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Construction Eukaryotic Express Vector of Human S100A13 Gene and Its Effect on Cell Cycle of HepG2

Author: LiuYingLan
Tutor: CaoRenXian
School: Nanhua University
Course: Oncology
Keywords: S100A13 gene HepG2 cells Cell cycle
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 23
Quote: 0
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Abstract


Objective: To construct the the S100A13 eukaryotic expression vector , the observed high expression of S100A13 gene on HepG2 cell cycle and to explore the role of S100A13 gene in hepatocellular carcinoma . Methods : pGEM-T-S100A13 vector extract preserved in the laboratory strains JM109 plasmid , design primers for PCR amplification . The PCR product was cloned to pcDNA3.2/V5-topo carrier , to build pcDNA3.2/V5-S100A13 eukaryotic expression vector , and transformed into E.coli Top10 after ampicillin screened positive clones , shake bacteria amplification , to extract plasmids were identified by PCR and insertion sequence identified by sequencing . Application of Lipofectamine 2000 the eukaryotic expression the carrier pcDNA3.2/V5-S100A13 and empty vector pcDNA3.2/V5 import HepG2 cell line , 48 hours after the indirect immunofluorescence staining S100A13 protein subcellular localization , and application of flow cytometry high express S100A13 gene on HepG2 cell cycle . Results: successfully constructed the the S100A13 eukaryotic expression vector pcDNA3.2/V5-S100A13 . The green fluorescence 48 hours after the observation showed that the transfected HepG2 HepG2- V5 - S100A13 group - green fluorescence evenly distributed throughout the cell , and the HepG2-V5 and HepG2 green fluorescent cytoplasmic distribution significantly reduced , mainly in the nucleus . Flow cytometry the HepG2 -V5 - S100A13 the HepG2- V5 and HepG2 three groups of cells , the proportion of S - phase cells ( 24.90 ± 0.66 ) % , ( 17.11 ± 0.23 ) % and ( 16.7 ± 0.36) % ( P lt ; 0.01) , the proportion of G2 / M phase of the cell were ( 12.19 ± 0.14 ) % and ( 7.26 ± 0.23 ) % and ( 7.51 ± 0.28 ) % (P lt ; 0.01) . Conclusion: We successfully constructed the the S100A13 gene eukaryotic expression vector of HepG2-V5-S100A13, high expression of the the S100A13 gene can promote HepG2 cells G1 to S and G2 / M phase transformation endogenous S100A13 are mainly located in the nucleus in HepG2 cells suits endogenous S100A13 is localized in the cytoplasm .

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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