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大鼠NRF-1基因的克隆、鉴定及功能分析

Keywords: NRF-1,pMD18-T,克隆

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Abstract:

目的克隆大鼠NRF-1基因并重组于载体pMD18-T,了解NRF-1基本结构和蛋白分子特征,预测可能的相关作用蛋白分子,为下一步研究NRF-1对心衰细胞能量代谢途径的作用机制提供实验材料和依据。方法通过重叠延伸PCR(SOEPCR)的方法合成目的基因序列,连接于载体pMD18-T,然后转化至感受态大肠杆菌DH5α。PCR及测序鉴定重组质粒NRF-1/pMD18-T序列正确性。并通过生物信息学软件分析NRF-1的氨基酸序列,蛋白质二级结构,修饰位点及蛋白质之间作用关系等,进行预测。结果大肠杆菌DH5α中保存的重组质粒NRF-1/pMD18-T经测序鉴定,其中的NRF-1基因序列与原始序列一致。生物信息学分析表明NRF-1的蛋白质分子量为57.28kD,分子式为C2498H3976N704O808S15,等电点为5.28,是稳定的亲水蛋白,α-螺旋和β-折叠结构较多,NRF-1与mtTFA,Akt1和Spastin等多个蛋白关系密切。结论本研究成功地克隆了NRF-1基因并重组于载体pMD18-T,为后续研究NRF-1对心衰心肌细胞能量代谢的调控机制提供了实验材料和研究思路。

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