| PrefaceThe molecular and cell biology technology which based on the gene recombination techniques is a useful instrument for people understanding the life and has contributed great accomplishment to the kinds of vocation including agriculture , medicine, industry et al. undoubtedly, gene therapy has been the new approach and focus in the medicine to investigate the treatment of morbus. Type 1 diabetes mellitus, which is caused by autoimmune destruction of pancreatic of P cell, and characterized by the only protein insulin deficiency is a ideal disease for gene therapy.The gene therapy of diabetes mellitus has been studied on the models of type 1 diabetes mellitus rats. But the emergency in the gene therapy is the regulated secreting of insulin and safely effective vector system .In our study, two regulating elements were inserted to the upstream of mutated human proinsulin on the basis of other relative studies. The two regulating elements are the glucose responsive element in the live - pyruvate kinase gene promoter and insulin responsive sequence in the insulin growth factor binding protein -1 gene promoter. We transferred the recombinant gene into retroviral vector and infected hepocytes . The ideal result is hepocytes infected with retro-viral vector excreting mature insulin under the stimulation of glucose and under the inhibition of insulin. Our purpose is to found stable base for the gene therapy of diabetes and its appliance in the clinical therapy.Materials and methods1. Multiplication and purification of pMDIS - T - INS and pLXSN - INS plasmids with the Mini Best plasmid Purification Kit(Takara biotechnology company).2. Identify the pUC - GI plasmid by sequencing with the autonucletide se-quenator.3. Analyzing the restriction site of mutated human proinsulin gene, insulin growth factor binding protein -1 gene, and liver pyruvate kinase gene, and choosing suitable endonuclease to construct target recombinants.4. Recombining the mutated human proinsulin gene, inserting the regulating element on the upstream of the proinsulin.(1) Cut pMDIS - T - INS plasmid with restriction endonuclease Xbal ,3" blunting 5 kination with the BKL kit( purchased from Takara) , then digested a-gain with endonuclease Sail ,the production ran on the 1% agarose gel in a 0.5 TBE buffer ,cut down and extracted the 418bp band.(2) Digested pUC - GI plasmid with enzyme SphI , 3 blunting 5 "kination, digested again with endonuclease Sail , then cut down and purified 3740bp band.(3 ) Ligation above production in the two procedure .(4) Ligation reaction solution transformed totally E coli competent cell JM109(Takara). pick out the positive colony,extracted and purified target DNA plasmid.(5 ) Identify the recombinant plasmid DNA by PCR and restriction endonuclease.5. Constructing the retroviral recombinant plasmid including the mutated human proinsulin and its two regulating elements.(1) Cut pUC - Gil plasmid with restriction endonuclease HindIII ,3i>lun-ting 5kination with the BKL kit( purchased from Takara) , then digested again with endonuclease EcoRI ,the production ran on the 1% agarose gel in a 0. 5 TBE buffer ,cut down and extracted and 1008bp target band.(2) Digested pLXSN - INS plasmid with endonuclease BamHI,3blunting 5 kination, digested again with endonuclease EcoRI,then cut down and purified 5900bp band.(3) Ligation the two production in the above procedure .(4) Ligation reaction solution transformed totally æ‹¢ coli competent cell JM109(Takara). Pick out the positive colony,extracted and purified target DNA plasmid.( 5 ) Identify the recombinant plasmid pLXSN - Gil by PCR and restriction endonuclease.Result1. The restriction site analysis of mutated human proinsulin gene, insulin growth factor binding protein - 1 gene and liver pyruvate kinase gene see pic-ture9.2. Restriction endonuclease Xbal, Sail lies on the two side respectively of the T - A colone site of T vector. After double digesting pMDIS - T - INS ,cut down the 418bp insulin agarose gel... |
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