【作者】 周利苹；

【导师】 金坚；

【作者基本信息】 江南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 C肽(C-Peptide,CP)是胰岛素A链与B链之间的连接肽,近年来研究发现CP通过与细胞膜上G蛋白耦联的受体结合,使Ca2+通道开放,激活Na+-K+-ATP酶活性和一氧化氮合酶的活性,增加血流量,扩张血管,从而改善糖尿病患者的肾脏、神经、微血管病变。为了加快CP的药物化进展,提高CP在体内的半衰期,我们构建了人血清白蛋白(HSA)-CP融合蛋白,将其在毕赤酵母系统中进行表达,并对表达的融合蛋白进行了鉴定、活性分析和初步的分离纯化。根据表达系统的密码子偏好性优化CP基因,PCR从T-CP质粒中扩增CP基因,酶切连接pBlue-HSA质粒(HSA1800bp)和CP(100bp)基因,构建pBlue-HSA-CP质粒。测序结果显示得到的融合基因HSA-CP序列与预期一致。用EcoRⅠ和NotⅠ双酶切重组质粒pBlue-HSA-CP,回收HSA-CP片段,插入分泌表达载体pPIC9k中,构建表达载体pPIC9k-HSA-CP。表达载体pPIC9k-HSA-CP经限制性内切酶分析和PCR验证表明,融合基因已经成功的插入到载体pPIC9k中。表达载体pPIC9k-HSA-CP经SalⅠ线性化后电击转化毕赤酵母GS115,用MM和MD平板筛选Mut+转化子,PCR鉴定后,用甲醇诱导摇瓶分泌表达。将表达量较高的重组子进行表达产物鉴定和活性分析,优化诱导时间和诱导剂浓度后,对融合蛋白进行初步的分离纯化。经G418筛选得到能耐受3 mg/mL G418重组菌8株,进一步摇瓶诱导筛选得到两株表达量120 mg/L的重组菌rHC1, rHC2。优化摇瓶培养条件后,重组菌表达量由120 mg/L提高到140 mg/L。Western blot鉴定显示表达的融合蛋白具有HSA和CP的抗原性,为HSA和CP的杂合分子,分子量约为70 kDa。细胞活性研究表明HSA-CP融合蛋白对人胚肾293细胞的生长具有一定的促进作用。更多还原

【Abstract】 C-peptide(CP), a linking chain with 31 amino acids in length, locates between A and B chain of insulin. The recent researches indicate that it elicits a number of cellular responses by specific binding to G-protein coupled receptor. Including increasing Ca2+ influx, stimulation of Na+-K+-ATPase and endothelial NO synthase activities resulting in the increase of blood flow and expansion of blood vessel. Therefore, CP can ameliorate sensory nerve dysfunction, nephropathy, neuropathy and microvascular dysfunction. In order to prolong the half-life of CP in vivo, we developed a potential long-acting CP(HSA-CP) by albumin fusion technology. The fused gene HSA-CP was expressed in pichia pastoris. Confirmed by Western blot and cell proliferation analysis, the fusion protein was purified preliminarily.To improve the expression of HSA-CP, the codon usage of the gene encoding for CP was optimized according to Pichia pastoris codon bias and reduced repetitive AT and GC content. The artificial CP gene was fused to the HSA cDNA in the same reading frame and then sequenced. The recombinant plasmid named pBlue-HSA-CP. The fused gene HSA-CP was recovered from pBlue-HSA-CP digested with DNA restriction enzyme EcoRⅠand NotⅠ, and then inserted into expression vector pPIC9k to construct recombinant plasmid pPIC9k-HSA-CP. The results showed that the cloned fused gene was in correspondence with our anticipation, and expression vector pPIC9k-HSA-CP was constructed correctly.The recombinant vector pPIC9k-HSA-CP was linearized by SalⅠ, then transformed into Pichia pastoris strain GS115 by electroporation. The Mut+ transformants were selected out from histidine-deficient medium plates and screening for Mut phenotype. Confirmed integration by PCR, The recombinant strains were induced to express fusion protein HSA-CP by methanol in shake. The genetic engineering Pichia pastoris whose expression level was relative high was identified by western bolt and cell proliferation analysis. After optimized the inducing time and concentration of inducer, the secreted fusion protein HSA-CP was purified. Finally, 8 strains were screened out from contained 3 mg/mL G418 MD plate, in which two high expression strain, named as rHC1, rHC2 could secrete 120 mg/L fusion protein was obtained with culturation in shakes. The expressed fusion protein has apparent molecular weight of 70 kDa observed by SDS-PAGE. It was specifically recognized by an anti-human HSA polyclonal antibody and CP polyclonal antibody in Western blot assay. After optimized the culture condition, the yield of the recombinant strain was raised up to 140mg/L determined by Microalbuminuria Immunoassay kit and C-peptide Chemilumin escence Immunoassay kit. Further studies on its cell activites, we found that C-peptide and HSA-CP in low concentration can effect on 293 cell proliferation.更多还原