[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ...[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.展开更多
Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/t...Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template,double stranded cDNA of HNG with FLAG in its C-terminal was obtained,which was cloned into the plasmid pcDNA3.1(-),and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time,the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG,25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h,then cell morphology,MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h,cell viability decreased to (65.8±5.3)%,and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG,Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast,pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.展开更多
[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according ...[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according to the gene sequence of ARV S2 gene in GenBank(accession number KF741763.1).With pMD18-T-σA recombinant vector as the template,the specific sequence ofσA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant plasmid.The cloning vector pMD18-T-σA and eukaryotic expression plasmid pEF1α-HA were double digested by restriction enzymes Kpn I and Not I.The purifiedσA gene was connected with pEF1α-HA to construct eukaryotic expression plasmid pEF1α-HA-σA.After colony PCR,double enzyme digestion and sequencing,the recombinant plasmid pEF1α-HA-σA was tansfected into HEK293T cells.The proteins were collected at 24 h after tansfection and verified by Western-blot.[Result]The ARVσA gene was successfully cloned in the test.The eukaryotic expression plasmid pEF1α-HA-σA was constructed,which could be expressed in HEK293T cells.[Conclusion]The protein could be accurately expressed in HEK293T cells.展开更多
Objective To construct the human interleukin 18 DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos 7 and D5.Methods Gene recombinant technique was used to construct hIL 18 ...Objective To construct the human interleukin 18 DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos 7 and D5.Methods Gene recombinant technique was used to construct hIL 18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL 18 eukaryotic expression vectors into Cos 7 and D5 cells. In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL 18 in Cos 7 and D5.Results The eukaryotic expression plasmid pVAX1 IL 18 was constructed successfully.hIL 18 was transiently expressed in Cos 7 and D5.Conclusion The eukaryotic expression plasmid pVAX1 IL 18 was constructed. In situ hybridization and Western Blot results proved the successful transient expression of pVAX1 IL 18 in Cos 7 and D5.Therefore,the work has settled the foundation for further biological research on hIL 18,including immunogene therapy through hIL 18.展开更多
Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by...Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase展开更多
Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
The eukaryotic expression vector of human arresten gene was constructed and its secretive expression in human embryonic kidney(HEK 293)cells was detected.Human arresten gene was amplified from recombinant plasmid pGEM-...The eukaryotic expression vector of human arresten gene was constructed and its secretive expression in human embryonic kidney(HEK 293)cells was detected.Human arresten gene was amplified from recombinant plasmid pGEM-Arr by polymerase chain reaction(PCR),and then digested with restriction endonucleases BamH I and EcoR I.The target fragment was inserted into the BamH I and EcoR I restriction sites of eukaryotic expression vector pSecTag2 to construct pST-AT.Restric-tion analysis and DNA sequencing indicated that the arresten gene was successfully inserted into pSecTag2.The recombinant plasmid was subsequently transfected into HEK 293 cells with LipofectAMINETM2000 Reagent,and the expression of the target gene was detected.Reverse transcription PCR(RT-PCR)revealed that the mRNA of the target gene was transcribed in the transfected HEK 293 cells.Western blot analysis verified that the recombinant protein in supernatants was correct.The supernatants of transfected cells were prepared,and 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide(MTT)assay was carried out to assess their effects on the proliferation of human umbilical vein endothelial cells,which showed that the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells in vitro.These results provided a solid foundation to explore the usage of arresten in tumor anti-angiogenic gene therapy.展开更多
Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmi...Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using展开更多
基金Supported by Anhui Provincial Natural Science Foundation of China(2008085MC65)Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Research Activities of Postdoctoral Researchers Foundation of Anhui Province,China(2020B470)。
文摘[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.
基金supported by Youth Foundation of Medical School of Xi an Jiaotong University (No.YQNO8O7)
文摘Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template,double stranded cDNA of HNG with FLAG in its C-terminal was obtained,which was cloned into the plasmid pcDNA3.1(-),and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time,the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG,25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h,then cell morphology,MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h,cell viability decreased to (65.8±5.3)%,and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG,Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast,pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.
基金Supported by Guangxi Science Base and Talents Special Program(AD17195083)Guangxi Science Great Special Program(AA17204057)+4 种基金National Natural Science Foundation of China(3166071531160512)Science and Technology Project of Guangxi Province(2018GXNSFAA138106)Guangxi Bagui Scholars Program Foundation(2019-79)National Ten-Thousand Talents Program of China(W02060083).
文摘[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according to the gene sequence of ARV S2 gene in GenBank(accession number KF741763.1).With pMD18-T-σA recombinant vector as the template,the specific sequence ofσA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant plasmid.The cloning vector pMD18-T-σA and eukaryotic expression plasmid pEF1α-HA were double digested by restriction enzymes Kpn I and Not I.The purifiedσA gene was connected with pEF1α-HA to construct eukaryotic expression plasmid pEF1α-HA-σA.After colony PCR,double enzyme digestion and sequencing,the recombinant plasmid pEF1α-HA-σA was tansfected into HEK293T cells.The proteins were collected at 24 h after tansfection and verified by Western-blot.[Result]The ARVσA gene was successfully cloned in the test.The eukaryotic expression plasmid pEF1α-HA-σA was constructed,which could be expressed in HEK293T cells.[Conclusion]The protein could be accurately expressed in HEK293T cells.
文摘Objective To construct the human interleukin 18 DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos 7 and D5.Methods Gene recombinant technique was used to construct hIL 18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL 18 eukaryotic expression vectors into Cos 7 and D5 cells. In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL 18 in Cos 7 and D5.Results The eukaryotic expression plasmid pVAX1 IL 18 was constructed successfully.hIL 18 was transiently expressed in Cos 7 and D5.Conclusion The eukaryotic expression plasmid pVAX1 IL 18 was constructed. In situ hybridization and Western Blot results proved the successful transient expression of pVAX1 IL 18 in Cos 7 and D5.Therefore,the work has settled the foundation for further biological research on hIL 18,including immunogene therapy through hIL 18.
文摘Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
基金supported by the National Natural Science Foundation of China(Grant Nos.30271242 and 30371396).
文摘The eukaryotic expression vector of human arresten gene was constructed and its secretive expression in human embryonic kidney(HEK 293)cells was detected.Human arresten gene was amplified from recombinant plasmid pGEM-Arr by polymerase chain reaction(PCR),and then digested with restriction endonucleases BamH I and EcoR I.The target fragment was inserted into the BamH I and EcoR I restriction sites of eukaryotic expression vector pSecTag2 to construct pST-AT.Restric-tion analysis and DNA sequencing indicated that the arresten gene was successfully inserted into pSecTag2.The recombinant plasmid was subsequently transfected into HEK 293 cells with LipofectAMINETM2000 Reagent,and the expression of the target gene was detected.Reverse transcription PCR(RT-PCR)revealed that the mRNA of the target gene was transcribed in the transfected HEK 293 cells.Western blot analysis verified that the recombinant protein in supernatants was correct.The supernatants of transfected cells were prepared,and 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide(MTT)assay was carried out to assess their effects on the proliferation of human umbilical vein endothelial cells,which showed that the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells in vitro.These results provided a solid foundation to explore the usage of arresten in tumor anti-angiogenic gene therapy.
文摘Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using
