NF2基因突变体真核表达载体的构建和表达

目的构建NF2突变体真核表达载体，并观察其在人胚肾细胞HEK293中的表达。方法采用分子生物学方法构建NF2突变体，将突变体克隆入真核表达载体pEGFP—N1中，进行筛选和鉴定，然后在脂质体介导下，将重组载体转染至真核细胞HEK293中，应用Western blot法检测蛋白的表达。结果通过使用重叠延伸PCR法定位突变，从野生型NF2基因cDNA模板中扩增得到预期大小的NF2突变体条带，DNA测序证实NF2突变体序列的正确性。重组载体转染HEK293后能产生merlin蛋白。结论本研究成功构建重组载体pEGFP—N1-NF2突变体，其转染HEK293后，能有效表达于HEK293中。

Construction of mutant NF2 eukaryotic expression vector and its expression

Objective To construct mutant NF2 eukaryotic expression vector and to express the protein in HEK293 cells. Methods Mutant NF2 gene was constructed and subcIoned into eukaryotic expression vector pEGFP-NI. After identified, the recombinant vector was transfected into HEK293 cells by lipofectamine method; the expression of protein was detected by Western blotting.Results Amplification fragment of mutant NF2 gene with the expected size was obtained from wild-type NF2 DNA template by site-directed mutagenesis, and the mutant NF2 was identified by DNA sequencing. After transfection of pEGFP-N1-NF2 into HEK293 cells, Western Blot detected the expression of merlin protein. Conclusion The mutant NF2 cDNA has been constructed into the eukaryotic expression vector successfully, and the merlin protein is expressed efficiently in HEK293 cells.