突变型和野生型MBL基因在COS7细胞中的瞬时表达

Transient expression of wild type and variant MBL genes in COS7 cells

采用PCR技术,从质粒pMBLm52、pMBLm54和pMBLm57中分别获取含CGT52TGT、GGC54GAC和GGA57GAA点突变的人甘露聚糖结合凝集素(MBL)突变体全长编码区cDNA序列,将其分别插入质粒pcDNA4/HisMax C中,构建成相应的3种真核表达载体。将各突变型MBL基因的真核表达载体和野生型MBL基因真核表达载体以脂质体法分别转染COS7细胞。72 h后,以双抗体夹心ELISA技术检测各细胞培养上清中目的蛋白的含量分别为0.980μg/ml、0.971μg/ml、0.900μg/ml和1.047μg/ml;用One-way ANOVA分析这些目的蛋白的含量无显著性差异(P>0.05)。因此,MBL结构基因的点突变并不影响MBL蛋白的合成与分泌。

The cDNA fragments of human mannan-binding lectin （MBL） containing CGT52TGT, GGC54GAC or GGA57GAA point mutations were amplified from the plasmid pMBLm52, pMBLm54 and pMBLm57 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C respectively. After confirmed by DNA sequencing, the recombinant expression vectors, pcDNA4/HisMax C-MBLm52, pcDNA4/HisMax C-MBLm54, pcDNA4/HisMax C-MBLm57 and a recombi nant expression vector containing wild type MBI. gene, pcDNA4/HisMax C MBLw, were transfected into COS7 cells by LipofectamineTM Reagent respectively. In each transfection, the concentration of the expression vectors and the number of COS7 cells were the same. 72 hours later, the expression product in each supernatant was quantitated by EI.ISA and analyzed by one way ANOVA. It was 0. 900μg/ml and 1. 047 the mass concentration of expression products in each supernatant is 0. 980 μg /ml, 0. 971 respectively and there are no obvious differences among the levels of four forms of MBI. proteins secreted by COS7 cells. Thus, it is concluded that the point mutations in the exon 1 of MBL gene might not the synthesis and secretion of MBL protein.