Construction And Package Of Human Proinsulin Gene Plasmid That Can Be Expressed In Non-β Cells

PrefaceType 1 diabetes mellitus is an autoimmune disease controlled by many genes. Autoimmune destruction of pancreatic β cells results in the loss of endogenous insulin secretion which can lead to hyperglycemia. Its inconvenience and painful injection exogenous insulin everyday and sometimes it can lead to serious hypoglycemia. In recent years, gene recombination techniques have developed rapidly. Type 1 diabetes mellitus, which is characterized by the deficiency of the only protein insulin is an ideal disease for gene therapy.In our study , we conducted an variant which replaced the histidine at position 10 in the B chain with an aspartic acid. When this mutation was introduced into the engineering - processed proinsulin cDNA, the resulting mutant insulin subsequently accumulated higher. Then we conducted an retroviral vector which contained the target DNA. Basing on recombinant plasmid conducted successfully, we introduced the recombinant plasmid into the genome of PA317 packing cell line. Then we determined the virus titer of every positive clone to screen one cell clone producing high titer virus. Our purpose is to explore a mature route for the gene therapy of diabetes mellitus.Materials and methods(1) Conduction of an variant which replaced the histidine at position 10 in the B chain with an aspartic acid.1. design a primer.2. PCR and receive the aim gene section by agrose swimming3- Blunting Kination of the aim gene and ligated it4. ligation reacting solution transformed totally E coli competent cell JM109.5. Picked out the positive colony, extracted and purified target DNA plas-mid.6. the aim gene was sent to be sequenced(2) construction of recombinate retroviral plasmid1. cut PUC118 - GII₃ plasmid with restriction endonuclease Hind III, 3' blunting 5'kination with the BKL kit (purchased from Takara) , then digested a-gain with endonuclease EcoR I , the production ran on the agarose gel, cut down and extracted the lOOObp band.2. cut PLXSN - INS plasmid with restriction endonuclease BamH 1,3' blunting 5' kination with the BKL kit (purchased from Takara) , then digested again with endonuclease EcoR I , the production ran on the agarose gel, cut down and extracted the 5900bp band.3. ligation of above production in the two procedure.4. ligation reactign solution transformed totally E coli competent cell JM109 (Takara).5. picked out the positive colony .6. identified the recombinant plasmid DNA by PCR7. extracted and purified target DNA plasmid. ( 3 ) package of the recombinant plasmid1. dispensing culture medium2. culture PA317 cells3. adopt liposome to introduce the recombinant plasmid into PA317 cells4. screen one cell clone producing high titer viruses5. abstract the DNA from PA317 cells that have been introduced the recom-binat plasmid6. identify the recombinant plasmid DNA by PCRResults1. PCR and receive the aim gene section by agrose swimming,see figure 1.2. sequence of target DNA3. agrose swimming of PUC118 - GII3 plasmid cut with restriction endonu-clease Hind III and EcoR I .4. agrose swimming of PLXSN - INS plasmid cut with restriction endonucle-ase BamH I and EcoR I .5. identify the recombinant plasmid DNA by PCR.6. normal PA317 cells7. clones of PA317 cells8. normal NIH3T3 cells9. clones of NIH3T3 cells10. identify the recombinant plasmid DNA by PCRDiscussionOf all the autoimmune diseases, none has been as popular a target of cell and gene - based prophylactic and therapeutic interventions as type I diabetes niellitus. In recent years, human proinsulin gene is cloned successfully and gene therapy of type I diabetes mellitus develops rapidly. Type I diabetes mellitus is caused by the destruction of pancreatic (3 cells, leading to insufficient insulin production . The emergency in the gene therapy is the regulated secreting of insulin and safely effective vector system.Using site - directed mutagenesis, we have introduced furin consensus cleavage sequence into the human proinsulin cDNA. Then, twe regulated elements were inserted to the upstream of mutated human proinsulin. The plasmid earring mutant human proinsulin gene can be expressed in non - β cells and secrete mature insulin out of the cells by transfection. The two regulating elements are the glucose responsive element in the liver pyruvate kinase gene promoter and insulin responsive sequence in the insulin growth factor binding protein -1...