EPHX2基因过表达质粒的构建及意义探讨

EPHX2 gene Overexpression Plasmid: Construction and Implications

目的:构建LETO、OLETF大鼠EPHX2基因过表达重组质粒,观察其在人胚肾细胞(human embryonic kidney293T,HEK293T)中的表达,及对HEK293T细胞NF-κBp65 mRNA表达的影响。方法:提取LETO、OLETF大鼠肾脏组织总RNA,RT-PCR方法扩增编码EPHX2基因的cDNA,克隆至T-载体并测序,经亚克隆至真核表达载体pcDNA3.1/V5-His,构建两型大鼠EPHX2过表达重组质粒,酶切鉴定并测序,采用磷酸钙共沉淀法转染HEK293T细胞系。Western Blot方法检测EPHX2融合蛋白的表达,RT-PCR方法检测EPHX2基因过表达对HEK293T细胞NF-κBp65表达水平的影响。结果:(1)两个重组质粒均含有完整EPHX2 cDNA,测序证实OLETF型大鼠较LETO型大鼠EPHX2重组质粒有五个核苷酸位点的改变。(2)转染HEK293T细胞后,Western Blot证实融合蛋白成功表达;(3)TNF-α刺激增加HEK293T细胞中NF-κBp65mRNA表达;(4)EPHX2基因过表达促进TNF-α刺激后的HEK293T细胞中NF-κBp65 mRNA表达升高(P<0.05)。结论:成功构建LETO型及OLETF型大鼠EPHX2过表达重组质粒(L-EPHX2/V5、O-EPHX2/V5),可在HEK293T细胞中过表达。为进一步探讨EPHX2基因在糖尿病肾病发生发展中的作用奠定了基础。

Objective： Contrust two kinds of EPHX2 plasmids （ LETO and OLETF） and evaluate their expression and effect on NF -Kbp65 in human embryo kidney 293T cells. Methods：EPHX2 eDNA was amplified from rat （LETO and OLETF） kidney to tal RNA by RT - PCR respectively. The amplified DNA fragments were cloned into T - vector, digested with restriction enzymes, se- quenced and sub - cloned into peDNA3, l/V5 - His. The recombinant plasmids DNA were transfected into HEK293T cell with calci- um phosphate. After 48 hours, HEK293T cells were stimulated by TNF - ctor not for 24h. The expression of exogenous EPHX2 in HEK293T cells was detected by Western Blot. The expression of NF - KBp65 in mRNA level was tested by RT - PCR. Results： （ 1 ） The two recombinant plasmids contained the complete eDNA of EPHX2 and five different bases. （2） Westem Blot showed that these recombinant plasmids DNA can express in HEK293T cell. （3）The Recombinant plasmids DNA increase HEK293T cell NF - KBp65 in mRNA level significantly compared with the control group. Conclusion：EPHX2 recombinant plasmids were constructed successfully and important to explore the relation between EPHX2 and diabetic nephropathy.