【作者】 周一江；

【导师】 张学成；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2005， 硕士

【摘要】 降钙素(Calcitonin,CT)是一种由32个氨基酸残基组成的多肽类激素,它广泛存在于哺乳动物的甲状腺和鱼类的后腮体中,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)的活性最高,约为人降钙素(hCT)的40倍。目前,鲑鱼降钙素已被广泛应用于临床。 为保证降钙素生物活性的同时尽量降低其在人体内的抗原性,本实验室人工合成了新型人/鲑降钙素嵌合基因(已申请专利)。为提高表达量,我们采用串联表达的方法。首先设计一小段柔性连接肽,经过SWISS-MODEL与3D-PSSM蛋白质结构预测,通过此短肽首尾相接的串联CT中降钙素的活性位点和抗原位点均可以得到充分暴露。因此,我们用同尾酶法构建了多拷贝串联基因表达质粒pLEX-nCT(n=1,2,3,4,5)(每段CT基因之间以编码连接肽的DNA连接),并导入大肠杆菌GI724中表达。表达产物经SDS-PAGE检测,1-5拷贝串联CT基因的表达均不明显。Western blotting检测结果显示,串联CT具有人源降钙素的抗原性。本工作为降钙素基因工程的进一步研究和应用提供了实验基础。更多还原

【Abstract】 Calcitonin (CT), a prominent hormone in the thyroid gland of mammal and in ultimobranchial body of fish, is composed of 32 amino acid residues. It’s principle function, reducing serum Ca level by suppressing osteoclastic resorption of bones in mammals. Salmon calcitonin (sCT) is already been used in clinical practice, since it’s promising hypocalcemic activity which is reported to as high as 40 times than that of human calcitonin.It was synthesized a new human/salmon chimeric CT gene(applied for the patent) in our lab, focussing on retaining hypocalcemic activity of calcitonin while depressing it’s antigenicity in the body. In order to enhance the expression quantity, tandem expression method was used. A passage of flexible linker was designed initially, the dimensional structure forecast of SWIIS-MODEL and 3D-PSSM showed, in the tandem CT that be joint by linker, the activity sites and the antigenicity sites could be exposed adequately. Therefore, by the method of isocaudarner, we constructed tandem expression plasmids pLEX-nCT (n=1,2,3,4,5) contained mulyicopy tandem CT genes, which were joint by a passage of DNA coding linker, and expressed them in E.coli GI724. Finnally, SDS-PAGE was used to test the expression product and it was shown that no obvious expression of tandem CT genes. In addition, the result of Western blotting showed that the tandem CT had the antigenicity of human CT. Our works provided experimental base for farther research and application of CT genetic engineering.更多还原