Construction And Identification Of Recombinant Retroviral Vector Carrying Modified Human Proinsulin Gene

Type 1 diabetes mellitus is caused by severe insulin deficiency secondary to the autoimmune destruction of pancreatic (3 cells. We can tranfer human proinsulin gene into diabetes mellitus patients, produce long - term , constant, physiobiological controll in the proposal of permanent therapy . So selecting the vector of gene transfer is threme important to gene therapy of diabetes mellitus.pLXSN, one new type of recombinant retroviral vectors, is based on MoMLV and possesses common digesting sites with the plasmid PMD18 -T vector carrying modified human proinsulin gene,so we can use standard molecular techniques to reconstruct new plasmid and to transfer MpINS gene into host cell for further research.One porpose of this trial is to master recombinant molecular techniques and identification methods of recombinant vector, the other is to construct recombinant retroviral vector carrying modified human proinsulin gene for the further research.MATERIALS AND METHODS1. Identify modified human proinsulin by sequence measure.2. The construction of pLXSN - MpINS:(1) Double digesting pLXSN with restriction enzymes Hpa I and BamHI.(2) After digesting the plasmid carrying modified human proinsulin with restriction enzyme Sal I and Blunting kination and ligation , digest it with restriction enzyme BamH I again.(3) Receive the aim gene sections in( 1) and(2) by agrose swimming.(4) Ligate digested pLXSN and the plasmid with TaKaRa ligation kit into pLXSN - MpINS.(5) Transform ligation mixture into E. coli HB101.( 6 ) Select positive recombination and purify it for further identification.3. Identify the desired recombinant plasmid :(1) confirm the recombination by PCR.(2) confirm orientation by restriction analysis with EcoR I and BamH I.RESULTS1. Measure sequence confirms that sequence of modified human proinsulin cDNA is correct.2. Construction of recombinant retroviral vector carrying modified human proinsulin gene:(1) Because Hpa I and BamH I respond 1477bp and 1491bp site respectively in the pLXSN(5. 9kb) , after double digesting , we can receive 5860bp section . See picture 5.(2) Sal I and BamH I respond 33 bp and 452bp site respectively in the PMD18 - T carrying MpINS, After digesting it with Sal I and BKL,digest it with BamH I, we can receive 418bp section,which is proved to be desired gene by standart Marker . See Picture 6.3. Identification of recombinant pLXSN - MpINS:(1) Identify recombinations by PCR: using pLXSN - MpINS cD-NA as model, amplificate and receive aim sections by PCR, agarose swimming identification. See picture 7.(2) Identify orientation by restriction analysis : the sections received 418bp and 5860bp sections through restriction enzymes BamHI and EcoR I are desired recombinations. See picture 8.DISSCUSSIONRetroviral gene transfer technology is a kind of efficient means through which target gene is transferred into host cells. RV can not only transfer high - efficiently but also integate aiming gene into genome of host cells ,and pass it to the next generation through cell division, which is often applied during gene therapy. pLXSN, a kind of new type RV vector, is based on MoMLV and is reconstructed; it results from the N2 generation of RV and belongs to the vector with interior promoter classfied by constructoin. Its structural genes such as gag, pol, env and so on are removed, and start codon ATG for translation of gag gene,is replaced by stop codon TAG,and 5bf MoMLV sequence is replaced by that of MoMSV sequence, thus further decreases rate of theheterologous recombination, which decreases the potent transferring diseases through RV and has higher safety. Some professors have succeeded in transferring and expressing ADA , clotting factor VIII , GDNF by pLXSN.Insulin is synthesized in the β cells within the islets of langerhans of the pancreas as a preprohormone. The signal sequence is cotransla-tionally removed in the endoplasmic reticulum followed by the trafficking of the prohormone to...