关键词：多药耐药基因；定量逆转录-聚合酶链反应；白血病
摘要 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MRP 292bp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。
Construction of the internal standard RNA and DNA templates for MRP gene quantitative RT-PCR analysis Cao Liping* ,Wang Bin, Yu Zhifei. Department of Hematology, Liuhua Bridge Hospital, Guangzhou 510010
Abstract 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.
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