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pEGFPN3,再用T4DNA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。
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.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第2期152-156,共5页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30972557)~~
作者简介
王素琴（1985-）,女（汉族）,在读博士. 研究方向：克山病发病的分子机制. E-mail： wangsuqin.198504@stu.xjtu.edu.cn
