Construction of eukaryotic expression vector pIRES_2-eGFP-Annexin A2 and expression of Annexin A2 in 293T cells
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Objective: To construct pIRES2-eGFP-Annexin A2 eukaryotic expression vector carrying human Annexin A2 gene and express Annexin A2 protein in 293T cell.Methods: Annexin A2 gene digested from pCMV5-Annexin A2 was ligated into eukaryotic expression vector pIRES2-eGFP.After restriction analysis and sequencing,the recombinant plasmid pIRES2-eGFP-Annexin A2 was transfected into 293T cells in the mediation of liposome.The expression of Annexin A2 was analyzed by western blot and real-time PCR. Results: The expression of Annexin A2 at both mRNA and protein levels were significantly increased when transfected with pIRES2-eGFP-Annexin A2 in 293T cells. Conclusion: The eukaryotic expression vector pIRES2-eGFP-Annexin A2 was correctly constructed and the Annexin A2 protein was successfully expressed in 293T cells.This will facilitate the following study on Annexin A2.Keywords:
Annexin A2
HEK 293 cells
Annexin A1
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AIM: To construct the eukaryotic expression vector of the c-Jun N-terminal kinases 3(JNK3)and stably transfect HEK293 cells with it,and to research the relationship between JNK3 and the cell apoptosis by using epirubicin induction.METHODS: The full-length JNK3 cDNA fragment was amplified by PCR from the pDBLeu-JNK3 plasmid and was inserted into eukaryotic expression vector pcDNA3.1/myc-His B.After identification by PCR and restriction digestion,the recombinant plasmid was transfected into HEK293 cells by lipofectamine.After screening culture by G418,a stably-transfected cell line was established,and the transcription and expression of the Myc tag JNK3-carrying fusion protein were identified by RT-PCR and Western Blot.The cell apoptosis induced by epirubicin was detected by flow cytometric analysis.RESULTS: The eukaryotic expression vector pcDNA3.1-JNK3 was successfully constructed and the JNK3 gene was transfected stably into HEK293 cells.The JNK3 gene was expressed successfully.Epirubicin significantly promoted the apoptosis of JNK3 gene transfected HEK293 cells.CONCLUSION: The study provides a solid experimental foundation for further studies on the function of the JNK3 gene.
Lipofectamine
HEK 293 cells
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To construct the eukaryotic expression vector pIRES2-EGFP-Axin, and to express Axin in C6 glioma cells.The Axin gene was amplified by PCR using pCMV5-HA-Axin as a template, and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-Axin was constructed by introducing Axin DNA fragment into the sites of Nhe I and Sal I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the Axin protein expression was detected by immunostaining using anti-Axin antibody.The eukaryotic expression vector pIRES2-EGFP-Axin was constructed and transfected successfully into C6 glioma cells. The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and Axin protein was only found in the plasma.The recombinant expression vector pIRES2-EGFP-Axin was constructed, and the EGFP and Axin gene could be co-expressed in the C6 cells. This study laid a foundation for the further research of the function of Axin in cell differentiation, growth and tumorigenesis.
Lipofectamine
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Objective To construct eukaryotic expression vector of pcDNA3.1(+)/hSp17 containing human sperm protein 17(hSp17) cDNA,so to provide basis for its clinical application.Methods hSp17 cDNA was obtained by PCR,and to construct eukaryotic expression vector pcDNA3.1(+)/GNLY by NheⅠ,KpnⅠdigestion and sticky end ligation techniques.The recombinant plasmid was transfected into H08910 cells with lipofectamine 2000.Expression of hSp17 in the transfected cells was observed by immunocytochemistry.Cells with stable expression of hSp17 were selected by G418.Results Sequencing and restriction enzyme digestion showed pcDNA3.1(+)/GNLY was correctly constructed.The stably hSp17 gene transfected HO8910 cell clones were obtained through G418 screening and immunocytochemistry assay were further performed to confirm the expression of hSp17 in the HO8910 cells.Conclusion An ovarian carcinoma cell model that stably expressed hSp17 can be successfully established;which can provide basis for immunotherapy of ovarian carcinoma.
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This study designed to construct and express original oncogene c-myc eukaryotic expression vector pIRES2-AcGFP1-Nuc-c-myc using gene engineering technique.The c-myc gene was obtained by PCR amplification from pET28a-c-myc,to constructed pIRES2-AcGFPl-Nuc eukaryotic expression vector,which was then confirmed by PCR method,restriction analysis and DNA sequencing.In addition,the recombinant plasmid pIRES2-AcGFPl-Nuc-c-myc was transfected to Hela cells,and green fluorescent protein was expressed successfully under fluorescence microscopy.Analysis of Western blotting showed that c-myc protein expression was increased in Hela cell,and MTT assay indicated c-myc protein could promote the proliferation of Hela cells.In conclusion,eukaryotic expression vector of c-myc gene was constructed and transfected,which lay foundation for the lamprey cell line research.
HeLa
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To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells.Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing. The hTERTpromoter-pIRES2-EGFP-TRAIL and CMV promoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were constructed respectively. The recombinant plasmids were transfected into ovarian carcinoma cell line, SKOV3, and the levels of mRNA were determined by RT-PCR. The cell cycle and apoptosis rate of SKOV3 cells were determined by FCM.The constructed two recombinant vectors were verified by restriction enzyme digestion analysis and DNA sequencing. After being transfected with two recombinant vectors, the growth of SKOV3 cells was strongly inhibited and apoptotic features appeared.The recombinant eukaryotic expression vector has been constructed successfully. TRAIL gene driven by hTERTpromoter can be obviously expressed in ovarian carcinoma SKOV3 cells, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.
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Objective To construct an eukaryotic expression vector of human vascular endothelial growth factor (VEGF 165) gene, and to investigate the transfection and expression of pCR3.hVEGF 165 eukaryotic expression vector in rat myocardial cells.Methods pCR3.hVEGF 165 eukaryotic expression vector was constructed. Primarily cultured rat cardiomyocytes were transfected with liposome/plasmid pCR3.hVEGF 165. RT-PCR, Southern blot, immunohistochemical method and ELISA were used to detect the expression and secretion of VEGFgene.Result There were significant increases in VEGFmRNA and protein in the myocardial cells transfected with pCR3.hVEGF 165.Conclusions The pCR3.hVEGF 165, an eukaryotic expression plasmid for hVEGF 165 gene,was constructed. High levels of VEGFmRNA and protein expression could be obtained in the myocardial cells transfected with pCR3.hVEGF 165 eukaryotic expression plasmid.
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Objective To construct a eukaryotic expression vector of microRNA-7 and study its effect on cell proliferation.Methods MicroRNA-7 was amplified from human genomic DNA isolated from whole blood and then inserted into pHRS-1cla-CMV-EGFP vector to generate pHRS-1cla-miR-7-CMV-EGFP vector.Subsequently,miR-7 expression vector pHRS-1cla-miR-7-CMV-EGFP and the blank vector pHRS-1cla-CMV-EGFP were transfected into 293FT cells.The expression of mature miR-7 was verified at the transcription level using RT-PCR and real-time RT-PCR.The two plasmids were transfected into the breast cancer cell line MCF-7 cells,followed by CCK-8 and BrdU incorporation assays to measure cell proliferation.Results Restriction enzyme digestion and plasmid sequencing confirmed the successful construction of pHRS-1cla-miR-7-CMV-EGFP vector.By real-time PCR,293FT cells increased miR-7 expression 72 hours after transfection(about 76.8 fold compared to the blank vector transfection cells),which suggested the high expression efficiency of the construct.The results of CCK-8 and BrdU assays showed that the overexpression of miR-7 in MCF-7 cells could significantly inhibit cell proliferation(P0.05).Conclusion The recombinant lentiviral eukaryotic expression vector pHRS-1cla-miR-7-CMV-EGFP is successfully constructed.RT-PCR and real-time PCR results indicate that the mature miR-7 is effectively expressed in 293FT cells.CCK-8 and BrdU incorporation results suggest that overexpressed miR-7 obviously inhibit cell proliferation.
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Objective To construct a eukaryotic expression vector for signal regulatory protein-α(SIRP-α) gene and express in human lung cancer A549 cells. Methods Total RNA was extracted from Hep G2 cells, with which SIRP-αgene was amplified by RT-PCR and inserted into eukaryotic expression vector pc DNA3. 1(+)-EGFP. A549 cells were transfected with the constructed recombinant plasmid pc DNA3. 1(+)-SIRP-α∕EGFP in mediation of Lipofectamine 2000,observed by fluorescent microscopy 24 h later, and determined for transcription level of SIRP-α gene by RT-PCR 48 h later and for expression level of SIRP-α by Western blot 72 h later. Results Restriction analysis and sequencing proved that recombinant plasmid pc DNA3. 1(+)-SIRP-α / EGFP was constructed correctly. Green fluorescence was observed in A549 cells 24 h after transfection with the plasmid. Target gene band at a length of 243 bp was proved by RT-PCR while the target protein band by Western blot. Conclusion Eukaryotic expression vector pc DNA3. 1(+)-SIRP-α / EGFP was successfully constructed and expressed effectively in A549 cells, which laid a foundation of further study on the rela-tionship of SIRP-α to lung cancer.
Lipofectamine
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[Objective] The aim of this paper was to construct and identify the siRNA eukaryotic expression vector targeting annexin A3.[Method]s Four shRNAs were designed according to the coding sequence of annexin A3 gene,and cloned into the downstream of H1 promoter of psiRNA-hH1-neo.The constructed recombinant was analyzed and identified by Ase Ⅰendonuclease digestion and DNA sequencing.[Result]s The constructed psiRNA plasmid digested with Ase Ⅰwas linearized.The sequencing result confirmed that the sequence of inserted fragment was correct.[Conclusion] Eukaryotic expression vector of siRNA targeting annexin A3 gene was successfully constructed.
Coding region
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Objective:To construct and express the eukaryotic expression vector of human autophagy related gene 12(ATG12) labeled with myc tag.Methods:Human ATG12 gene was obtained from human mammary gland cDNA library by PCR and cloned into myc vector.Human 293 T cells were transfected with the recombinant plasmid of myc-ATG12 and the expression was detected by Western blot.In addition,immunoprecipitation assay was applied to determine the interaction between myc-ATG12 and flag-ATG3.Results:ATG12 eukaryotic expression vector labeled with myc tag was successfully constructed by double digestion identification.The inserted fragment was confirmed correct by sequencing.The expression of human ATG12 protein in human 293 T cells was identified by Western blot.The immunoprecipitation assay results showed that human ATG12 protein could interact with Flag-ATG3 in vivo.Conclusion:The eukaryotic expression vector of myc-ATG12 was successfully constructed and expressed in human 293 T cells,which had laid foundation for the further study of the role of ATG12 in autophagy.
ATG12
Immunoprecipitation
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