共信号分子CD40/CD40L质粒标准品的构建  

Construction of co-stimulatory molecular CD40 and CD40 ligand plasmid standards

作　　者：王亮 史进方[2] 蔡惠芬[2] 蒋敏[2] 顾国浩[2] 

机构地区：[1]苏州市木渎医院检验科,江苏省苏州市215101 [2]苏州大学附属第一医院检验科,江苏省临床免疫学实验室,江苏省苏州市215006

出　　处：《中国组织工程研究与临床康复》2009年第44期8675-8679,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:目前,对CD40/D40L的研究主要集中在可溶性和蛋白方面,对其在转录水平上的研究文献不多,可能与其无商品化质粒标准品有关。目的:构建检测CD40/CD40L mRNA表达水平差异的标准品。设计、时间及地点:单一样本实验,于2009-04在苏州大学附属第一医院检验科实验室完成。材料:取手术切除的子宫肌瘤组织,经患者知情同意。方法:以手术切除的子宫肌瘤组织细胞的总RNA为模板、Random6mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人CD40/CD40L基因相应的cDNA片断,构建PMD-18-CD40/CD40L重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线。主要观察指标:①RNA质量检测结果。②扩增的目的片段电泳结果。③CD40/CD40L基因测序结果。④标准品的扩增情况。结果:组织提取的总RNA完整性良好,构建的CD40/CD40L质粒经PCR扩增后分别得到136bp,200bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.66×1013、2.45×1013copies/mL,倍比稀释至2.0×106copies/mL均能得到良好的标准曲线(R2=1)。结论:实验成功构建了CD40/CD40L基因荧光定量PCR标准质粒。BACKGROUND：At present,the researches on CD40 and CD40 ligand mainly concentrated in their soluble and protein,and only a few references focus on their transcription level,which may be related to there is no commercial plasmid standard.OBJECTIVE：To construct CD40 and CD40 ligand plasmids standard in sequence.DESIGN,TIME AND SETTING：An experiment of single simple was performed in the First Hospital Affiliated to Soochow University in April 2009.MARERIALS：Uterus myomas were obtained after hysteromyomectomy.Informed consent was obtained from all patients.METHODS：Total RNA extracted from the uterus myomas was as template and Random 6mers was as primer.RNA was reverse transcribed to cDNA.The cDNA was as template to amplify the fragments of human CD40 and CD40 ligand.PMD-18-CD40/CD40L reconstruction plasmids were produced by using this cDNA fragments.The standard curve was perfect by fluorescence quantitative polymerase chain reaction after identify and sequence of plasmids.MAIN OUTCOME MEASURES：① RNA quality.②Electrophoresis results of reverse transcription-polymerase chain reaction products.③The results of CD40 and CD40 ligand after DNA sequence.④The amplified condition of CD40 and CD40 ligand standard.RESULTS：Total RNA extracted from the uterus myomas was perfected.After polymerase chain reaction,the CD40 and CD40 ligand plasmids were presented with 136 and 200 bp clear stripes,respectively.And the DNA sequencing of CD40 and CD40 ligand were according with aimed fragment（100%）.The original concentrations of plasmids were 2.66×1013 and 2.45×1013 copies/mL.When they were diluted to 2.0×106 copies/mL with multiple proportions,perfected standard curve could be all received（R2=1）.CONCLUSION：The standard plasmids containing human CD40 and CD40 ligand cDNA have been constructed successfully by fluorescence quantitative polymerase chain reaction.

关 键 词：CD40/CD40L T-A克隆 荧光定量PCR 

分 类 号：R318[医药卫生—生物医学工程]