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Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein
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作者 CHEN Nan LI Xiao-yue +6 位作者 GU Xin-yu WU Tong-xin ZHANG Ru LI Yun TANG Xiang-ping DAI Jin YI Yong-xiang 《Journal of Hainan Medical University》 CAS 2023年第10期1-7,共7页
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
关键词 Human soluble DSG2 extracellular domain protein Eukaryotic expression PURIFICATION Activity characterization
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Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells
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作者 Jinwu CHEN Yi LI +4 位作者 Mei LIU Sainan WANG Zilong XIAO Junjie XIA Lulu QI 《Agricultural Biotechnology》 CAS 2023年第3期50-54,共5页
[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
关键词 Recombinant human coagulation factor X(rhFX) Eukaryotic expression MTX Affinity chromatography
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Construction and Preliminary Identification of Eukaryotic Expression Vector of Cryptosporidium parvum miR-2980
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作者 呼高伟 程天印 +5 位作者 米荣升 秦培兰 黄燕 周鹏 曹薇 陈兆国 《Agricultural Science & Technology》 CAS 2012年第5期1093-1096,共4页
[Objective] This study aimed to construct and preliminarily identify the eu- karyotic expression vector of Cryptosporidium parvum miR-2980. [Method] The cp-miR- 2980 precursor was amplified from C. parvum genomic DNA ... [Objective] This study aimed to construct and preliminarily identify the eu- karyotic expression vector of Cryptosporidium parvum miR-2980. [Method] The cp-miR- 2980 precursor was amplified from C. parvum genomic DNA and cloned into pMD18- T vector. The amplified precursor was then subcloned into pVAX I vector and identi- fied with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX-miR2980 was transfected into HCT-8 cells. Total RNA was extracted and the expression of cp-miR-2980 was evaluated by RT-PCR detection. [Result] The results showed that the recombinant eukaryotic expression vector pVAX-miR2980 was suc- cessfully constructed, which can express cp-miR-2980 in HCT-8 cell. [Conclusion] This study laid the foundation for further exploring the biological function of cp-miR-2980. 展开更多
关键词 cp-miR-2980 PRECURSOR Eukaryotic expression vector RT-PCR
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Construction of Eukaryotic Expression Vector with Partial Encoding Sequence of Actin from Cryptosporidium andersoni
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作者 陈健 胡进平 +6 位作者 宫鹏涛 李建华 杨举 李赫 张国才 张西臣 任文陟 《Agricultural Science & Technology》 CAS 2012年第3期641-643,655,共4页
[Objective] To clone the actin gene of Cryptosporidium andersoni, and to study its eukaryotic expression in Hela cells. [Methed] Specific primers were designed for the partial encoding sequence of actin, which were ob... [Objective] To clone the actin gene of Cryptosporidium andersoni, and to study its eukaryotic expression in Hela cells. [Methed] Specific primers were designed for the partial encoding sequence of actin, which were obtained by screening the T7 phage display library of Cryptosporidium andersoni, and the actin gene CA42 was amplified by PCR. Recombinant eukaryotic expression plasmid pVAX1-CA42 was constructed and transfected to Hela cells with lipofection strategy. Indirect im- munofluorescence staining, SDS-PAGE and Western blotting analysis were used to detect the expression of recombinant protein in Hela cells. [Result] CA42 protein was successfully expressed in Hela cells, and the expression products had reactogenicity. [Conclusion] The partial encoding sequence of actin from Cryptosporidium andersoni has been successfully cloned, and it can be stably expressed in Hela Cells 展开更多
关键词 Cryptosporidium anderssonr ACTIN Eukaryotic expression
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Expression and Purification of Arabidopsis High Mobility Group B Protein Gene At2G34450 in Pichia pastoris
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作者 冀芦沙 肖庆振 +1 位作者 王曰文 王洪霞 《Agricultural Science & Technology》 CAS 2012年第4期731-734,共4页
[Objective] The aim was to study the expression of Arabidopsis gene A/2G34450 in Pichia pastoris and to obtain recombinant Arabidopsis HMGB protein. [Method] The At2G34450 gene was cloned into yeast expression vector ... [Objective] The aim was to study the expression of Arabidopsis gene A/2G34450 in Pichia pastoris and to obtain recombinant Arabidopsis HMGB protein. [Method] The At2G34450 gene was cloned into yeast expression vector pPIC9K containing AOXl promoter and the sequences of secreting α-signal peptides. Recombinant plasmid was linearized by Sal l and transformed into P. pastoris GSl15 competent cells by electroporation. Positive integrated clones were screened out, and the At2G34450 protein was expressed under the induction of methanol. [Result] The At2G34450 protein was expressed in yeast medium through methanol induction. SDS-PAGE results showed that recombination product was At2G34450 protein. [Conclusion] At2G34450 protein was successfully expressed in the P. pastoris system for the first time, which paves a direct path to further research on the functions of HMGB family members. 展开更多
关键词 ARABIDOPSIS High mobility group protein Pichia pastoris Eukaryotic expression
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Construction of Eukaryotic Expression Vector for Pig Ghrelin Gene
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作者 曹月胜 陈俏俏 孙金海 《Agricultural Science & Technology》 CAS 2012年第6期1184-1185,1197,共3页
[Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pi... [Objective] This study aimed to investigate the functions of transgenic growth related gene in pig growth. [Method] A pair of primers containing Nhe I and Hind Ⅲ restriction sites were designed by referring to the pig Ghrelin mRNA sequence published in Genbank. Total RNA was extracted from the small intestine tissue of 13/17 Robertson translocation heterozygous pig, and then was purified and used as the template in later RT-PCR reaction to amplify the full-length pig Ghrelin gene. The correct pig Ghrelin gene fragment was cloned into the pMD19-T simple vector for sequencing analysis. The obtained full-length cDNA of pig Ghrelin gene fragment was digested with both Nhe I and Hind Ⅲ, and then was linked into the eukaryotic expression vector pEGFP-N1 to obtain the recombinant plasmid pEGFPGhrelin. The recombinant plasmid was transected into the fibroblast cells to detect the fluorescence labeled gene expression. [Result] The nucleotide sequence extracted from 13/17 Robertson translocation heterozygous pig was the same as expected; and the eukaryotic expression vector pEGFP-Ghrelin was successfully constructed. [Conclusion] The eukaryotic expression vector constructed in this study can be further used in research on transgenic pigs, but also lays foundation for research on the regulatory mechanism of Ghrelin gene. 展开更多
关键词 Porcine growth hormone gene Eukaryotic expression vector TRANSGENIC
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Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells 被引量:6
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作者 Yuan Chen Peng Zhang +2 位作者 Yuanguo Wang Shangwen Dong Yimei Liu 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2013年第5期493-499,共7页
Objective:PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells (TE13),and evaluate t... Objective:PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells (TE13),and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.Methods:First,mRNA was extracted from human esophageal cancer tissue by RT-PCR,then reversetranscribed to cDNA.After amplifying from the DNA template,PR domain was linked to T vector.Second,after extraction,PR domain was cut using enzyme and linked to pcDNA3.1(+).Then,the plasmid was transfered to Trans1-T1 phage resistant competent cells,following by extracting the ultrapure plasmid,and transfecting into TE13 cells.In the end,the protein expression of pcDNA3.1(+)/PR domain in TE13 was detected by Western blot,and the apoptosis of TE 13 by technique of flow cytometry.Results:More than 5,000 bp purposed band of pcDNA3.1(+)/PR domain plasmid was found by agarose gel electrophoresis.After transfection,the PR domain (molecular weight of about 28 Da) was found only in 3,4 and 5 groups by Western blot.Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group (P<0.05).Conclusions:The PR domain eukaryotic expression vector was constructed successfully.The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection,and a single PR domain could promote apoptosis of TE13 cells. 展开更多
关键词 Esophageal cancer RIZ1 PR domain eukaryotic expression vector
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Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles 被引量:5
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作者 Zhong-Jun Wu, Xiao-Hong Yang, Shu-Sen Zheng, Su-Fen Yang and De Shi Organ Transplant Center, First Affiliated Hospital,Zhejiang University School of Medicine, Hangzhou 310003, China Department of General Surgery, Affiliated Hospital of ZunyiMedical College, Zunyi 563003 , China and Department ofVascular Surgery, Chongqing Medical University, Chongqing 400016 , Chi-na 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2004年第3期355-359,共5页
BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth fa... BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ. 展开更多
关键词 eukaryotic expression plasmid human vascular endothelial growth factor vascular smooth muscle cell gene transfer organ transplant
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CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE 被引量:4
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作者 郑秋红 郑天荣 +2 位作者 谢云青 卢林 陈晖 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2000年第2期125-127,共3页
Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA fr... Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct. 展开更多
关键词 Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) Reverse transcription and polymerse chain reaction (RT-PCR) Eukaryotic expression
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Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells 被引量:2
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作者 Organ Grafting Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003 , China Department of Cardiothoracic Surgery, the General Hospital of Daqing Oil Field, Daqing 163001 , China and Department of Vascular Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2005年第3期364-369,共6页
After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular ... After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ. 展开更多
关键词 eukaryotic expression plasmid vascular endothelial grow factor 165 vascular endothelial cell gene transfer organ transplantation
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Construction of recombinant plasmid pEGFP-C2-L539fs/47 and its expression in HEK293 cells 被引量:2
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作者 Lue Ying Zhang Aifeng +6 位作者 Han Wenqi Li Guoliang Zhang Junbo Gao Jie Pan Junqiang Zhang Yong Sun Chaofeng 《Journal of Medical Colleges of PLA(China)》 CAS 2012年第3期125-133,共9页
Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of... Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47. 展开更多
关键词 HERG gene Nonsense mutations Eukaryotic expression vector PEGFP HEK293 cells
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Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells 被引量:1
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作者 尚丹 郑启昌 +3 位作者 宋自芳 李毅清 汪谢丹 郭兴军 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2006年第2期202-205,共4页
The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukar... The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40. 154, P〈0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments. 展开更多
关键词 ARRESTEN eukaryotic expression vascular smooth muscle cells cell proliferation
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Overexpression of the mTERT gene by adenoviral vectors promotes the proliferation of neuronal stem cells in vitro and stimulates neurogenesis in the hippocampus of mice 被引量:1
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作者 Mengying Liu Yao Hu +4 位作者 Lijuan Zhu Chen Chen Yu Zhang Weixiang Sun Qigang Zhou 《The Journal of Biomedical Research》 CAS 2012年第5期381-388,共8页
We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase(mTERT),as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was... We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase(mTERT),as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells. 展开更多
关键词 TELOMERASE construct eukaryotic expression vector adenoviral vector PROLIFERATION neuronal stemcells
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EXPRESSION OF HUMAN BETA-DEFENSIN 3 IN COS-7 CELL 被引量:1
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作者 Xiao-yeTuo Ming-daXu +2 位作者 BiChen Jia-keChai Zhi-yongSheng 《Chinese Medical Sciences Journal》 CAS CSCD 2004年第3期207-211,共5页
To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector... To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line. 展开更多
关键词 human beta-defensin 3 eukaryotic expression gene transfection
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EXPRESSION OF RHGM-CSF GENE IN EUKARYOCYTE BY LIPOFECTION 被引量:1
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作者 郑天荣 郑秋红 +2 位作者 谢云青 卢林 陈晖 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2001年第1期35-38,共4页
Objective: To recombinant the nearly natural human granulocyte-macrophage-colony stimulating factor (GM-CSF) for supplying more safe and steady expressed cytokine in clinic. Method: The eukaryotic recombinant pcDNA3.1... Objective: To recombinant the nearly natural human granulocyte-macrophage-colony stimulating factor (GM-CSF) for supplying more safe and steady expressed cytokine in clinic. Method: The eukaryotic recombinant pcDNA3.1-GM-CSF plasmid which was controlled by the CMV promoter was transferred into CHO cell by lipofectamine, selected by G418 and the positive clones was got. The recombinant vector which was rejoined into the groups of DNA of CHO was identified by PCR. Results: The results showed that the protein of rhGM-CSF was about 28 KD by using ELISA, SDS-PAGE and Western blot. Conclusion: rhGM-CSF was expressed steadily and highly. The rhGM-CSF will be of more use value. 展开更多
关键词 Human granulocyte-macrophage colony stimulating factor (hGM-CSF) LIPOFECTAMINE Eukaryotic expression
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THE CONSTRUCTION OF HPV16-L1 RECOMBINANT PLASMID AND ITS EXPRESSION IN MAMMALIAN CELLS 被引量:1
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作者 Sun Xiangle),Si Lüsheng,Cao Zansun Department of Obstetrics & Gynecology,Wang Yili,Liu Tianju,Guo Jianfen,Song Jianming Institute of Immunopathology, Xi′an Medical University, Xi′an 710061 《Journal of Pharmaceutical Analysis》 CAS 1999年第2期116-119,共4页
Infection with high risk human papillomavirus is regarded as the major risk factor in the development of cervical cancer. In this study, HPV16 L1 eukaryotic expression plasmids pcDNA LI were constructed, which were... Infection with high risk human papillomavirus is regarded as the major risk factor in the development of cervical cancer. In this study, HPV16 L1 eukaryotic expression plasmids pcDNA LI were constructed, which were transfected into mammalian cells Cos 7. The expression of HPV16 L1 in transfected cells were identified by in situ hybridization, immunospot and immunocytochemistry. HPV16 L1 mRNA transcription and L1 protein expression were found in recombinant plasmid transfected cells. This expression system will provide us with plentiful resource for HPV16 L1 immunological study and will be helpful for the design of HPV16 prophylactic vaccine. 展开更多
关键词 human papillomavirus type 16(HPV16 L1) eukaryotic expression cervical carcinoma
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CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR HUMAN CCL21 AND CHARACTERIZATION OF ITS CHEMOTACTIC ACTIVITY
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作者 侯丽 刘奇 +6 位作者 焦玉莲 张捷 王来城 马春燕 崔彬 张雪 赵跃然 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2006年第4期246-250,共5页
Objective: To obtain recombinant human CCL21 with biological activity from eukary0tic expression system for further use in cancer gene therapy. Methods: A fragment of human CCL21 gene was obtained from pSK-hCCL21 pl... Objective: To obtain recombinant human CCL21 with biological activity from eukary0tic expression system for further use in cancer gene therapy. Methods: A fragment of human CCL21 gene was obtained from pSK-hCCL21 plasmid digested by Xho I and BamH I, inserted into the responding sites of eukaryotic expression vector pVAX1, and then transfected into COS-7 cells by electroporation method. The expression of hCCL21 protein was detected by western blotting analysis. The in vitro chemotaxis assay was used to test the chemotactic function of the expression product to lymphocytes. Results: Human CCL21 protein was expressed by transfected COS-7 cells with recombinant plasmid containing hCCL21 gene, and was verified by western blotting. The in vitro chemotaxis assay demonstrated that human CCL21 protein had a potent chemotactic function to lymphocytes. Conclusion: Human CCL21 was successfully and transiently expressed in eukaryotic cells, which lays some foundation for the study of CCL21 gene therapy in murine tumor models. 展开更多
关键词 CC chemokine ligand 21 Eukaryotic expression Chemotaxis assay Gene therapy
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Science Letters:Construction of a eukaryotic expression plasmid of Humanin
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作者 罗本燕 陈祥明 +2 位作者 唐敏 陈峰 陈智 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2005年第1期11-13,共3页
Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments... Objective:To construct a eukaryotic expression plasmid pcDNA3.1(-)-Humanin.Methods:The recombinant plasmidpGEMEX-1-Humanin was digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ and the Humanin gene fragments,about100 bp length,were obtained.Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1(-)andthe recombinant plasmids pcDNA3.1(-)-Humanin were identified by sequencing.Results:Recombinant plasmid DNA success-fully produced a band which had the same size as that of the thimauin positive control.The sequence of recombinant plasmidsaccorded with the Humnain gene sequence.Conclusions:A eukaryotic expression plasmid of Humanin was successfully con-structed. 展开更多
关键词 HUMANIN Alzheimer's disease Eukaryotic expression
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Cloning of Humanα-defensin-1(HNP-1) Gene and Construction of Its Eukaryotic Expression Vector
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作者 Hua-Hua CHEN Jing-Ping Ou YANG Bao-Hua WANG Yue Yang Han-Qiao ZHENG(Pathophysiology Department of Medical Institute of Wuhan University, Wuhan 430071, China) 《生物医学工程学杂志》 EI CAS CSCD 北大核心 2005年第S1期97-98,共2页
关键词 HNP-1 Gene and Construction of Its Eukaryotic expression Vector defensin-1 Cloning of Human
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Eukaryotic expression and biological activity analysis of neuroprotective peptide [Gly14]-Humanin
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作者 Hui Jin,Hai-Tao Hu ,Wei-Xi Wang,Gai-Feng Feng,Zhao-Hui Liu,Wei-Na YangDepartment of Human Anatomy and Histoembryology,Medical School of Xi’an Jiaotong University,Xi’an 710061,China. 《Journal of Pharmaceutical Analysis》 SCIE CAS 2010年第2期105-110,共6页
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
关键词 Alzheimer's disease [Gly14]-Humanin eukaryotic expression biological activity
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