Conditional RNA interference achieved by Oct-1 POU/rtTA fusion protein activator and a modified TRE-mouse U6 promoter
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POU domain
Objective To study the construction and expression of anti HCC single chain immunotoxin (sFv TNF α) and GFP fusion proteins.Methods The single chain antibody(sFv) gene derived from a monoclonal antibody with high specifity and affinity binding to hepatocellular carcinoma (HCC) was first added leader sequence by polymerase chain reaction(PCR) and then fused inframe with human tumour necrosis factor α(TNF α)gene. The recombinant sFv TNF α gene was cloned into an expression vector pEGFP N3 containing the report gene of GFP and the recombinant vector was first identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. To investigate whether mammalian cells could produce fusion protein, human HCC cell line SMMC 7721 cells were transfected with the plasmid pEGFP ST by Calcium Phosphate method.Results The recombinant vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. Bright green fluorescence in the cytoplasm of the transfected SMMC 7721 cells was observed under the fluorescence microscope 20 hours after transfection of the recombinant vector. Conclusions The gene of single chain immunotoxin was constructed. The fusion proteins of anti HCC single chain immunotoxin and GFP were successfully expressed in SMMC 7721 cells. It is suggested that mammalian cells can express immunotoxin fusion proteins, which may be theoretically necessary for our next step of study in a gene therapy.
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Objective Through constructing prokaryotic expression vector pET-30a-GPI-B7-1, to gain purified GPI-B7-1 fusion protein so as to confirm the tumor immune effect. Methods The DNA fragment encoding the signal for GPI-anchor attachment of hPLAP-1 and the cDNA encoding the human costimulatory molecule CD80 ( BT-1 ) were cloned from fresh placenta and human peripheral blood monocytes (PBMC) respectively. The two fragment were annealed to form a fusion gene (GPI-BT-1) by PCR. Then the fusion gene was inserted into the prokaryotic expression vector pET-30a, resulting in pET-30a-GPI-BT-1. Transfer to E. coli BL21, purified fusion protein were analysed by SDS-PAGE and Western blot. Results Agarose gel electrophoresis map of GPI and BT-1 PCR products show that GPI goal gene strap was seen at 133bp region and BT-1 goal gene strap at 792 region. Identification of recombinant pET-30a-GPI-B7-1 by restriction enzyme and PCR illustrate two goal fragment for 5000 bp and 900 bp, to realize the expression of fusion gene ( GPI-B7-1 ) at the E. coli BL21. The fusion protein was successfully produced in the pET expression system induced by IPTG and purified by Ni2 + -NTA agarose column. By SDS-PAGE and Western blot analysis, the observed molecular weight of the fusion protein was 38 kDa. Conclusion The purified GPI-B7-1 fusion protein can be obtained from E. coli BL21 transfered by prokaryotic expression vector pET-30a-GPI-B7-1, which will prove useful tool for the study of tumor immune therapy.
Key words:
Gene expression; Glycosylphos phatidylinositols
Agarose
Agarose gel electrophoresis
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AIM:To track the trace of Leptin in vivo ,designing constructing the Leptin fluoresent protein probe.METHOD:With polymerase chain reaction(PCR),constructing the LG fusion gene,which the Leptin cDNA was connected with 5′end of the cDNA of green fluoresent protein gene(GFP).The LG fusion gene was cloned into pET3c expression vector.The recombinant plasmid was transformed into BL21(DE3) for expressing the aim protein.Using the Western bloting to analyze the immunogenic of the Leptin,and using the fluorescence microscope to observe the green fluorescence of the fusion protein and the cell after induceing.RESULT:It was confirmed that the DNA sequence of the synthetic fusion gene was identical with that of the designed gene by DNA sequencing.The recombinant was expressed in BL21(DE3) successfully.The expression level of the recombinant LG in BL21(DE3) was above 40% of the total protein.Western blot analysis shows that the recombinant protein has the immunocompetence of leptin.Fusion protein and the cell after induceing can emit the green fluorescence.CONCLUSION:Recombinant LG gets overexpression in E.coli BL21(DE3) successfully.The fusion protein of expressed has the immunocompetence of Leptin and the normal function of the GFP.
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AIM To construct a recombinant eukaryotic expression vector containing pcDNA3.1-mOX40-Ig fusion gene and obtain mOX40-Ig fusion protein with bioactivity by transfecting CHO cells. METHODS The gene fragment encoding the human IgG1Fc was amplified by RT-PCR and the eukaryotic expression vector pcDNA3.1-hIgG1Fc was constructed. After sequencing, mOX40 extracellular gene was cloned from pIRES2-EGFP-OX40 by PCR and then inserted into the recombinant vector pcDNA3.1-hIgG1Fc. The right recombinant was transfected into CHO cells with lipofectin Ragent and its expression was detected by RT-PCR and sandwich-ELISA. After purified by protein A affinity column chromatography, the mOX40-Ig fusion protein was identified by SDS-PAGE and its effect on the proliferation of B cells in vitro was studied by (3)H-TdR method. RESULTS The hIgG1Fc, mOX40 extracellular gene and mOX40-Ig gene were consistent with DNA sequencing.The expression of mOX40-Ig fusion protein in CHO cells was confirmed by RT-PCR, sandwich-ELISA and SDS-PAGE. (3)H-TdR analysis showed the mOX40-Ig fusion protein stimulated the proliferation of B cells in vitro. CONCLUSION A eukaryotic expression vector containing pcDNA3.1-mOX40-Ig has been constructed successfully and the stable expression of mOX40-Ig fusion protein with bioactivity has been acquired, which lays a solid basis for further study of the application related to OX40.
Myc-tag
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A hydrophobic polypeptide linker (Gly_(4)Ser)_(3) was used to splice two different gene fragments of p40 and p35 subunits of hIL-12 (hIL-12 p40-linker-p35) by recombinant PCR for constructing rhscIL-12 fusion gene in vitro. After sequencing, rhscIL-12 fusion gene was inserted into pcDNA3.1(+) eukaryotic expressing plasmid and then transfected into COS-7 cell by lipofection. The fusion protein expressed was analysed with Western blot. Sequence analysis showed that the splicing order, the direction and the sequence in hscIL-12 fusion gene were completely correct. Expressed hscIL-12 fusion protein in COS-7 cells combined with mouse-anti-human IL-12 monoclonal antibody specifically. Western blot showed the apearance of 70 000u band of fusion protein. The result indicates that recombinant PCR is a very effective and reliable method for gene recombination in vitro.
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To construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function.RCAS1 encoding gene was amplified by RT-PCR from total RNA extract of MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases BamH I and EcoR I. Then the ligated products were inserted into competence JM109 E. Coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. Coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-RCAS1 (N-18) and anti-RCAS1 (C-20) polyclonal antibody. The apoptosis of activated T cells induced by GST-RCAS1 fusion protein was detected by flow cytometry with Annexin V and propidium iodide (PI) staining.A 642 bp product was cloned by RT-PCR and the recombinant plasmid was constructed successfully. The GST-RCAS1 fusion protein was recognized by GST monoclonal antibody and RCAS1(N-18 and C-20) polyclonal antibody. FACS analysis showed that GST-RCAS1 fusion protein induced apoptosis in activated T cells.The recombinant plasmid of RCAS1 has been successfully constructed and the GST-RCAS1 fusion protein expressed and purified. The apoptosis inducing effect of GST-RCAS1 fusion protein on activated T cells is demonstrated.
Polyclonal antibodies
Propidium iodide
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Finding putative transcription factor binding sites in the upstream sequences of similarly expressed genes has recently become a subject of intensive studies. In this paper we investigate how much gene expression regulation can be attributed to the presence of various binding sites in the gene promoters by correlating the binding sites and the changes in gene expression resulting from gene disruptions (e.g. knockouts).We have developed a data analysis method for comparing mRNA measurements of gene disruption experiments with information about gene promoters. The method was applied to a well-known dataset to uncover correlations between known transcription factor binding site motifs in the upstream regions of all S. cerevisiae genes and the gene expression changes in various gene disruption experiments. The possible explanations of the correlations were categorized and analyzed using e.g. expression cascades. Several correlations turned out to be consistent with existing biological knowledge while some new ones suggest themselves for further study.The resulting tables are available at http://www.cs.helsinki.fi/u/kpalin/CorrDisrupt/.
DNA binding site
Gene prediction
Gene knockout
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Objective: To construct and express a fusion protein of a single chain antibody(scFv) to human inte-grin αvβ3 and soluble human tissue factor(sTF). Methods: The scFv gene was amplified by overlapping extension PCR and the sTF gene was synthesized by assembly PCR technique, and they were joined together as a fusion gene and were cloned into the prokaryotic expression vector pQE80L to construct the expression plasmid pQE80L-scFv-sTF, which was expressed in E.coli M15. Results: SDS-PAGE analysis showed that the expressed recombined protein had a molecular weight of 55 kD. Western blot analysis confirmed that the protein was tagged with 6×His. The scFv-sTF protein was expressed as soluble protein under the condition of low concentration IPTG induction at low temperature. The reconbinant protein was purified and ELISA suggested that the recombinant fusion protein could bind to the human integrin αvβ3. Conclusion: The fusion protein scFv-sTF have been expressed, which lays the foundation for the analasis of its anti-tumor activities.
Protein A/G
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AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein(anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells.METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene.To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP,we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A.The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid.After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4,the recombinant virus was collected to infect Tn-5B1-4.SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4.The fusion protein was purified with Ni2+-NTA affinity chromatography.The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7.The binding effects on the surface of breast cancer cells were observed under laser confocal microscope.RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp.The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope.A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting.Under laser confocal microscope,strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3.However,no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7.Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS.CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4,and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.
SKBR3
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