摘要
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 express the carbohydrate recognition domain （CRD） of Balb/C mouse mannan binding lectin A （MBL-A） in E.coli. Methods The target gene fragment was obtained by PCR from the plasmid pmMBL-A harboring mouse MBL-A gene. The PCR product was recombined with the prokaryotic expression vector pET-41a （＋） and the resulting recombinant plasmid was identified by PCR, restriction analysis and sequencing before transformation into E.coli BL21 （DE3） cell for expression of the target protein. After washing and renaturation, the protein was purified on GST-Tag purification resins and analyzed by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay （ELISA）. Results A DNA fragment of about 450 bp was amplified by PCR and the recombinant plasmid pET41a-mMBL-A-CRD was constructed by linking the fragment with pET41a（＋） vector. The result of restriction enzyme analysis and sequencing of the selected clones were consistent with those by computer analysis. The recombinant vector was expressed in E.coli BL21 （DE3）, and the expressed protein existed mainly as inclusion bodies, whose relative molecular mass was about 47 000 by SDS-PAGE analysis. After washing, renaturation and purification, the purity of recombinant protein was about 90%. Western blotting suggested immunoreactivity of the purified protein with anti-GST antibody, and its sugar binding activity was verified by ELISA. Conclusion We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2009年第2期267-270,共4页
Journal of Southern Medical University
基金
广东省自然科学基金研究团队项目(015003)
关键词
甘露聚糖结合凝集素
糖识别域
原核表达
天然免疫
mannan binding lectin
carbohydrate recognition domain
prokaryotic expression
innate immunity
