人B细胞激活因子受体-BAFF-R基因mRNA实时荧光定量PCR标准品的构建  

Construction of the standards for detecting human B lymphocyte stimulator receptor-BAFF-R mRNA with real-time fluorenscence quantitative Polymerase chainreaction

作　　者：赵丽[1] 张冬雷[2] 孙竹华[1] 鞠少卿[3] 

机构地区：[1]江苏省南通市第四人民医院检验科,南通226005 [2]南通大学附属医院酶学研究室,南通226005 [3]南通大学附属医院检验医学中心,南通226005

出　　处：《医学检验与临床》2006年第2期9-10,25,共3页Medical Laboratory Science and Clinics

摘　　要：目的构建实时荧光定量PCR(RFQ,-PCR的标准品以定量检测人B细胞激活因子受体。BAFF-R基因mRNA的表达。方法在BAFF-R基因高保守区设计了特异性的引物和荧光探针,用RT-PCR法从人外周血单个核细胞的总mR-NA中逆转录扩增BAJFF-R的cDNA,将纯化的BAFF-R cDNA与PGEM-T载体进行连接,转化宿主菌DH-5α,然后提取重组质粒DNA,用限制性核酸内切酶EcoR I进行酶切鉴定并测序分析,最后对质粒标推进行实时荧光定量PCR检测。纯化质粒并检测260nm吸光度,确定原被的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品。结果酶切鉴定、PCR扩增及测序分析均证实TNF-αcDNAn重组到PGEM-T载体上。结论成功克隆了BAFF-R实时荧光PCR定量标准。Objective To construction a series of standards for detecting the expression of human B lymphocyte stimulator receptor-BAFF-R mRNA with real-time fluorenscence Polymerase chain reaction(RFQ-PCR).Methods Human peripheral blood mononuclear cells(PBMC)were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation.Total RNA extracted from the PBMC and was revrerse transcribed to cDNA.BAFF-R cDNA was ligated with pGEM-T vector and transformed to bacterium DH-5α.Plasnid DNA extracted positive clones was digested with endonucleases EcoR I and sequenced.BAFF-R was amplified by real-time fluorescence quantitative PCR from the plasmid DNA.The concentration of DNA template purified was detected by analysing absorbancee in 260nm and then was diluted to series standard concentrations of BAFF-R recombined plasmid for RFQ-PCR.Results That BAFF-RcDNA Was recombhined with pGEM-T vector was proved by digestion and PCR amplification and sequsece analysis.Conclusion A series of standards for real-time PCR analysis have beenconstructed successfully.

关 键 词：人外周血单个核细胞 激活因子受体 基因 实时荧光定量 标准品 构建 重组质粒 酶切鉴定 测序分析 定量检测 逆转录扩增 核酸内切酶 载体 荧光探针 梯度浓度 高保守区 定量标准 纯化 限制性 吸光度 

分 类 号：R450[医药卫生—治疗学]