几种海水鱼降钙素基因克隆及表达的研究-海洋生物学专业论文.docx

几种海水鱼降钙素基因克隆及表达的研究几种海水鱼降钙素基日克隆及表达的研究 几种海水鱼降钙素基因克隆及表达的研究 几种海水鱼降钙素基日克隆及表达的研究 内容提要 (降钙素(Calcitonin)是由32个氨基酸残基组成的一种多肽类激素，其主 要生理功能是参与体内的钙磷代谢，通过抑制破骨细胞的活性，促进钙在骨中 的沉淀，从而降低血钙含量。天然降钙素中鲑鱼降钙素(sCT)生物活性最高， 约是人降钙素活性的40倍，目前，鲑鱼降钙素已被应用于临床。4 本文用PCR方法克隆了野生鲑鱼、养殖鲑鱼的降钙素基因，并应用体外 克隆PCR的方法首次克隆出鲈鱼、六线鱼降钙素的部分基因序列，通过对克 隆的降钙素序列的比较研究，结果显示同一科的鱼降钙索序列保守性较高，同 时，根据降钙素的部分氨基酸序列进行了降钙素相似性的比较研究，结果显示 在分类学上，分类地位较远的鱼，其降钙素相似性较低，分类地位越接近的鱼， 其降钙素相似性越高；我们利用谷胱甘肽s诤}移酶(Glutathions S-transferase， GST)融合表达载体pGEX-4T．X对克隆的鲑鱼降钙素基因进行了融合表达研 究，应用表达PCR的方法对降钙素基因的独立表达进行了初步的研究探索， 并将纯化的融合蛋白(GST．sCT)作为抗原，获得了高效价的兔抗鲑鱼降钙素 免疫血清；生物活性研究表明，大肠杆菌表达的融合蛋白(GST．sCT)具有显 著的降血钙作用。本研究结果为降钙素基因工程迸一步研究和降钙索的应用提 供了系统的实验证据。 关键词降钙素j基因克隆j表昏融合蛋白j抗血清j生物活性检测 几种海水鱼降钙紊基因克隆及表达的研究Studies 几种海水鱼降钙紊基因克隆及表达的研究 Studies on c|oning and expression of caicitonin gene of some marine fishes Calcitonin(C’I)iS a hormone，composed of 32 amino acid residues．The main function of CT is to suppress osteoclastlt resorption of bones followed by decreases in serum Ca level in mammals．Hypocalcemic activity of salmon calcitonin(seT)is the highest in all of nature calcitonins，which is 40 times higher than human calcitonin．At present，seT has already been applied to clinical practice． The different fishes which are employed in the present studies are wild—type salmon，cultured salmon of freshwater and seawater,sea perch and fat greenling． The complete CT gene sequences of salmons are obtained by PeR amplification． The partial CT sequences of sea perch and fat greenling are obtained by in vitro cloning PeR method，Alignment of obtained CT sequences witll other fish CT shows that CT appears to be well conserved among the sanle family．And the relative in taxonomy iS far away，the similarity of different fish CIs iS low．On the contrary,the closer the relative in taxonomy is，the higher the similarity of different fish CTs is．The seT is expressed in pGEX一4T—X by recombinant form(GST—seT)． We also succeed in the research of seT expression alone by expression PeR．In addition，sCT antiserum is obtained