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目的检测白血病细胞株KG-1中Mxil基因SID和bHLHzip区突变情况并构建Mxil基因的野生型和突变型真核表达载体,观察其转染真核细胞后基因表达的情况。方法应用RT-PCR和SSCP分析KG-1细胞株Mxil基因的突变,将Mxil野生型/突变型基因cDNA插入到pDs-red2-N1质粒中,构建pDs-red2-N1/Mxil(野生型/突变型)真核表达载体,采用脂质体方法将质粒转染COS-7细胞,流式细胞仪检测红色荧光蛋白表达并计算转染效率。RT-PCR检测转染后Mxil基因的表达。结果 Mxil基因突变位于HelixⅡ区第82编码子的CAT/TAT(His/Tyr)。成功构建pDs-red2-N1/Mxil(野生型/突变型)真核表达载体,经酶切、电泳可以看到在4 700、1845 bp存在两条电泳条带,Mxil基因在COS-7细胞中得到表达,转染后COS-7细胞红色荧光蛋白表达的阳性细胞为18.07%,表达高峰出现在转染后第3天,并可持续表达6d左右。结论 Mxil基因的高度保守区域bHLHzip存在基因突变,Mxil突变基因的真核表达载体构建成功,并成功转染真核细胞COS-7。
Objective To detect the mutation of Mxil gene in the SID and bHLHzip region of leukemia cell line KG-1 and to construct the wild-type and mutant eukaryotic expression vectors of Mxil gene and to observe the gene expression after transfection of eukaryotic cells. Methods Mutations of Mxil gene in KG-1 cell line were detected by RT-PCR and SSCP. The wild type / mutant of pDs-red2-N1 / Mxil was constructed by inserting Mxil wild-type / mutant gene cDNA into pDs- Type) eukaryotic expression vector, the plasmid was transfected into COS-7 cells by lipofectamine and the expression of red fluorescent protein was detected by flow cytometry and the transfection efficiency was calculated. The expression of Mxil gene was detected by RT-PCR. Results The Mxil gene was mutated at CAT / TAT (His / Tyr) at codon 82 of Helix II region. The eukaryotic expression vector pDs-red2-N1 / Mxil (wild-type / mutant) was successfully constructed. After electrophoresis, two electrophoresis bands at 4 700 and 1845 bp were found. The Mxil gene was expressed in COS-7 cells The expression of red fluorescent protein in COS-7 cells after transfection was 18.07%. The peak of expression appeared on the 3rd day after transfection, and the expression of red fluorescent protein was about 6 days. Conclusion The gene mutation in bHLHzip is highly conserved in Mxil gene. The eukaryotic expression vector of Mxil gene was successfully constructed and transfected into COS-7 cells successfully.