MBL突变体在CHO细胞中表达及其产物分析

Expression of GGC54GAC MBL mutant in CHO cells and analysis of expression product

采用PCR技术,从质粒pMBLm54中获得含GGC54GAC点突变的甘露聚糖结合凝集素(MBL)基因,将其插入真核表达载体构建重组表达载体pcDNA4/HisMax C-MBLm54,DNA测序验证序列正确后将其电转染入CHO细胞。以Zeocin筛选并维持培养转染后CHO细胞,获得2株稳定高效表达目的蛋白的单克隆细胞株。采用Ni^(2+)-NTA agarose层析柱纯化表达产物,获得54Asp突变MBL蛋白,SDS-PAGE和Western blot分析表明,54Asp突变MBL蛋白主要为相对分子质量为60000的成分,而重组野生型MBL蛋白则主要是相对分子质量为200000和>200000的分子。配体结合试验发现,天然MBL和重组野生型MBL能与甘露聚糖结合,而54Asp则否。结果表明,54Asp突变MBL蛋白不能形成正常的MBL结构单位和寡聚体,并进而影响其配体结合活性。

The mannan-binding lectin （MBL） gene containing GGC54GAC point mutation was amplified from the plasmid pMBLm54 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. Confirmed by DNA sequen cing, the recombinant expression vector, pcDNA4/HisMax CMBLm54, was transfected into Chinese-hamster ovary （.CHO） cells by electroporation. Zeocin was used to select transfected CHO cells and two monoclonal cell lines that express the objective protein stably and efficiently were gained. The recombinant protein, 54Asp mutant MBL protein, was purified by Ni^2＋ -NTA agarose chromatography from CHO-MBLm54 cell culture supernatant. Analyzed by SDS-PAGE and Western blot, it was found that the 54Asp mutant MBL protein appears mainly at the site of （Mr） 60 000 and the recombinant wild type MBL protein, at the site of （Mr） 200 000 and above 200 000. Analyzed by the ligand-binding assay, it was demonstrated that the plasma-derived natural MBL and the recombinant wild type MBL proteins can efficiently bind with mannan, but the 54Asp mutant MBL protein had no this activity. The data showed that 54Asp mutant MBL protein can＇t form normal structural units and oligomers, which affects its ligand-binding activity.