| 摘要: |
| 目的：分别构建抑癌基因NF2的42位和47位点突变的2种真核表达载体，并在大鼠神经鞘瘤细胞RT4-D6P2T中诱导表达。方法：用定点突变法分别将pcDNA3.1-NF2第42位赖氨酸(Lys)突变为脯氨酸(Pro)，第47位苯丙氨酸(Phe)突变为亮氨酸(Leu)构建2种NF2突变子（pcDNA3.1-NF2ΔLys42Pro和pcDNA3.1-NF2ΔPhe47Leu），将3 种NF2质粒用脂质体法分别转染RT4细胞，RT-PCR和Western印迹法分析NF2基因及蛋白表达，MTT法检测转染后RT4细胞的增殖情况。结果：测序证实定点突变成功，构建的重组真核表达载体pcDNA3.1-NF2ΔLys42Pro和pcDNA3.1-NF2ΔPhe47Leu均能表达相应的蛋白和mRNA，这2种NF2突变体对RT4细胞抑制率明显低于野生型NF2组，差异有统计学意义(P<0.05)。结论：成功构建了2种能够在RT4中表达的单个点突变的NF2基因真核表达载体。 |
| 关键词: NF2 点突变 真核表达载体 |
| DOI：10.3724/SP.J.1008.2008.00546 |
| 投稿时间：2008-03-05修订日期：2008-04-11 |
| 基金项目:上海市科委启明星跟踪计划（04qmh1414），上海市科委自然科学基金（06ZR14064）. |
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| Construction of eukaryotic expression vectors of two NF2 mutants and their expression in rat schwannoma cell line |
| HE Hua1,PENG Biao2,BIAN Liu-guan1*,SUN Qing-fang1,SHEN Jian-kang1,HE Zi-jian3,LIN Yi-hai4 |
| (1.Department of Neurosurgery，Ruijin Hospital，Shanghai Jiaotong University School of Medicine，Shanghai 200025,China;2.Department of Neurosurgery,The First People’s Hospital of Guangzhou,Guangzhou 510180;3.Department of Neurosurgery,Minhang Central Hospital of Shanghai,Shanghai 201100;4.Department of Neurosurgery,Central Hospital of Taizhou,Taizhou 318000) |
| Abstract: |
| Objective：To construct the eukaryotic expression vectors of two NF2 mutants (neurofibromatosisⅡ) and to study their expression in rat schwannoma cell line (RT4).Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Lys42 in NF2 into Pro in pcDNA3.1-NF2ΔLys42Pro， and convert the codons for Phe 47 into that of Leu.RT4 cells were transiently transfected with the 3 kinds of plasmids containing the mutations and wide-type NF2 via lipofectin separately,then the expression levels of NF2 mRNA and protein were determined by RT-PCR and Western blotting in 3 groups.The cell proliferation was determined by the MTT after transfection.Results: DNA sequence analysis demonstrated the two-step mutagenesis was successful and the two plasmids of pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu were obtained,both of the transfectants could produce merlin protein and mRNA.The two mutants had a significantly lower inhibitory rate for RT4 cells compared with wide-type NF2 (P<0.05).Conclusion: The recombinant plasmids pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu have been successfully constructed and they can be efficiently expressed in RT4 cells. |
| Key words: NF2 point mutation eukaryotic expression vector |
