人腺苷酸环化酶激活多肽cDNA克隆和序列分析  

The Cloning and Sequencing of Adenylate CyclaseActivating Polypeptide(ACAP) cDNA from Human Testis

作　　者：余焱林[1] 刘飞鹏[1] 

机构地区：[1]第一军医大学细胞生物学与医学遗传学教研室

出　　处：《中国生物化学与分子生物学报》1998年第3期248-253,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金;广东省科学基金

摘　　要：以人睾丸组织总ＲＮＡ为材料，用ＲＴ－ＰＣＲ方法合成了人腺苷酸环化酶激活多肽（ＡＣＡＰ）编码区（５３０ｂｐ）和全长ｃＤＮＡ（１９３０ｂｐ）片段．并分别将这些ｃＤＮＡ片段克隆入ｐＵＣ１８载体的ＳｍａⅠ限制性内切酶位点．对重组质粒分别采用直接ＤＮＡ双链末端终止法和在核酸外切酶Ⅲ和核酸酶Ｓ１作用下连续缺失ＤＮＡ后，相继克隆，构成一系列连续缺失的缺失体的方法，测定了全部核苷酸顺序．结果表明：ＡＣＡＰ编码区的ｃＤＮＡ顺序与已报道的有１２处碱基的改变，其中１１处碱基顺序的改变不引起编码的氨基酸变化，只有第３８５位的Ｔ→Ａ后，才引起其编码的氨基酸由Ｓｅｒ→Ｔｈｒ，但由于Ｓｅｒ和Ｔｈｒ的理化性质极其相似，这一变化可能并不导致蛋白质的生物活性的变化．这些改变可能是由于种族、群体或个体的差异．Adenylate cyclase activating polypeptide (ACAP) is a recently isolated bioactive polypeptide in which many researchers are interested to understand its function and acting mechanism.It is too difficult to isolate enongh amount of ACAP from tissue.ACAP gene was cloned in an attempt to express its recombinant peptide for experimental and clinical application.The full length cDNA and its coding region of a gene encoding ACAP were synthesized by RTPCR method from total RNA of human testis.These cDNA fragments were ligated into SmaⅠ site of pUC18 vector.The nucleotide sequences of the coding region cDNA were determined by directional dideoxynucleotide chainterminator method and the full length cDNA was deleted in different length by exonuclease and S1 endonuclease and constructed nine deletion mutants for DNA sequencing.Results show that the nucleotide sequence in the coding region cDNA changed at 12 sites compared with Ohkubos report,but the amino acid sequence of the protein deduced only changed at one site,which is probably due to diversity of population or individuals.

关 键 词：腺苷酸环化酶 激活多肽 PCR DNA重组 序列分析 

分 类 号：Q516[生物学—生物化学] Q78