摘要
利用酸性异硫氰酸胍－酚－氯仿一步法从人胎儿基底前脑中提取总ＲＮＡ，用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法，扩增出人神经生长因子低亲和力受体ｐ^（７５）ＮＧＦＲ基因ｃＤＮＡ，在限制性内切酶ＳｍａⅠ存在下的连接体系中，将扩增出的ｃＤＮＡ片段克隆入ｐＵＣ１２的ＳｍａⅠ位或，经限制性内切酶ＥｃｏＲⅠ和ＰｓｔⅠ酶切鉴定是否插入以及ＨｉｎｄⅢ酶切鉴定方向。将重组质粒中的ｐ^（７５）ＮＧＦＲ的ｃＤＮＡ再次亚克隆至ｐＵＣ１２载体中后，以其双链ＤＮＡ为模板，用末端终止法测出其全部核苷酸顺序，证实其核苷酸编码的ｐ^（７５）ＮＧＦＲ除两个碱基突变外，其余与文献完全一致。完整的ｐ^（７５）ＮＧＦＲ的ｃＤＮＡ分两步克隆到逆转录病毒表达载体ｐＸＴ－１，经ＰＡ３１７包装细胞株体外包装后、收集病毒上清转染条件不死性大鼠小脑神经细胞系Ｒ２．初步结果表明转染了ｐ^（７５）ＮＧＦＲ的Ｒ２细胞株去除ＮＧＦ培养时出现程序化死亡的典型特征梯型ＤＮＡ带。
In order to study the mechanism of physiological and pathological action of human P￣(75) NGFR gene cDNA. Total RNA was isolated from human fetal basal forebrain with the singlestep method of acid guanidinium thiocyanate phenol-chloroform (AGPC) extraction. The cDNA encoding human P￣(75) NGFR was amplified by reverse transcription-polymerase chain reaction(RTPCR)method using directly the isolated fetal basal forebrain total RNA as the template. The amplified cDNA fragment was inserted into pUC12 plasmid which was digested with restriction endonuclease Sma I in the ligation system containing Sma I. After transformation the recombinant plasmids were analysed by restriction endonucleases to determine the direction of the cloned cDNA fragment,and the subcloning was made. The nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination method. There are two nucleotides mutations in 1108 and 1 168 positions. P￣(75) NGFR was cloned into retrovirus expressinon vector pXT-1. After being packaged by PA317 cells, the infected pseudoviruses conditionally immortalized rat cerebellar neural cell line R2. Primary result indicated that the stable transfected R2 cells showed clear DNA ladders-the significant characteristics of apoptosis after deprived NGF during cultivation.
基金
国家自然科学基金
卫生部青年基金
