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背景:卵巢颗粒细胞的过度凋亡是诱发卵巢早衰的根本途径,bcl-2基因具有抗细胞凋亡的特性。但实验表明,大鼠bcl-2基因序列中鸟嘌呤与胞嘧啶含量高达60.6%,应用常规聚合酶链反应方法,无法扩增出此基因。目的:拟克隆大鼠基因组中高鸟嘌呤与胞嘧啶含量的bcl-2基因编码序列。设计、时间及地点:开放性实验,于2007-05/12在中山大学生命科学院医药分子生物学实验室完成。材料:Wistar大鼠购自中山大学实验动物中心生产供应部,大肠杆菌DH5α由中山大学分子医药生物学实验室保种,载体pMD18-T购自TakaRa大连宝生物技术公司。方法:采用改进的反转录-聚合酶链反应方法从大鼠脾脏组织中扩增出鸟嘌呤与胞嘧啶含量为60.6%的bcl-2cDNA编码序列,将扩增产物克隆至pMD18-T载体中,用单菌活聚合酶链反应扩增快速鉴定DNA片段后进行序列分析。主要观察指标:①大鼠bcl-2基因cDNA克隆与纯化。②bcl-2基因cDNA与pMD18-T载体的连接、转化、鉴定阳性克隆及测序结果。结果:在筛选的阳性克隆中扩增到大鼠bcl-2基因,DNA序列分析结果与Genebank中序列相比,同源性为99%。结论:高鸟嘌呤与胞嘧啶含量基因的扩增属于困难聚合酶链反应,实验采用改进技术成功克隆了大鼠bcl-2基因,此技术也适用于其他富含鸟嘌呤与胞嘧啶的基因快速扩增。
BACKGROUND: Overexpression of granulosa cells is the fundamental pathway of premature ovarian failure. The bcl-2 gene has anti-apoptotic properties. However, experiments showed that the content of guanine and cytosine in rat bcl-2 gene was as high as 60.6%. This gene could not be amplified by conventional polymerase chain reaction. OBJECTIVE: To clone the bcl-2 gene coding sequence of high guanine and cytosine content in rat genome. DESIGN, TIME AND SETTING: The open experiment was performed at the Laboratory of Molecular Biology of Medicine, School of Life Sciences, Sun Yat-sen University from October to December 2007. MATERIALS: Wistar rats were purchased from Production and Supply Department of Experimental Animal Center of Sun Yat-sen University. Escherichia coli DH5α was bred from Sun Yat-sen University Molecular Medicine and Biology Laboratory. Vector pMD18-T was purchased from TakaRa Dalian Bao Biotech Company. Methods: The bcl-2 cDNA coding sequence with a guanine and cytosine content of 60.6% was amplified from rat spleen tissue by modified reverse transcription-polymerase chain reaction. The amplified product was cloned into pMD18-T vector , With a single live PCR amplification of DNA fragments identified after rapid sequence analysis. MAIN OUTCOME MEASURES: ① Cloning and purification of rat bcl-2 gene cDNA. ② The connection of bcl-2 cDNA with pMD18-T vector, transformation and identification of positive clones and sequencing results. Results: The rat bcl-2 gene was amplified in the positive clones screened. The DNA sequence analysis showed 99% homology with that in Genebank. CONCLUSION: Amplification of high guanine and cytosine genes is a difficult polymerase chain reaction. The bcl-2 gene was cloned successfully using improved techniques. This technique is also applicable to other genes rich in guanine and cytosine Amplify.