【作者】 裘锋平；

【导师】 詹金彪；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 糖尿病是由多种病因引起以慢性高血糖为特征的代谢紊乱,在发达国家中它是继心血管病和肿瘤之后的第三大非传染病,已经成为严重威胁人类健康的世界性公共卫生问题。胰岛素是由51个氨基酸组成的多肽,有A、B两链,两链之间由两个二硫键连接(A7-B7,A20-B19),其作为治疗糖尿病的特效药物是通过与胰岛素受体的结合来发挥复杂的生物学功能的。从进化过程来看,各种动物的胰岛素结构大体相似,差不多有一半的氨基酸残基的排列顺序保持不变,只是在某些氨基酸组成和排列上有不同程度的差异。比如,六个半胱氨酸残基在两条肽链上的位置不变,这是维持胰岛素独特空间结构的关键。而比较人胰岛素与猪胰岛素,两者仅有一个氨基酸之差,比较牛胰岛素与人胰岛素也只差三个氨基酸。故一些携带重要氨基酸或者能够形成类似空间构象的小肽或许具有类胰岛素的活性。 本研究旨在通过噬菌体展示技术,筛选随机九肽文库(C7C),寻找能与靶分子特异性结合的噬菌体,测序获得小肽序列,并合成相应的短肽,进一步研究这些短肽的生物学活性。若能够找到合适的短肽代替胰岛素发挥其生物学功能,这为研制类胰岛素的新型肽类药物奠定了一定的基础,对研究胰岛素与其受体结合的作用位点也有一定意义。 一、筛选胰岛素相关肽 1.噬菌体展示肽库的亲和筛选更多还原

【Abstract】 Diabetes mellitus is a metabolic disorder that results from complex interactions of multiple factors and is characterized by the hyperglycemia. It is the third non-infectious disease followed by the cardiology and cancer in the industrialized country. And now it is growing in prevalence worldwide and becoming a serious public health problem.Insulin, the peptide of 51 amino acids, consists of the A chain and the B chain, and there are two disulfide bonds between them (A7-B7, A20-B19). It is regarded as the specific medicine for diabetes, and its physiological function in vivo depends on its binding to insulin receptor (IR). According to the process of evolution, the arrangement of the amino acid residues of insulin almost unchanged. For example, the six cysteines are still in the same location, and these are the key to keep the unique spatial structure of insulin. Among the animals, there are some amino acids differed from the human insulin. In the pig, the alanine substituted the threonine in the B30. In the cattle, besides the same change to the pig, the alanine substituted the threonine in the A8 and the valine substituted the isoleucine in the A10. The peptide bearing the hotspot or the spatial structure can bind to the insulin receptor specifically and has the similar biological activity of insulin.Using the phage-displayed library C7C, we attempted to identify peptides that mimic the epitopes involved in protein-protein interaction between insulin and the insulin receptor. Many researches demonstrate that short peptide ligands can stimulatethe function of the corresponding whole protein molecules. So seeking small analogs for making oral surrogate of insulin is of great importance. Also it is very significant for study the interaction of insulin and its receptor.1. Isolate the peptides mimetic insulin by screening a phage-displayed library(1) The screening of phage-displayed libraryPhage clones binding to the polyclonal antibody of insulin were screening by incubation of the random peptide library C7C with the polyclonal antibody of insulin. During bio-panning, the progressively increasing the concentration of TBST and strict washing conditions lead to selective pressure so that non-binding and weakly binding phages are removed. After three rounds of bio-panning, the phage clones with high affinity and great specificity were thereby enriched.(2) Evaluation of binding specificity of phage clones to target by ELISAAfter three rounds of bio-panning, 25 clones were randomly picked and amplified in Escherichia coli. The binding specificity of those phages was determined by ELISA. The results revealed that different phages bound with different affinities to the target. Among them, 15 clones showed the high affinity and great specificity than the primary library.(3) Sequence analysis of insulin mimetics15 phage clones were selected from the 25 phage clones of insulin mimetices to DNA sequence in order to deduce amino acid sequences of peptides. The reverse primer 5’-CCCTCATAGTTAGCGTAACG-3’ was used for sequencing. Eight out of the 15 clones carried an identical sequence CPTSQANSC (referred as ZJ1) and the other seven clones had the corresponding sequence CVQPSHSSC (referred as ZJ2). The two peptides had no sequence homology with insulin.2. The biological activities of insulin mimetices presenting phage(1) The synthesis of the insulin mimeticesAccording to the sequence deduced by the DNA sequence, the insulin mimetices were synthesized by the Chinese Peptide Company, Hangzhou, China. In order to the further study, the peptide purity was ^85%.(2) Inhibition of binding of insulin to its receptor by peptideThe relative receptor-binding affinities were obtained with the human hepatomacell line (HepG2). HepG2 cells were cultured at 37 °C in a 5% C02 humidified atmosphere and the effect of inhibition was determined by the cell-ELISA. The binding of insulin to its receptor was competitively inhibited by the peptides at the concentration range of 10’12 to 10"6 M. The 50% inhibition concentration of ZJ1 and ZJ2 was approximately 6.5 X 10"10 M and 9.7 X10’10 M. These data demonstrated that the two peptides could inhibit the binding of insulin to its receptor.(3) The glucose-lowering effect of peptide in normal male ICR miceMale ICR mice were randomly grouped and treated with the peptides, insulin and normal saline (NS). After fasting for 12 h, the blood samples were collected from the tail vein and blood sugar was measured by the method of o-Toluidine. The peptide was injected to mice via the tail vein at a dose of 100 u g in a final volume of 100 u 1 of the sterile NS. Insulin and NS were conducted in the same way. Plasma glucose was determined in a group of animals after 45 min. At the end of the treatment, we compared the changes of the blood sugar before and after the injection. Peptide (ZJ1) at about 4 mg/kg had obvious effect on reducing blood sugar levels and was amounted to a decrease of about 32% of initial value. Peptide (ZJ2) at about 4 mg/kg had slightly increased blood sugar of about 24% of initial value.(4) The effect of administration of peptide to alloxan-induced diabetic miceAfter a 12 h fast, some mice were induced to diabetic models by a single intraperitoneal injection of 1% alloxan (200 mg/kg body weight). Alloxan was dissolved in normal saline (NS) immediately before administration. Diabetic models were assessed by blood sampling from the tail vein. The diabetic models were randomly grouped into the treated group and the control group according to the weight and FBG After a 12 h fast, the peptide (ZJ1) was injected to mice via the tail vein at a dose of 1 mg/ml and NS were conducted in the same way. The blood sugar was measured by the method of o-Toluidine. The results showed that blood sugar levels in the control showed no variation. The peptide(ZJl) treated group showed a significant reduction in serum glucose levels in the diabetic mice and was amounted to a decrease of about 23% of initial value.hi summary, insulin mimetic was obtained by phage display technique. Based on competitive ELISA studies, the peptide CPTSQANSC has been identified to bind to asimilar hotspot of the insulin receptor on the cell surface. The biological activity was measured by injecting i.v. the synthetic peptide into normal and alloxan-induced diabetic mice. The blood sugar was determined to evaluate the insulin-like function. The results showed that the peptide had insulin-like activity. In conclusion, the short peptide CPTSQANSC has glucose-lowering effects, which may have potential clinical application.更多还原