大鼠ETFβ基因过表达对NADPH氧化酶基因表达的影响

Effects of Overexpression of Rat ETFβ on Gene Expression of NADPH Oxidase

目的:构建大鼠ETFβ的重组质粒并检测其在人胚胎肾293T细胞中的表达和对NADPH氧化酶的影响。方法:应用RT-PCR方法从大鼠肾脏组织总RNA中扩增出编码ETFβ的cDNA,克隆至pUM-T载体并测序,然后亚克隆至真核表达载体pcDNA3.1/V5-His,酶切鉴定后测序,测序正确后用磷酸钙共沉淀法瞬时转染HEK293T细胞,使用RT-PCR方法检测空载体组和重组质粒转染组的NADPH氧化酶各亚基mRNA水平的表达变化。结果:(1)测序结果证实PCR扩增得到编码ETFβ的cDNA序列正确;(2)磷酸钙共沉淀法转染HEK293T细胞后,ETFβ融合蛋白成功表达;(3)RT-PCR结果显示重组质粒转染组的NADPH氧化酶5个亚基中的NOX3,NOX4的mRNA表达降低(P<0.05),空载体组和重组质粒转染组之间比较NOX1,NOX2,NOX5的mRNA表达差异无统计学意义。结论:成功构建大鼠ETFβ的重组质粒,该重组质粒可在HEK293T中过表达,并且降低了NADPH氧化酶NOX3,NOX4的mRNA表达水平。

Objective：Constructing rat ETFβ cDNA in eukaryotic expression vector for testifying its expression in human embryo kidney 293T cell （ HEK 293T） and elucidating the effects of ETF on NADPH oxidase expression. Methods：Rat ETFβ eDNA was amplified from rat kidney total RNA by RT- PCR. The amplified DNA fragment was cloned into vector pUM -T, digested with restriction enzymes, sequenced, then sub - cloned into the eukaryotic expression vector pcDNA3, l/V5 - His. After restriction en- zymes digestion and sequence, the recombinant plasmid DNA was transiently transfeeted into HEK 293T cell with calcium phosphate. The expression of rat ETFI3 in 293T cells was detected by Western Blot. The expression levels of the subunits of NADPH oxidase were tested by RT - PCR. Results ： （ 1 ） Rat ETFβ eDNA with the eukaryotie expression vector was successfully constructed. （2） This recombinant plasmid DNA can express in HEK293T cell. （3）RT- PCR showed that overexpression protein ETFβdecreased the expression of NOX3, NOX4. Conclusion ：The eukaryotic expression vector containing ETFβ were constructed and it can express ETFβ in HEK 293T cell, and ETFβ participated in the genesis of NOX3 ,NOX4.