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Objective To construct siRNA expression vector of XIAP,and study its effect on XIAP expression in Hep3B cells. MethodsThree XIAP siRNA sequences were designed,synthesized,and cloned to pRNAT-U6.1/Neo.The successfully constructed recombinant plasmid was determined by sequence analysis,and will be transfected into Hep3B.The best interference plasmid were analyzed by RTPCR,Western blot,and immunohistochemistry.Results The plasmid of pRNAT-U6.1/Neo-XIAP was constructed successfully,the trans-fected with different plasmid of siRNA XIAP can lower significantly XIAP.Conclusions The siRNA vector of XIAP gene was constructed successfully.It will be a basis for the study of XIAP function in apoptosis regulation of the Hepatoma cells.
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Objective To investigate the inhibitory effect of downregulation of hepatitis B virus (HBV) core gene (HBcAg) expression by RNA interference and magnetic nanoparticles on both HBV DNA replication and expression in vitro. Methods HepG2 2.2.15 cells were transfected with U6 promoter plasmids coding for small interfering RNA (siRNA) targeting HBV core gene using magnetic nanoparticles. RT-PCR and Western blot were used to assess the mRNA and protein expression HBV core antigen. Real-time PCR was used to evaluate the suppression efficiency of HBV-DNA replication and expression; and radioimmunoassay was used for HBV surface antigen (HBsAg), core antigen (HBcAg), and e antigen (HBeAg) detection. Results We successfully constructed nanoparticles with siRNA plasmid targeting HBV core antigen; HBcAg mRNA and HBV core antigen protein levels were significantly reduced in the transfected cells. HBV-DNA downregulation was estimated at 4-5 logs and the HBsAg and HBeAg levels were also reduced compared with the controls. Conclusion Downregulation of HBV core gene using RNAi technology and magnetic nanoparticles can potentially be used as a therapeutic strategy for Hepatitis B.
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Objective To provide a technique of preparation recombinant hALR by gene engineering in larger scale.Methods Construct hALR(human augmenter of liver regeneration) protein expression vector pGEX-3X-hALR,induce the expreesion of hALR,and pruified the protein by affinity chromatography.Results Succeed in constructing the vector pGEX-3X-hALR and obtained the recombinant hALR protein.Conclusion The mothed could be used for preparation recombinant hALR by gene engineering in larger scale.
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Objective To construct ICAM-1 recombinant eukaryotic expression vector. Methods Human intercellular adhesion molecule-1 (ICAM-1) cDNA was obtained by RT-PCR of totol RNA extracted from human hepatocellular carcinoma tissue. Amplified ICAM-1 cDNA fragment was cloned into pGEM-T easy vector to construct pGEM-ICAM-1 vector. Then ICAM-1 cDNA from pGEM-ICAM-1 vector was cloned into eukaryotic expression pcDNA3.1hisB to construct recombinant pcDNA3.1hisB-ICAM-1 vector. Restriction endonuclease digestion and DNA sequencing were used to confirm the recombinant vector. Results 1622bp ICAM-1 cDNA was obtained by RT-PCR. The PCR product was successfully ligated with pGEM-I easy vector. Restriction endonuclease digestion analysis and DNA sequencing showed that recombinant pcDNA3.1HisB-ICAM-1 was successfully constructed. Conclusion Eukaryotic expression recombinant vector pCDNA3.1hisB-ICAM-1 was contructed.
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0.05).Conclusion The significantly reduced serum IGFBP-3 level is helpful for the diagnosis of HCC,especially in patients without chronic hepatitis and cirrhosis.
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Objective:To evaluate the expression of apoptosis related gene Fas Ligand(FasL) in human hepatocellular carcinoma(HCC) cells HepG2 and its significance in apoptosis.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1hisB- FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells were detected by immune histochemistry. After stained by annexin V and propidium iodine, cells were passed throw flow cytometer and examined by fluorescence microscope and sym-focus laser scaning microscope.Results:In comparison with untransfected cells,the soluble FasL could be detected in the supernatant of transfected cells, Fas L can be expressed in the membrane and cytoplasm of transfected cells. The apoptotic cell rate in transfected cells was 36.30%, as the control, untransfected cells was 11.53%.Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine stain.Conclusion:This supports a novel pathway of HCC cells were apoptotic itself via the CD95-CD95 ligand system without involvement of immune cells.
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Abstract Objective:To observe and compare the effects of immune responses of HBV DNA caccine by insertion IL-2 gene.Try to ex-Plore the efficient way to enhance the immune effect of DNA vaccines. Methods:The recombinant expression plasmid vector peDNA3.1-S/IL-2with fusion gene of HBsAg and IL-2 cloned was constructed by PCR method and DNA recombinant technique.Then, it was transfered into Cos-7 cells by cation lyposomes and was detected its transient expressing product in vitro.At last,observed and compared the cellular and humoralimmune responses to HBsAg in C57BL/6 mice by intramuscular injection.Results:The pcDNA3. 1-S/IL-2 was constructed successfully by themethod of restriction endonucleases digestion, RCR and DNA sequencing. It can increase the titers of anti-HBs antibody. The bioactivity of IL-2and lymphocyte proliferation responses to HBsAg induced by the mouse spleen immunized with psDNA3.1-S/IL-2 were improved also. Conclu-sion:These results showed the expression of HBsAg-IL-2 fused gene can enhance the immune responses and suggested the insertion of IL-2 maybe a practicable way to enhance the DNA vaccine efficacy.