| 摘要: |
| ［摘要］ 目的 制备用于人KIM-1基因mRNA实时荧光定量PCR检测的标准品质粒。方法 通过PCR扩增出目的片断，纯化PCR产物与pMD18-T载体连接，转化宿主菌E.coli DH5α，获得阳性克隆；通过PCR扩增鉴定和测序分析，确认重组质粒完整正确。提取重组质粒测定DNA浓度后，10倍倍比稀释，建立KIM-1质粒标准品。结果 KIM-1基因目的片段成功重组至pMD18-T载体上，扩增目的片段长度为131 bp，测序结果证实所构建的质粒序列与NCBI基因库KIM-1序列性一致。结论 成功构建KIM-1基因实时荧光定量PCR标准品质粒。 |
| 关键词: KIM-1基因 实时荧光定量PCR 重组质粒 标准品 |
| DOI：10.3969/j.issn.1674-3806.2016.08.04 |
| 分类号:R 346 |
| 基金项目:广西自然科学基金资助项目（编号：2012GXNSFBA053084）；广西卫计委科研课题（编号：Z2013403） |
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| Constructing a plasmid standard for detecting human KIM-1 gene mRNA with real-time fluorescence quantitative PCR |
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CHEN Zhi-zhong, QING Ji-lin, YANG Wen-hui, et al.
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Constructing a plasmid standard for detecting human KIM-1 gene mRNA with real-time fluorescence quantitative PCR
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| Abstract: |
| ［Abstract］ Objective To construct the plasmid recombinant plasmid pMDl8-KIM-1 as a standard for detecting the mRNA expression of KIM-1 by real-time fluorescence quantitative PCR.Methods A 131 bp DNA segment of KIM-1 gene cDNA was amplified by PCR, and purified KIM-1 PCR fragments from RT-PCR cloned into pMDl8-T vector, then transformed into bacterium DH5α. The recombinant plasmid DNA extracted from positive clones was identified by PCR amplification and sequence determination. Positive clones were purified and accurately quantified, and a 10-fold serial dilution of the recombinant plasmid DNAs was used as the calibrator.Results The target segment was successfully recombined into pMDl8-T vector, and the length of the amplified target fragment of KIM-1 was 131 bp. The objective gene sequence was in accordance with that provided by the NCBI genbank.Conclusion The recombined standard plasmid for KIM-1 gene real-time PCR analysis is constructed successfully. |
| Key words: KIM-1 gene Real-time fluorescence quantitative PCR Recombinant plasmid Reference standards |
