[Construction, eukaryotic expression and biological activities of a recombinant human single chain interleukin-12 fusion gene].
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To explore the feasibility of recombinant human interleukin 12 (IL-12) with biological activities by molecular biological techniques.Both p40 and p35 subunits cDNA of human IL-12 were cloned from mRNA extracted from NC-37 cell line by using RT-PCR, and the fusion gene (p40-linker-p35) of recombinant human single chain IL-12 (rhscIL-12) was constructed by using a polypeptide linker (Gly(4)Ser)(3). rhscIL-12 eukaryotic expressing vector pcDNA3.1 (+)-hscIL-12 was constructed by inserting the rhscIL-12 fusion gene into pcDNA3.1 (+) eukaryotic expressing plasmid. COS-7 cells were transfected with pcDNA3.1 (+)-hscIL-12 plasmid.A stable rhscIL-12 expressing cell line COS-rhscIL-12 was obtained by G418 selection. Western blot showed the presence of a 70 x 10(3) band of the fusion protein, which specifically bond to mouse-anti-human IL-12 monoclonal antibody. The assays of biological functions showed that the fusion protein had strong bioactivities in stimulating the lymphocyte proliferation, enhancing the NK cell cytotoxicity and increasing the IFN-gamma production.The constructed rhscIL-12 fusion gene could express biologically functional rhscIL-2 fusion protein.Keywords:
Cell fusion
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To explore the feasibility of recombinant human interleukin 12 (IL-12) with biological activities by molecular biological techniques.Both p40 and p35 subunits cDNA of human IL-12 were cloned from mRNA extracted from NC-37 cell line by using RT-PCR, and the fusion gene (p40-linker-p35) of recombinant human single chain IL-12 (rhscIL-12) was constructed by using a polypeptide linker (Gly(4)Ser)(3). rhscIL-12 eukaryotic expressing vector pcDNA3.1 (+)-hscIL-12 was constructed by inserting the rhscIL-12 fusion gene into pcDNA3.1 (+) eukaryotic expressing plasmid. COS-7 cells were transfected with pcDNA3.1 (+)-hscIL-12 plasmid.A stable rhscIL-12 expressing cell line COS-rhscIL-12 was obtained by G418 selection. Western blot showed the presence of a 70 x 10(3) band of the fusion protein, which specifically bond to mouse-anti-human IL-12 monoclonal antibody. The assays of biological functions showed that the fusion protein had strong bioactivities in stimulating the lymphocyte proliferation, enhancing the NK cell cytotoxicity and increasing the IFN-gamma production.The constructed rhscIL-12 fusion gene could express biologically functional rhscIL-2 fusion protein.
Cell fusion
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1762 Objectives To investigate the changes of iodide uptake by transfection of a recombinant eukaryotic expression plasmid pcDNA3.1/hTSHR-cDNA in FTC-133 cell lines. Methods The recombinant plasmid pcDNA3.1/hTSHR was verified by restriction enzyme digestion and DNA sequencing. Then it was transfected into FTC-133 cell lines by Lipofectin method. Target protein expression was detected by immunofluorescence and the mRNAs were detected by real-time PCR. Radioiodine uptake was measured with a gamma counter. Statistical analyses was performed by t-test analysis using SPSS 13.0 software. A value of P Results The sequence analysis confirmed that pcDNA3.1/hTSHR had been constructed successfully. After transfection with a recombinant plasmid pcDNA3.1/hTSHR cDNA, expression of the hTSHR protein in the FTC-133 was detected by immunofluorescence which was localized at the cell surface and cytoplasm. Radioiodine uptake of experiment group after infection was 2.9 times more (t=28.63, P Conclusions Radioiodide uptake was triggered by transfection of the recombinant plasmid pcDNA3.1/hTSHR in FTC-133 cell lines. It outlined the potential of this novel gene therapy approach for radiotherapy of thyriod cancer. Research Support This work was supported by Shanghai Leading Academic Discipline Project
Immunofluorescence
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Objective To construct an eukaryotic expression vector of RNF6(ring finger protein 6)gene and identify its recombinant protein expression and localization.Methods Total mRNA was extracted from HEK-293 cells,cDNA was formed by reverse transcripton.The hRNF6 coding sequence was amplified by polymerase chain reaction(PCR) method and cloned into pcDNA3.1-myc-his A vector and pEGFP-C1 vector.After the target region was sequenced,the plasmid was transfected into HEK-293 cell lines.The expression of the recombinant plasmid in HEK-293 cells was proved by Western blot.The localization of pEGFP-RNF6 in CV-1 cell and gastric cancer cell SGC-7901 was observed by using laser scanning confocal microscopy.Results hRNF6 had been constructed into expressing vector pCDNA3.1-myc-his A and pEGFP-C1 successfully.The length of the fragment was 2 058 bp,identified by restriction enzymes digestion.The expression of myc-RNF6 fusion protein was detected by Western blot,with a molecular weight 78 kDa.The pEGFP-RNF6 protein was localized more in the nucleus,less in the cytoplasm.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,the pEGFP-RNF6 fusion protein was expressed mainly in the nucleus.
HEK 293 cells
Coding region
Myc-tag
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Objective To construct a recombinant expression plasmid with rat Sirt1 cDNA and to obtain the fusion protein.Methods A fragment of rat Sirt1 cDNA containing BamHI/XhoI were amplified by RT-PCR.The fragment was cloned into pGEM-T easy vector and subcloned into pET41 vector.Subsequently,E.coli BL21 cells were transformed by the recombinant plasmid.The fusion protein,which was induced by IPTG was purified and was analyzed by SDS-PAGE.Results A fragment of 506 bp of rau Sirt1 cDNA was amplified by RT-PCR.The construction of the recombinant plasmid pET41-Sirt1 was confirmed by sequencing.The fusion protein of 48 kD was expressed and purified.Conclusions The recombinant plasmid has been successfully constructed.The fusion protein of rat Sirt1 can be expressed in E.coli BL21 cells with the expected molecular weight.
XhoI
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The IL-4 gene of porcine was subcloned into the eukaryotic expression vector pEGFP-N1 to construct the recombinant plasmid pEGFP-PIL-4,which was transfected into the CHO-K1 cells by the method of using Lipofectin,The expression of PIL-4 was determined by fluoroscopy,RT-PCR and Western-blot.After 24 hours and 48 hours,the Green fluorescence can be observed under fluorescence microscope and a 339 bp cDNA was detected by RT-PCR after screening 14 days with G418,39 000 protein was detected by Western-blot.The results indicate that fusion protein of PoIL-4 and EGFP was successfully expressed in CHO-K1 cells.And expressed protein had obviously biological activity.
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Objective To construct the expression vector of pEGFP-N1-TFF3 fusion protein and explore the expression and localization of pEGFP-N1-TFF3 fusion protein in COS7 cells.Methods Total RNA was extracted from human epithelial cells.The hTFF3(286bp-462bp) gene sequence was amplified from the cDNA of HT29 cells by polymerase chain reaction(PCR) method and subcloned into pEGFP-N1 vector by EcoRⅠand XhoⅠ.After DNA sequencing was employed,the recombinant plasmid was transfected into HEK293 cells,and recombinant protein was identified by Western blot.Then the localization of recombinant protein in COS 7 cells was observed by laser scanning confocal microscopy.Results The hTFF3 gene was successfully cloned into pEGFP-N1 vector,then confirmed by enzyme digestion and sequencing.GFP-TFF3 fusion protein was detected by Western blot,corresponding to a molecular weight about 34kDa.GFP-TFF3 fusion protein was more located in the nucleus,less in the cytoplasm of COS 7 cells.Conclusion The eukaryotic expression plasmid of pEGFP-N1-TFF3 was successfully constructed and the expression of its fusion protein was in the nucleus and cytoplasm of COS 7 cells.
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Objective: To study the efficient expression of GST-Tat protein in Escherichia coli BZ21 (DE3). Methods: HIV-l Tat gene was amplified by PCR from cDNA library of HIV and was inserted into vector of pEGx-KG. The recombinant plasmid was transferred and expressed in E. coli BL21 (DE3). The expressed products were identified by SDS-page and Western-blot. Results: HIV-1 Tat gene was amplified successfully by PCR. The recombinant plasmid was expressed efficiently in E. coli (BL21). SDS-page and Western-blot analyses showed the expressed Tat fusion protein with relative molecular weight was 38.9 kDa. Conclusion: HIV-1 Tat gene can be cloned and GST-Tat fusion protein can be expressed efficiently in E. coli, which may contribute to further research of anti-AIDS.
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Objective To clone recombinant human IL-1B gene and express with a prokaryotic expression system and to prepare the monoclonal antibody against it.Methods The cDNA of HL-60 cell line was used as a template,the human IL-1β gene was detected by PCR.IL-1β expressed by a prokaryotic expression system was purified.Mice were immunized with purified fusion protein IL-1β for three times.The specificity and sensitivity of anti-human IL-1β monoclonal antibody were characterized by Western blot and indirect ELISA.Results Fusion protein IL-1β was highly expressed in E.coli with a molecular weight of about 51kd,its purity was 95%.Western blot and FACS results showed that the monoclonal antibodies could specifically recognize the target protein expressed in the E.coli expression system and HL-60 cell line.Conclusions The IL-1βsene is successfully cloned,the fusion protein of which and the mAb against recombinant human IL-1β are prepared,which provids a useful tool for laboratory and clinical research.
clone (Java method)
Cloning (programming)
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Objective: To construct eukaryotic green fluorescent protein(GFP) expressing recombinant plasmids, pEGFP-C2-p100, which contain human p100 full length cDNA. Methods: The p100 full length cDNA was purified after pSG5-p100 plasmids were digested by EcoR I and BamH I. And then they were inserted into pEGFP-C2 fluorescent expressing vector. We detected the p100 fragment by double digestion. This pEGFP-C2-p100 recombinant plasmids was transfected into HeLa cell line and the expression of green fluorescent protein was observed. Results:(1)The p100 fragment was detected in the products of the restriction enzyme digestion. (2)The green fluorescent protein could be observed by fluorescence microscope after the transfection. Conclusion:(1)The full length of p100 cDNA fragment is inserted into the pEGFP-C2 vector. (2)The fluorescent expressing recombinant plasmids pEGFP-C2-p100 could express p100-GFP fusion proteins successfully.
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Objective To construct recombinant expression plasmid of human estrogen recptor α(hERα) gene and identify its protein expression and localization. Methods The hERα coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into pEGFP-C1 vector. After the target region was sequenced,the plasmid was transfected into MCF-7 cell lines. The expression of the recombinant plasmid in breast cancer cells(MCF-7 cells) was proved by Western blot and the localization of pEGFP-hERα C was observed by using laser scanning confocal microscopy. Results HERαwas constructed into expressing vector pEGFP-C1 successfully,the length of the fragment was 1800bp identified by restriction enzymes digestion. The expression of pEGFP-hERα fusion protein was detected by Western blot in MCF-7 cells,with a molecular weight 95KD,and localized in the nucleus of MCF-7 cells. Conclusion The recombinant plasmid of pEGFP-hERαwas constructed successfully and the fusion protein was localized in nucleus of MCF-7 cells.
Coding region
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