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人PIF1全长基因及其截短体真核表达载体的构建及鉴定 被引量：1

Construction and identification of the human PIF1 gene full-length and its truncated eukaryotic expression vector
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摘要 目的构建人PIF1基因5端、3端及全长基因的真核表达载体。方法从pCR2.1-TOPO-PIF1原始质粒中,采用PCR方法扩增目的基因PIF1,PIF1N及PIF1C,将目的基因和目的载体pcDNA3.1(-)分别酶切;纯化酶切产物后定向连接,并将其转化大肠杆菌DH5α,对生长出的克隆行特异限制性内切酶酶切鉴定,鉴定为阳性的克隆送样测序。结果凝胶成像结果显示重组基因pcDNA3.1(-)-PIF1,pcDNA3.1(-)-PIF1N及pcDNA3.1(-)-PIF1C酶切后条带大小分别为1 926,540,1 428bp。结论成功构建hPIF1基因5端、3端及全长基因表达载体,分别为pcDNA3.1(-)-PIF1N,pcDNA3.1(-)-PIF1C及pcDNA3.1(-)-PIF1。 Objective To construct human pcDNA3.1（-）-PIF1,pcDNA3.1（-）-PIF1N and pcDNA3.1（-）-PIF1C eukaryoutic expressing plasmid.Methods The aim fragments（PIF1,PIF1N and PIF1C） were PCR-amplified,digested by Hind Ⅲ and XhoⅠ,and ligated into purified pcDNA3.1（-） vector.These recombinant plasmids were transfected into E.coli DH5α by means of heat shock.The recombinant plasmid was identified by digestion,and the positive clonings were identified by DNA sequence analysis.Results Gel imaging results showed that the digested band sizes of the recombinant pcDNA3.1（-）-PIF1,pcDNA3.1（-）-PIF1N and pcDNA3.1（-）-PIF1C were 1926bp,548bp and1428bp.Conclusion The recombinant pcDNA3.1（-）-PIF1 N,pcDNA3.1（-）-PIF1C and pcDNA3.1（-）-PIF1 plasmid is constructed successfully,which lays the foundation for further study on the structure and function of human PIF1.
作者 王攀 赵德根 徐涛 顾永清
机构地区 石河子大学医学院生物化学教研室
出处 《中华实用诊断与治疗杂志》 2012年第5期430-432,共3页 Journal of Chinese Practical Diagnosis and Therapy
基金 国家自然科学基金(30960093) 国家自然科学基金(301160183) 教育部留学人员科技活动项目启动经费(2009LX005) 石河子大学高层次人才启动项目(RCZX200920)
关键词 解螺旋酶 人PIF1 质粒构建 pcDNA3.1(-) Helicase human PIF1 plasmid constructed pcDNA3.1（-）
分类号 R346 [医药卫生—基础医学]
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参考文献8

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中华实用诊断与治疗杂志
2012年 第5期

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