| PREFACEType 1 diabetes mellitus is caused by severe insulin deficiency secondary to the autoimmune destruction of pancreatic p cells.Type 1 diabetes mellitus ( DM) is usually precipitated by autoimmune destruction of pancreatic (3 cells, leading to insufficient insulin-simple protein production. Its right a logical target for gene replacement therapy. But unregulated insulin secretion cant keep blood glucose in steady physical range, and e-ven produce lethal hypoglycemia. By using an inhibitory insulin response element of the insulin-like growth factor binding protein-1 (IGFBP-1) basal promoter inserted directly downstream of three copies of a stimulatory glucose responsive element ( GLRE)from the rats liver pyruvate kinase(rL-PK)gene, We constructed a insulin gene regulated system,, which can been stimulated by glucose and inhibited by insulin, so as to lay groundwork for constructing regulated insulin expression vector.One propose of this trial is to master recombinant molecular techniques and identification methods of recombinant vector, second is to master DNA abstraction techniques and PCR amplification techniques, and another propose is to contract recombinant vector carrying human proinsulin gene regulating system for the further research.MATERIALS AND METHODS1. Abstracting genome DNA from SD rat liver by saturated sodium chloride method.2. Amplifying IGFBP-1 promoter gene from genome DNA by PCR. , marked as P0 after receiving the aim gene sections by agar electrophoresis.3. The construction of pUC118( GLUE)3-IGFBP-1 .3. 1 Double digesting the pUC118(GLRE)3 and P0 with restriction enzymes Kpn I and Xba I overnight.3.2 Ligate digested P0and pUC118(GLRE)3 with TaKaRa ligation kit into pUCl 18 ( GLRE)3-IGFBP-1 after purified separately, then transform ligation mixture into E. coli JM109.3. 3 Select positive recombination and purify it for further identification.4. Identify the desired recombinant plasmid.4.1 Conform the recombination by PCR.4. 2 Conform orientation by restriction analysis with Kpn I ,Xba I . 4. 3 Identify the recombination by sequence measure.Results1 Abstracting the aim gene DNA from SD rat liver.1. 1 Abstracting genome DNA from SD rat liver. Agar electrophoresis with lul. See picture 1.1.2 Amplifying IGFBP-1 promoter gene from genome DNA by PCR. Receive the aim gene and agar electrophoresis with lul. See picture 2.2 Construction of pUC118 ( GLRE )3-IGFBP-1:2.1 Purified separately after double digesting the pUCl 18 (GLRE)3 and P0 with restriction enzymes Kpn I and Xba I overnight. Agar electrophoresis with lul. See picture 3 and 4.2.2 Select positive recombination of pUCl 18 ( GLRE )3-IGFBP-1 ,and identify by PCR, agar electrophoresis with 5ul. We got 720bp aim section. See pic-ture 5.2.3 Abstract plasmid by purify kit, and agar electrophoresis with lul. See picture 6.3 Identification of recombinant pUC118(GLRE)3-IGFBP-l:3.1 Conform the recombination by PCR: using pUC118(GLRE)3-IGFBP-l cDNA as model, amplify and receive aim by PCR, agar electrophoresis with 5ul, compare with pUC118(GLRE)3, See picture7.3.2 Conform orientation by restriction analysis with Kpn I ,Xba I : the received 420bp and 3.3kb sections through restriction enzymes Kpn I and Xba I are desired recombination. See picture 8.3.3 Identify the recombination by sequence measure; Measure sequence confirms that sequence of recombinant pUC118 ( GLRE)3-IGFBP-1 is correct. See sequencing.DISSCUSSIONGene treatment for type 1 diabetes mellitus requires insulin secretion adapt to biologic metabolism. To achieve it, we designed an insulin regulating system by an inhibitory insulin response element of the insulin-like growth factor binding protein-1 (IGFBP-1) basal promoter inserted directly downstream of a promoter composed of three copies of a stimulatory glucose responsive element ( GLRE) from the rat liver pyruvate kinase ( rL-PK) gene. Following gene transfer into hepatocytes, insulin production is stimulated by glucose, while promot... |
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