| Type 1 diabetes mellitus is caused by severe insulin deficiency secondary to the auto immune destruction of pancreatic β- cells. We can trafer human proinsulin gene into diabetes mellitus patients, produce long - term, constant, physiological control in the proposal of permanent therapy.Insulin is normally processed into the mature, active A and B chain complex in the secretory vesicles of pancreatic β- cells which contain the requisite - processing enzymes ( PC2 and PC3). To create most non - β- cells with the ability to process human proinsulin to mature insulin, we took advantage of the fact that there are processing enzyme furin in them. Using site - directed mutagenesis, we engineered proinsulin to be a substrate for furin by introducing new cleavage sites at both the B - C and C - A junctions of human proinsulin gene. Following transfection, the non - β - cells can process mature insulin and secret it out of the cells. This provides a experimental basis for diabetes gene therapy. One of proposes of this trial is to master PCR techeniques and identification methods, the other is to construct mutant human proinsulin plasmids for further reseach of diabetes gene therapy.Materials and Methods1 Drugs and ChemicalsHuman proinsulin gene plasmid, PCR Fragment Recovery kit, Site - directed mutagenesis kit2 Instruments PCR equipment3 MethodsUsing site - directed mutagenesis, we introduce new cleavages sites at both the B - C and C- A junctions of human proinsulin gene. (1)the plasmid is amplified (2)design the primers needed(3)PCR(4)Receive the aim gene section in (3) by agarose swimming (5)Blunting Kination Ligation the aim gene, then Ligate it. (6)Transform ligation mixture into E. coli HB101 (7)the aim gene is sent to be sequenced.ConclusionDiseases caused by a single protein deficiency are logical targets for gene replacement therapy. Animal studies suggest gene therapy may become therapeutic for a number of such metabolic diseases. Type 1 diabetes mellitus(DM) is usually precipitated by auto immune destruction of pancreatic β- cells, leading to insufficient insulin production. Since clinical symptoms are caused by diminished production of a single protein, diabetes is a natural candidate for treatment bygene therapy.Proteins are processed and secreted by one of two means in eu-karyotic cells: the constitutive or the regulated pathway ( CPSP and RPSP). Insulin is normally processed into the mature, active A and B chain complex in the secretory vesicles of pancreatic β- cells which contain the requisite processing enzymes ( PC2 and PC3). During processing, the C peptide, which resides between the B and A peptides in proinsulin, is excised by enzymes that make two separate cleavages : one at the B - C junction ( Arg - Arg dibasic site) , and one at the C - A junction ( Lys - Arg dibasic site ).In the past model of gene therapy about diabetes mellitus, the non - β - cells were transfected with human proinsulin gene, which maily processed proinsulin and low mature insulin that can not be secreted out of the cells.In most non - β - cells contain the endogenous, Golgi - anchored , processing enzyme furin. Although furin, like PC2 and PC3, cleaves at the pairs of basic residues, its substrate specificity is different. Human proinsulin is not effectively coverted to mature insulin in non -β- cells.Our goal in this study is to develop a means by which non -β -cells can produce mature, active human insulin. To create non - endocrine cells with the ability to correctly and efficiently process human proinsulin to mature insulin, we took advantage of the fact that furin was expressed in most cells. Using site - directed mutagenesis, we have introduced furin consensus cleavage sequences ( Arg - X - Lys -Arg) into the human proinsulin cDNA. We engineered proinsulin to be a substrate for furin by introducing new cleavage sites at both B - C and the C - A junctions of human proinsulin gene.Sites I B - C junction: Lys Thr Arg Arg - Arg Thr Lys Arg Sites II C -... |
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