IRES序列连接的人GDNF基因和EGFP基因逆转录病毒真核表达载体的构建

发布时间：2018-05-22 18:04

本文选题：胶质细胞源性神经营养因子 + 基因序列　；参考：《山东大学》2005年硕士论文

【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
[Abstract]:Objective: to clone the full length gene of human glial cell derived neurotrophic factor (GDNF) and to construct a retroviral eukaryotic expression vector, which is connected with the retroviral vector pLXSN connected with IRES2-EGFP, and lay the foundation for the treatment of Parkinson's disease and cerebrovascular disease.
Methods: (1) the total RNA was extracted from the human glioblastoma tissue. According to the molecular cloning guide, cDNA 1,2 chain was synthesized and the target gene was amplified by primer PCR. After the target fragment of 558bp was obtained, the recombinant pMD18T-GDNF. was cloned and the IERS2-EGFP gene fragment was amplified by the same method, and the recombinant pM was cloned to obtain the recombinant pM. The recombinant pM was cloned and the recombinant pM was obtained. The recombinant pM was cloned and the recombinant pM was obtained. D18T-IRES2-EGFP. (3) pMD18T-GDNF, pMD18T-IRES2-EGFP, pLXSN retrovirus vectors were cut, connected, and the expression plasmids were constructed. (4) the recombinant plasmid was identified by enzyme digestion and sequenced. 5. The recombinant retrovirus recombinant plasmid was sequenced by electroporation, and transferred into the PT67 expression of the packaged cell, flow cytometry and fluorescence display. The transfection efficiency was observed under microscopically.
Results: after the total RNA was back transcriptional cDNA, the GDNF target gene of PCR amplification was 500 to 600bp, IRES2 EGFP gene fragment, and the target gene of PCR amplification was 1200 ~ 1500bp. The sequence of 558bp and 1308bp was obtained by enzyme digestion and sequence identification. The base mutation, which alone does not affect the late fluorescent protein expression.GDNF and the 636bp strip published on the Genebank, is missing 78 bases, referring to the related literature. The deletion of the 78 base gene fragment still retained the function of the protein, which was the result of the protein selective shear, and further sequencing found the sequence of the preprotein in this gene fragment. A base mutation was found, which was found to be a synonymous mutation, so it did not affect the expression of protein, indicating that the GDNF gene and the IRES2-EGFP gene were cloned successfully. The results of the enzyme digestion were consistent with the expected assumption. After electrotransfection of PT67 cells, the fluorescence microscopy and flow cytometry showed that the transfected PT67 cells were fluorescent after transfection. Under the microscope, a large number of PT67 cells were observed to show green fluorescence in the cytoplasm. At the same time, the transfection efficiency was measured by flow cytometry. The result was about 24.4%, suggesting the further expression of EGFP gene and GDNF gene, indicating that the target gene fragment had been successfully transferred into the packed cell.
Conclusion: (1) the whole sequence of human GDNF gene was cloned completely by molecular biology technology, and verified by sequencing, which showed that the fragment conformed to the complete sequence of GDNF gene fragment and connected human GDNF gene to IRES2-EGFP
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【引证文献】