中文摘要：

目的 构建含有人GPx-1基因Pro198Leu多态的真核表达载体,为深入研究GPx-1基因Pro198Leu多态在相关疾病中的作用与机制提供依据.方法采用全基因合成的方法合成含多态位点的人GPx-1基因cDNA片段,同时在基因5′端和3′端分别引入BamHⅠ和NotⅠ酶切位点,通过限制性内切酶酶切目的 片段和真核表达载体pEGFP-N3,再用T4 DNA连接酶将含有目的 基因的片段定向插入到载体pEGFP-N3真核人巨细胞病毒启动子下游.连接产物转化至DH5α中.挑取克隆、提取质粒进行鉴定.将hGPx-1-198Leu真核表达载体转染HEK293细胞,观察转染效率,并采用RT-PCR检测转染重组载体后细胞中GPx-1的mRNA表达水平.重组载体转染H9C2细胞,观察补硒之后的GPx-1表达情况.结果 获得目的 基因cDNA片段全长869bp,PCR和测序鉴定载体中含有人GPx-1-198Leu基因cDNA.重组载体转染HEK293细胞后检测到GPx-1的mRNA水平比未转染组及空质粒转染组明显升高,为深入研究GPx-1基因Pro198Leu多态在相关疾病中的作用与机制奠定了基础.H9C2细胞在转染后GPx-1蛋白水平升高并且随着硒浓度的升高而升高.结论 成功构建了含Pro198Leu多态位点的hGPx-1真核表达载体,并且也插入了保证该基因有效表达的硒代半胱氨酸插入序列.

英文摘要：

Objective To construct and identify a recombinant hGPx-1-198Leu cDNA eukaryotic expression vector in order to lay foundation for studying the function of the mutated human GPx-1 gene. Methods First, we synthesized the mutated human GPx-1 cDNA. Meanwhile, two restriction endonucleases BamH I and Not It sites were introduced in 5＇and 3＇ ends. Second, the GPx-1 cDNA and pEGFP-N3 plasmid were digested by BamH T and Not It restriction endonucleases, and then the CoPx-1 eDNA was directly inserted into the downstream of the human cytomegalovirus promoter of the pEGFP-N3 plasmid by T4 DNA ligase. Third, the ligation products were transformed into DH5a; the positive colonies that survived in the LB4- Kanamycin medium were selected, and then plasmid DNA was abstracted and identified by polymerase chain reaction （PCR） and sequencing. The hGPx-1- 198Leu eukaryotic expression vector was transfected into HEK293 cells, and the transfection efficiency was observed by fluorescence microscope. H9C2 cells were also transfected with hGPx-l-198Leu construct and then interfered with sodium selenite. Results The total length of the synthesized gene products was 869bp. Both PCR and sequencing analysis showed that the mutated GPx-1 gene was successfully inserted into the pEGFP-N3 plasmid, which laid foundation for further studying the function of Pro198Leu polymorphism of GPx-1 gene. The expression level of GPx-1 in the H9C2 cells transfected with hGPx-l-198Leu construct was significantly higher than that of the other two group cells; the higher the concentration of selenium, the higher the level of GPx-1 protein. Ooncltlsion The hGPx-1-Pro198Leu eukaryotic expression vector was successfully constructed and the selenocystenine insertion sequence was also included in the vector, which can make GPx-1 express successfully.