Study on expression of recombinant plasmid PcDNA3-AMG for human amelogenin in COS-1 cell line
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Objective To investigate the amelogenin expression of recombinant plasmid PcDNA3-AMG in COS-1 cell line.Methods PcDNA3-AMG was used to transfect COS-1 cells firstly.An appropriate concentration of Zeoin was chosen for the selection of stably transfected cell colonies.The ELISA technique was applied to detect the expression of recombinant amelogenin.Results The amelogenin was detected in the stably transfected COS-1 cells in both cell culture supernatant and cell homogenate,and the concentration of expressed amelogenin in PcDNA3-AMG transfected cell homogenate was as high as 0.253 μg/ml.Conclusions The results provided basis for the preparation of recombinant AMG by PcDNA3-AMG transfected eukaryrotic cell line COS-1.Keywords:
Amelogenin
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Objective To study VEGF165 gene expression and the activity of the product in mammalian cells transfected by a recombinant plasmid pcDNA3.1(+)-VEGF165.Methods VEGF165 gene was obtained by PCR and was constructed into the expression vector pcDNA3.1(+).The pcDNA3.1(+)-VEGF165 plasmid was transfected into NIH 3T3 cells by Lipofectamine 2000.The positive clones were screened by G418.The expressed product was identified by sandwich ELISA,SDS-PAGE and Western blotting.The biological activities of the product were analyzed by cell ELISA and MTT assay.Results The expression vector pcDNA3.1(+)-VEGF165 was successfully constructed and transfected into NIH 3T3 cells.After screened by G418,the resistant cells were cultured and passaged.The VEGF165 gene expressed in the recombinant NIH 3T3 cells.The product could bind to the receptor cells ECV304 and stimulate the proliferation of ECV304.Conclusion NIH 3T3 cells transfected by the pcDNA3.1(+)-VEGF165 plasmid can express active VEGF165 protein.
Lipofectamine
3T3 cells
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The full length cDNA of human hepatocyte growth factor (hHGF) was recombinanted into pEE14 plasmid to construct eukaryotic stable expression vector. The vector was transfected into CHO K1 cells with the help of lipofectin liposome. The positive cell clone was selected under the pressure of methionine sulfoximine(MSX). RT PCR showed that positive cells expressed hHGF mRNA. The culture supernatant obtained from above system enhanced 3H thymidine incorperation into rat primarily cultured liver cells. ELISA showed that rhHGF in the culture supernatant had a concentration over 8 μg/L.
clone (Java method)
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Objective To establish the K562 eukaryotic cell line for stable expression of Ag85A protein and lay a foundation of further study on immunogenicity of oral vaccine using cationic liposome as a delivery carrier.Methods The constructed recombinant plasmid pCDNA3.1+ /Ag85A was encapsulated with LipofectamineTM 2000,a cationic liposome,and transfected to K562 cells.The expressed Ag85A protein was analyzed by SDS-PAGE and Western blot.The cell strains stably expressing Ag85A gene were screened with G418,and the expression and location of Ag85A protein were determined by flow cytometry and IFA.Results The optimal ratio of recombinant plasmid pCDN A3.1+ /Ag85A to liposome for transfection of K562 cells was 2 μg:2 μl.The expressed Ag85A protein,with a relative molecular mass of about 32 000,showed good reactogenicity.FITC markers were observed in 42.37% of cells 3 months after stable transfection with recombinant plasmid pCDNA3.1+ /Ag85A.The expressions of Ag85A were observed in cytoplasm and on cell membrane.Conclusion The K562 cells stably expressing Ag85A protein was established,which might be used for preparation of Ag85A protein in a large quantity and study on antibody against Ag85A.
K562 cells
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To construct a plasmid expressing human imprinted gene PEG10 and to study the effect of overexpression of PEG10 in a stable transfected human normal liver cell line L02 and in non-liver derived cell line 293.Full length cDNA of PEG10 open reading frame 1 was amplified and subcloned into a mammalian expression vector pcDNA3.1hisC. Recombinant plasmid was stably transfected into L02 cells and control cells via Lipofectamine 2000. The expression and the function of PEG10 in L02 cells and control group cells were examined using RT-PCR, Western blot, MTT and TUNEL.Recombinant plasmid was successfully constructed and confirmed through DNA sequencing and restriction digesting. PEG10 gene accelerated the growth of L02 cells and inhibited their apoptosis but it had no conspicuous effect on the non-liver derived cells.The constructed expressing vector pcDNA3.1hisC-PEG10 provides a useful tool for further study on the effects and mechanisms of PEG10. Over-expression of PEG10 may promote L02 cells' proliferation and inhibit their apoptosis, but not in the non-liver derived cell line 293.
Lipofectamine
HEK 293 cells
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Objective To compare the differences of expression of BMP-2 cDNA in COS cells and B16F1 cells.Methods BMP-2 cDNA was cloned into AD1-1 vector under the control of CMV promoter to construct the expression plasmid BMP-2/AD1-1.The expression vector BMP2/AD1-1 was then transfected into COS cells and B16F1 cells respectively using cationic lipid as gene delivery vector.At 72 h post-transfection,100 μL of culture medium was taken respectively from two transfected cells for Western blot to determine the expressed products of BMP-2.Results Two specifically expressed products,39 and 36,of BMP-2 were detected by western blot in culture medium of the transfected COS cells.Only one specifically expressed products,15,of BMP-2 was detected in culture medium of B16F1 cells.Conclusion COS cells express different size proteins of BMP-2,compared with B16F1 cells.
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Objective To investigate the amelogenin expression of recombinant plasmids PsecTaq2A-AMG in COS1-cell line. Methods PsecTaq2A-AMG was used to transfect COS-1 cells firstly. Appropriate concentration of Zeoin was chosen for the selection of stably transfected cell colonies. The immunohistochemistry and ELISA techniques were applied to detect the expression of recombinant amelogenin. Results The amelogenin was detected in the stably trasfected COS-1 cells in both cell culture supernatant and cell homogenate, the concentration of expressed amelogenin in PsecTaq2A-AMG transfected cell homogenate is as high as 0.877 μg/ml. Conclusion The results thus provided bases for the preparation of recombinant amelogenin by PsecTaq2A-AMG transfected eukaryotic cell line COS-1.
Amelogenin
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Objective To construct a eukaryotic expression vector for human P115 gene and investigate the effect of the expression on proliferative activity of human hepatocarcinoma HepG2 cells.Methods Human P115 gene was amplified by RT-PCR from HepG2 cells and inserted into plasmid pEGFP-N1 Vector(+).The constructed recombinant plasmid pEGFP-N1 Vector(+)-USO1 was transfected to HepG2 cells.The transfection efficacy was determined by IFA and flow cytometry.The transfected cells were determined for expressions of P115 mRNA and protein by RT-PCR and Western blot,for proliferative activity by MTT method,and for cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant plasmid pEGFP-N1 Vector(+)-USO1 was constructed correctly.The transfection efficacy of HepG2 cells with the recombinant plasmid was 84.83%.The expression levels of P115 gene and protein as well as proliferative activity of HepG2 cells transfected with the recombinant plasmid were significantly higher than those with empty vector and untransfeted.The percentage of transfected cells at G1 / G2 phases decreased significantly,while that at S phase increased significantly.Conclusion A recombinant eukaryotic expression vector for human P115 gene was successfully constructed and over-expressed in hepatocarcinoma cells,and the expressed P115 promoted the proliferation of hepatocarcinoma cells.
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Objective To construct a recombinant eukaryotic expressing vector pcDNA3.1-DCN and make it express in HepG2 hepatoma cell so as to investigate the effect of resisting tumor.Methods Using PCR,the full length of decorin cDNA was amplified.The recombinant plasmid-decorin was constructed and transformed into the bacteria.Recombinant eukaryotic expressing vector pcDNA3.1-DCN was transfected into HepG2 hepatoma cell mediated by liposome,established stable transfected cell line using G418 screening and detect recombinant secreting decorin expression using RT-PCR and immunochemistry methods.The cell activity was detected by MTT method,cell cycle was analyzed by flow cytometry.Results DCN mRNA expression was significantly increasing in transfected group using RT-PCR method.DCN protein expression also was high in transfected group using immunochemistry method.Cell growth curve displayed cell growth was slow in transfected group.The percentage of G1 phase in transfected cells was higher than in control cells.Conclusion We successfully construct the stable transfected HepG2 cell line with DCN and verify DCN can inhibit HepG2 cell line growth.
Lipofectamine
Immunochemistry
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Evidence-Based Medicine
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Objective To construct the recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/ EGFP containing gene of endostatin and promoter of heat shock protein 70 gene, and to study its expression in HepG2 cells.Methods The HSP70 promoter (350 bp) was cloned by methods of polymerase chain reaction.Fusion gene of Endo/EGFP was cut from plasmid of pSP-Endo/EGFP using restriction endonucleases of EcoR Ⅰ and Sal Ⅰ , and cloned into multiple clone site of pcDNA3.1 (+), obtaining recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/EGFP.HepG2 cells were transfected with the recombinant plasmid using lipofectine2000.Following the thermal induction at 37,39,41,43,45 ℃ for 30 min,the expression of EGFP was detected by fluorescence microscope and flow cytometry.The expression of endostatin mRNA was tested by RT-PCR.The concentration of endostatin protein in supernatant was tested by ELISA.Results The subsequence of HSP70 promoter was confirmed with that of AL671762 in GenBank.Hypothermia could induce the up-regulation of Endo/EGFP genes in HepG2 cells, especially heated at 43 ℃,its EGFP expression reached 3.3-fold of 37 ℃ group.The expression of endostatin mRNA in HepG2 cells with thermal induction was higher than that in those without thermal induction.The concentration of endostatin protein in supernatant in 43 ℃ group was (177±28) μg/L,higher than that of 37 ℃ group [(43 ± 11)μg/L].Conclusion Recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/EGFP was constructed.Hypothermia could induce HSP70 promoter to up-regulate the expression of Endo gene in HepG2 cells.
Key words:
Carcinoma,hepatocellular; Gene therapy; Heat shock protein
Endostatin
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