克隆高鸟嘌呤与胞嘧啶含量大鼠bcl-2基因的编码序列

Cloning and sequencing of rat bcl-2 gene riched guanine and cytosine

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： Excessive apoptosis of ovary granulosa cell is a dominant cause of premature ovarian failure and bcl-2 gene is able to inhibit cell apoptosis. But studies demonstrate that, the guanine and cytosine （GC） content reaches 60% in the rat bcl-2 gene sequence. This gene cannot be amplified using routine polymerase chain reaction method. OBJECTIVE： To clone and identify the bcl-2 gene riched GC. DESIGN, TIME AND SETTING： Open experiment was finished in the Laboratory of Medicine and Molecular Biology, Life Science School of Sun Yat-sen University from May to December in 2007. MATERIALS： Wistar rats were purchased from Experimental Animal Center of Sun Yat-sen University. Ecoli DH5 a was preserved by Laboratory of Medicine and Molecular Biology, Life Science School of Sun Yat-sen University; pMD18-T vector was purchased from Takara Biotechnology （Dalian） Co., Ltd. METHODS： The bcl-2-cDNA, in which GC accounted for 60.6%, was obtained by modified reverse transcription- polymerase chain reaction from kidney tissue of Wistar rats, and was cloned into vector-pMD 18-T. Characterizations and sequencing of the pMD 18-T-bcl-2 were carried out by polymerase chain reaction screening of individual bacterial colonies. MAIN OUTCOME MEASURES： Cloning and purification of bcl-2 gene cDNA; results after connecting bcl-2 gene cDNA to pMD 18-T vector and transducting Ecoli DH5 a, identification of positive clone and results of sequencing. RESULTS： The bcl-2 gene was identified by the clone and DNA sequencing. DNA sequence analysis was consistent with Genebank sequence, with a 99% homology. CONCLUSION： The gene riched GC is difficult to be amplified, bcl-2-cDNA can be cloned and constructed into cloning vector pMD 18-T successfully by the efficient technique for other genes riched GC.