| 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. |
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