【作者】 陈睿；

【导师】 刘雪平；

【作者基本信息】 山东大学 ， 老年医学， 2005， 硕士

【摘要】 目的:克隆人胶质细胞源性神经营养因子(GDNF)全长基因,与连接有IRES2-EGFP的逆转录病毒载体pLXSN相连,构建逆转录病毒真核细胞表达载体,为治疗帕金森氏病、脑血管病等神经系统疾病奠定基础。 方法:①从人脑胶质细胞瘤组织中提取总RNA,参照分子克隆指南,合成cDNA第1、2链,加入引物PCR扩增目的基因,得到558bp的目的片段后,克隆到pMD18-T载体中,得到重组pMD18T-GDNF。②同样方法扩增IERS2-EGFP基因片段,克隆到pMD18-T载体中,得到重组pMD18T-IRES2-EGFP。③分别对pMD18T-GDNF,pMD18T-IRES2-EGFP,pLXSN逆转录病毒载体进行酶切、连接,构建表达质粒。④对重组质粒进行酶切鉴定并测序验证。⑤将测序后的逆转录病毒重组质粒以电穿孔的方法,转入包装细胞PT67表达,流式细胞仪及荧光显微镜下观察转染效率。 结果:提取的总RNA反转录cDNA后,PCR扩增的GDNF目的基因为500～600bp,IRES2—EGFP基因片段,PCR扩增的目的基因为1200～1500bp,经酶切鉴定和序列鉴定分析,分别得到558bp和1308bp的序列,与Genebank做对照,发现IRES2—EGFP的片段上IRES2序列上有一个碱基突变,单它不会影响到后期荧光蛋白表达。GDNF与Genebank上发布的636bp条带相比,缺失了78个碱基,参考相关文献,此缺失78碱基的基因片段仍保留原蛋白的功能,是蛋白选择性剪切的结果,并且进一步测序发现,在此基因片段前蛋白的序列上出现了一个碱基突变,经过查证,属于同义突变,故不影响蛋白质的表达,表明GDNF基因和IRES2-EGFP基因克隆成功,酶切鉴定结果与预期设想一致。电转染PT67细胞后,经绿色荧光显微镜观察和流式细胞仪测定发现,转染后PT67细胞在荧光显微镜下经蓝光激发,可以观察到大量的PT67细胞胞浆内呈现绿色荧光,同时做流式细胞仪测定转染效率,结果约为24.4%,提示EGFP基因及GDNF基因的进一步表达,说明目的基因片段已成功转入包装细胞内。 结论:①利用分子生物学技术,完整地克隆了人GDNF基因全序列,通过测序加以验证,表明该片段符合人完整的GDNF基因片段序列并将人GDNF基因连接于含IRES2-EGFP更多还原

【Abstract】 Objectives: To clone human glial cell line-derived neurotrophic factor(GDNF) total gene and combine with Retroviral vector pLXSN which contained Internal Ribsome Entry Site (IRES) Sequence and EGFP gene sequence to construct expression plasmid for prokaryotic cell, to make preparations for treatment of nervous system disease including Parkinson’ s disease, cerebrovascular disease and so on.Methods: (1)Total RNA is extracted from glioma of human brain tissues, According to molecular cloning: a laboratory manual the first and second cDNA chains are synthesized and added PCR primers to amplify the target gene. After amplified with PCR, the section about 558bp is obtained. this section is cloned into carrier pMD-18T so that we got reconstructive pMD18T-GDNF. (2)Amplify the IERS2-EGFP gene with PCR which cloned into carrier pMD-18T so that we can get reconstructive pMD18T-IERS2-EGFP. (3)After using restriction endonuclease to shear pMD18T-GDNF , pMD18T-IRES2-EGFP and Retroviral vector pLXSN, joining them to construct reconstructive Retroviral vector, (4)The recombinant is identified by digestion and confirmed by sequencing. (5)The recombinant plasmid is then transfected into PT67 by electroporation for transient expression, the efficiency of gene transfer was detected by flow cytometry and fluorescence microscope.Results: Obtaining cDNA by reverse transcription from total RNA. We acquired 500~600bp of GDNF target gene, 1200~1500bp of IRES2-EGFP gene segment, after PCR amplification. The gene segment is identified by digestion and confirmedby sequencing, we achieve 558bp and 1308bp, respectively, contrasted with Genebank, finding that there is a base mutation on IRES2 sequence of pIRES2— EGFP, but it will not influence expression of Green Fluorescence Protein; and there is 78 base deletion of GDNF gene, contrasted with Genebank. According to some related reference, this 558bp section still reserve the protein function. We detect a same-sense mutation on preprotein sequence of GDNF gene, but it still will not influence expression of GDNF gene. We clone GDNF gene and IRES2-EGFP gene , successfully. Confirmed by digestion of restriction endonuclease, we get the gene which expected before. After transfected PT67 cell, we detected the efficiency of transfection by flow cytometry and fluorescence microscope. Being excitated by blue fluorescence under fluorescence microscope, a large quantity PT67 cell’ s endochylema present green fluorescence .Meanwhile , we can get a result about 24.4% by detecting the efficiency of transfection by flow cytometry. The consequence shows that EGFP gene and GDNF gene have been expressed in cell’ s endochylema concomitance and target gene has been integrated in cells, successfully. Conclusions: (T)We have cloned the total sequence of human GDNF with molecular biological technology and checked the sequence by sequencing.it shows that we obtained the integrity human GDNF gene fragement correctly. GDNF gene has been linked with Retroviral vector pLXSN containing IRES2-EGFP gene fragment. ? Target gene has been integrated in cells by electroporation . we can detect the expression of IRES-EGFP-GDNF gene fragment in PT67 cells . We have constructed recombinant expression plasmid for prokaryotic cell and thus lay the foundation for further obtaining GDNF protein of recombination activity and in gene therapy research and its clinical utility in the future for nervous system disease. (3) Retroviral vector pLXSN is an appropritate carrier in our experiment which has many advantages such as avoiding rearrangement of target gene, high transfection efficiency to quickly fissile cells,stabile gene expression in daughter cells , and so on. ?EGFP is a kind of valuable reportgene which has many characteristics such as no need to add any substrate, stable expression of fluorescence , low molecular weight, convenience to apply, observation fixed and timed to vivi-cells, preparing for transfecting MSCs and then implanting to Parkinson’ s disease mouse model.更多还原