Ⅱ型神经纤维瘤基因突变型的构建及生物学功能研究

目的 构建Ⅱ型神经纤维瘤(NF2)抑癌基因546位点突变的真核表达载体,并在大鼠神经鞘瘤细胞(RT4)中诱导表达.方法 用定点突变法将pEGFP-N1-NF2第546位异亮氨酸(Ⅱe)突变为蛋氨酸(Met)从而构建NF2突变子pEGFP-N1-NF2~(△Ⅱe546Met),经酶切和测序鉴定后用脂质体法将其转染至RT4细胞,同时设pEGFP-N1-NF2转染组做为对照,Westem blot法检测细胞pEGFP-NF2蛋白的表达,MTT法检测转染后细胞的增殖情况. 结果 酶切测序证实定点突变成功,Western blot结果显示RT4细胞能表达pEGFP-NF2蛋白,pEGFP-N1-NF2~(△Ⅱe546Met)转染组RT4细胞抑制率低于pEGFP-N1-NF2转染组,差异有统计学意义(P<0.05).结论 成功构建了1个能够在RT4细胞中表达的单个点突变的NF2基因真核表达载体.

Construction and characterization of mutants in the neurofibromatosis type 2 tumor suppressor gene

Objective To construct the eukaryotic expression vector of mutants in the neurofibromatosis type 2 (NF2) tumor suppressor gene and observe its expression in rat schwannoma cell line RT4. Methods Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Ile 546 in pEGFP-N1-NF2 into Met to construct the muton of pEGFP-N1-NF2~(△Ⅱe546Met). After lipofectin-mediated transient transformation of RT4 with the plasmids containing the mutation and the one without mutation, respectively, the mRNA and protein expression levels of NF2 were determined using fluorescence imaging and Western blotting. The cell proliferation was determined by the MTT assay. Results DNA sequence analysis confirmed the success of site-directed mutagenesis and Western blotting showed that pEGFP-NF2 protein could be expressed in the RT4 cells. The RT4 cell inhibition rate in the pEGFP-N1-NF2~(△Ⅱe546Met) transfection group was statistically lower than that in the pEGFP-N1-NF2 transfected group (P<0.05).Conclusion The recombinant plasmids pEGFP-N1-NF2~(△Ⅱe546Met) has been successfully constructed with efficient expressions in RT4.