Zuo Da-ming, Zhang Li-yun, Lu Xiao, Chen Zheng-liang
Department of Immunology, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Feb;29(2):267-70.
To express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli.
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).
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.
We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.
在大肠杆菌中表达Balb/C小鼠甘露聚糖结合凝集素A（MBL-A）的碳水化合物识别结构域（CRD）。
通过PCR从携带小鼠MBL-A基因的质粒pmMBL-A中获得目标基因片段。将PCR产物与原核表达载体pET-41a(+)重组，所得重组质粒经PCR、酶切分析和测序鉴定后，转化至大肠杆菌BL21(DE3)细胞中表达目标蛋白。洗涤和复性后，在GST标签纯化树脂上纯化该蛋白，并通过SDS-PAGE、Western印迹和酶联免疫吸附测定（ELISA）进行分析。
PCR扩增出约450 bp的DNA片段，将该片段与pET41a(+)载体连接构建重组质粒pET41a-mMBL-A-CRD。对筛选出的克隆进行酶切分析和测序结果与计算机分析结果一致。重组载体在大肠杆菌BL21(DE3)中表达，表达的蛋白主要以包涵体形式存在，SDS-PAGE分析其相对分子质量约为47,000。经洗涤、复性和纯化后，重组蛋白纯度约为90%。Western印迹显示纯化蛋白与抗GST抗体具有免疫反应性，ELISA验证了其糖结合活性。
成功获得小鼠MBL-A CRD蛋白，为进一步研究MBL-A分子的功能奠定了基础。
