)经洗涤和复性处理后,利用GST柱纯化,以SDS-PAGE、Westernblotting和ELISA进行分析鉴定。结果从pmMBL-A中扩增到约450bp的DNA片段,构建重组载体pET41a-mMBL-A-CRD,经酶切和测序鉴定后,将其导入大肠杆菌BL21(DE3)中表达,表达产物主要以包涵体形式存在,其相对分子质量约47000。包涵体经洗涤及复性处理,GST柱纯化后,融合蛋白的纯度达90%以上。Westernblotting显示重组融合蛋白与抗GST抗体特异性反应,糖结合试验表明表达产物具有生物学活性。结论成功获得了具有生物学活性的Balb/C小鼠MBL-ACRD蛋白,为MBL-A分子的研究奠定了一定基础。
Objective To obtain the biologically active Balb / C mouse serum-type mannan-binding lectin (MBL-A) glycoprotein domain (CRD) protein. Methods The target gene fragment was amplified by PCR from the pmMBL-A plasmid containing the MBL-A gene of Balb / C mouse and cloned into prokaryotic expression vector pET41a (+). The recombinant protein was expressed in E. coli BL21 (DE3) . The expression product (inclusion body) was washed and refolded, then purified by GST column and analyzed by SDS-PAGE, Western blotting and ELISA. Results The recombinant plasmid pET41a-mMBL-A-CRD was amplified from pmMBL-A to about 450bp. The recombinant plasmid pET41a-mMBL-A-CRD was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was introduced into E. coli BL21 (DE3) Inclusion body exists, the relative molecular mass of about 47000. Inclusion bodies were washed and refolded, and purified by GST column, the purity of the fusion protein was over 90%. Western blotting showed that the recombinant fusion protein specifically reacted with anti-GST antibody, and the carbohydrate binding assay showed that the expressed product was biologically active. Conclusion The biological activity of MBL-ACRD in Balb / C mice was successfully obtained, which laid the foundation for the study of MBL-A molecules.