| Carbamyl Phosphate Synthetase I (CPSI ) is the first key-enzyme,mainly located in the hepatocyte mitochondria. In this experiment, the two CPSI subclones containing modified 0.587kb and 0.712kb fragments was first prepared ,for favouring recombination of CPSI cDNA with pSV2 vector and suiting its structure to gene expression.Using bal31 and S1 nuclease, from 5'terminus we deleted DNA fragments recovered from PHN3491 plasmid digested by EcorRI which contain full-length CPSI cDNA fragments.A mumber of the shorter CPSI cDNA fragments were recovered from the mixture of CPSI cDNA deletion fragments digested with bamHI and then inserted these fragment into pBSKS plasmids at EcoRV/BamHI cloning site.By analysis of RE mapping and double strand DNA template sequencing .We picked out pBS-CPS05 and pBS07recombinant plasmids, which contain 0.587kb and 0.712kb CPSI cDNA fragments. Both of two CPSI fragment have ATG codens and are added a hindIII site at their 5'terminus. The 0.712kb CPSI DNA have a complete leader sequence but 0.587kb CPSI gene only has 8 bp ones.There are no other changesinvolving in CPSI structure.Sequentialy, we reconstructed PSV vector into PSV2E- and PSV2HEB by destroying EcoR I site and adding two EcoRI-BamHI adaptors at BgIII cloning site for increasing its cloning adaptorbility.These modify was confirmed to have no effect on function of PSV2 by mammalian gene expression.Basing on the previous works, we constructed pSV2CPS505 and pSV2-CPS507 plasmids by recovering 0.587kb and 0.712kb CPSI cDNA fragments with hindIII and BamHI digestion and inserting directly them to pSV2 vector at HindIII/BglII region.The results of multiple RE mapping and Southern blot analysis demonstrate that these two plasmids have correct cloning orientation and perfect structure. Using DNA Calcium phosphate co-recipetation mediated gene transfection, we transferred pSV2-CPS505 and pSV2-CPS507 into cells, and the transformed pSV2-CPS505-NIH/3T3 and pSV2-CPS507-NIH/3T3 cells were determined to express specific CPSI mRNA and pepitides by methods of indirect immuno-fluorescent, iinmuno enzyme-coupled antibody staining and run-off transcription assay.Then we set up pSV2 CPS505 and pSV2-CPS507stable expression cell lines by screening with G418 In construction of pSV2-CPScF, we used 0.712kb and 3.9kb CPSI cDNA fragments as foreign genes and the modified PSV2HEB as vectors.The structure of 0.712kb CPSI cDNA gene have been proved to be suitable to gene expression The 3.9kb CPSI cDNA ,which contain residual structure gene sequence except 0.7kb CPSI cDNA and "polyA" sequence, is recovered from pHN3491 by BamHI/EcoRI digestion.By multiple DNA fragment recombination, we linked sequentially and directly the two CPSI cDNA fragments with PSV2HEB .Using 0.712kb and 3.9kb CPSI cDNA as probes respectively, we select several positive colonies from transformed bacterials.By multiple RE mapping and Southern blot.we demonstrated that PSV2-CPScF expression plasmids have correct cloning orientation and perfect structure agreeing with our design.By methods of DNA-Calcium phosphate co-precipitate, we transferred PSV2-neo into CHO,NIH/3T3 and 7402 hepatoma cells , and identified CPSI expression by indirect immunoflurenscent, immunoenzyme-coupled antibody staining, Northern blot and run off methods.The results show that full-length CPSI gene express successfully within all three cell lines.Then we established PSV2-CPSIcF-NIH/3T3, PSV2-CPSI-CHO and PSV2-CPSIcF-7402 stable expression systems which express CPSI by screening with G418.AU of these results give the following suggestions expression of CPSI cDNA gene of different length is possible in non-liver cell lines including hepatoma.The leader sequence of CPSI gene have no relation to CPSI tissue-specific and differentiation-peoriodic gene expression.The fact of decrease of CPSI activity during oncogenesis due to decrease of CPSI transcriptional activity is further confirmed.PSV2 vectors is able to express approximate 5.0kb in full length cDNA gene.Our expression systems offer a mo... |
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