【作者】 朱书峰；

【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】 粒细胞集落刺激因子(G-CSF)主要来源于巨噬细胞、内皮细胞及纤维母细胞,具有高度特异性的刺激中性白细胞系的功能。G-CSF作为药物分子已广泛应用于临床,其主要的不足有生物活性低、体内稳定性差和体内半衰期短等。本文设计G-CSF突变体期望提高G-CSF的生物活性,采用白蛋白融合表达技术提高G-CSF的体内稳定性,为长效G-CSF的研究创造条件。依据G-CSF的结晶结构信息,我们利用Swiss-pdb Viewer 3.7软件的同源建模功能构建出融合蛋白突变体的三维结构。在Hyper Chem软件中计算各融合蛋白突变体的单点能,并与突变前作比较。单点能计算显示,对远离活性位点的5个氨基酸残基进行的突变得到的结构分子能量最小。分析各突变体的G-CSF骨架结构与野生型G-CSF骨架结构的差异,表明那些能保留活性作用结构的突变体其突变位点均接近N端,与单点能计算结果综合分析,认为M8突变体生物特性与天然构象相似,活性位点的两个重要氢键(19-Glu-O与173-Tyr-O、22-Arg-N与238-His-O)得到保留。选取活性和半衰期有望提高的突变方案M8,将其在毕赤酵母系统中进行表达,并对融合蛋白进行了鉴定,验证理论计算的合理性和可行性。全合成人G-CSF基因突变体的序列(hG-CSF~m),插入克隆载体pBlu2KSP-HSA中,构建重组质粒pBlu2KSP-HSA-hGCSF~m。测序结果显示克隆得到的hG-CSF~m基因与突变方案的序列完全一致。用EcoRⅠ和NotⅠ双酶切重组质粒,回收HSA-hGCSF~m片段,插入到毕赤酵母表达载体pPIC9k中,构建表达载体pPIC9K- HSA-hGCSF~m。经限制性酶切分析证明融合基因已经成功地插入到载体pPIC9k中,测序结果表明HSA-hGCSF~m融合基因与预期的一致。表达载体经SalⅠ线性化后电击转化毕赤酵母GS115,以MD平板进行初筛,得到的重组子进行诱导表达分析。将表达量较高的重组毕赤酵母进行表达产物鉴定和G-CSF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。诱导表达筛选得到1株表达200 mg/L目标蛋白的重组菌32。Western杂交显示表达产物具有很好的HSA抗原性,分子量约84 kDa;NFS-60细胞/MTT比色法测定表达产物具有G-CSF的生物学活性,野生型融合蛋白的比活性为5×104IU/mg,突变后提高为1.5×106IU/mg,说明重组菌32可成功表达具有G-CSF生物活性的HSA-hGCSF~m融合蛋白。更多还原

【Abstract】 The granulocyte colony stimulating factor (G-CSF) mainly comes from macrophagocyle,endothelial cell and fibroblast. It can highly specially stimulate functional active factor of neutral leukocyte strain and is widely used in clinical treatment while the low biological activity, lack of stability, and short half life are major shortcomings. In order to enhance the biological activity of G-CSF, site-directed mutagenesis was employed to design several mutants of G-CSF. They were fused with human serum albumin (HSA) to prolong the half-life of the biological activity of G-CSF in vivo. Lasting effective G-CSF research will be based on such methods.3-D conformation of HSA-hGCSF~m was built up by homology modeling in Swiss-pdb Viewer 3.7 while X-ray diffraction structure of G-CSF was used as a model. Single point energies were calculated and compared with HSA-hGCSF in HyperChem. Results showed that the mutant with the 5 amino acids away from contact site substituted had the minmax total energy among 8 mutants. Biological characters were predicted by theoretical analysis of backbone difference of them, envidences are distinct that natura configuration was maintained when mutation located in N-end, in accord with single point energy results. A candidate M8 whose two necessary H-bonds was well reserved in simulating structure was therefor believed to be near to the wild type G-CSF in biological activity, and was chosen to expresse in GS115 strain of pichia pastoris. The fusion protein HSA-hGCSF~m was identified by western blot and NFS-60 cell proliferation experiment. The results are used to verify and improve the method of theoretical forecast.The chemical synthetical cDNA of human G-CSF~m gene (about 541 bp) was inserted into plasmid pBlu2KSP-HSA by SauⅠa nd NotⅠrestriction enzyme digestion to achieve the fusion gene. The sequence of hG-CSF~m gene was sequenced to be in correspondence with the design in advance.The HSA-hGCSF~m fusion gene was recovered with EcoRⅠand NotⅠfrom the recombinant plasmid (pBlu2KSP-HSA-hGCSF~m) and inserted into pPIC9k to construct expression vector pPIC9k-HSA-hGCSF~m. The sequence of the fusion gene was confirmed by sequencing.The pPIC9k-HSA-hGCSF~m was linearized with SalⅠand transformed into GS115 strain of pichia pastoris by electroporation. A strain named 32 with high productivity which could secrete 200mg/L fusion protein was obtained after screening, and the molecular weight of the fusion protein was 84kDa. Western blot data showed that the fusion protein was a hybrid compound composed of HSA and hG-CSF~m and the biological activity of 1.5×106IU/mg , compared with 5×104IU/mg of wide type fusion protein, has been proved to be 30 times higher. Rational design with the help of computer confirmed its feasibility and impotance in drug molecular discover and modification.更多还原