【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p MD18-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p MD18-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。更多还原

【Abstract】 Objective To construct the plasmid recombinant plasmid p MDl8-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 c DNA was amplified by PCR,and purified KIM-1 PCR fragments from RT-PCR cloned into p MDl8-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 p MDl8-T vector,and the length of the amplified target fragment of KIM-1 was131 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.更多还原