MRP基因定量RT-PCR内标准DNA模板和RNA模板的构建

Construction of the internal standard RNA and DNA templates for MRP gene quantitative RTPCR analysis

目的：构建定量逆转录聚合酶链反应（ＲＴＰＣＲ）的内标准ＤＮＡ模板和ＲＮＡ模板，定量检测多药耐药相关蛋白（ＭＲＰ）基因表达的ｍＲＮＡ。方法和结果：利用现有的ＭＲＰ全基因质粒和分子克隆技术经２次克隆将庚型肝炎病毒的一段２３８ｂｐ的核苷酸序列插入ＭＲＰ２９２ｂｐ的目的ｃＤＮＡ片段，构建了插入突变型ＭＲＰｃＤＮＡ重组体作为定量ＰＣＲ的ＤＮＡ竞争模板；再经第３次克隆构建了插入突变型ＭＲＰＲＮＡ重组体，最后经体外转录出５３０ｎｔ的正链ＲＮＡ作为逆转录的ＲＮＡ竞争模板。结论：所建立的ＲＴＰＣＲ技术简便、快速、灵敏，可检出１ｆｇ水平的ｍＲＮＡ量。用ＤＮＡ竞争模板定量检测比用ＲＮＡ竞争模板更简便、经济、可靠。

Objective:To construct the internal standard RNA and DNA templates for quantitative reverse transcriptionpolymerase chain reaction (RTPCR) and quantitatively measure the mRNA expression of MRP gene. Methods and Results: A 292bp fragment was amplified by PCR from plasmid pRC/RSVMRP containing fulllength MRP cDNA and inserted into the SmaⅠ site of pUC19 vector, constructing a recombinant plasmidpUMRP292. A 238bp HGV fragment was amplified by PCR from plasmid pUHGV and inserted into the ClaⅠ site of pUMRP292, constructing a new recombinant plasmidpUMRP 292/HGV as the internal DNA competitive template for quantitative PCR of MRP. A 530 fragment containing the 292bp of MRP and the 238bp of HGV was cut down by EcoRⅠ and XbaⅠ pUMRP292/HGV and cloned into the transcriptional vector pSP72 by the same enzyme sites, constructing a recombinant plasmidpSMRP292/HGV, and then pSMRP292/HGV was cleaved with EcoRⅤ and transcripted in vitro by SP6 RNA polymerase, obtaining a 530bp mutant MRPRNA positive strand as the internal RNA competitive template for quantitative RTPCR. Conclusion: The established quantitative RTPCR assay is simple, rapid and sensitive, and the DNA competitive template is more simple, economic and reliable than the RNA one.