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The Construction and Identification of Eukaryotic Expression Vector pEGFP-N1-hTERT 被引量:5
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作者 李勇 李军 +1 位作者 吕长荣 窦忠英 《Agricultural Science & Technology》 CAS 2008年第5期50-54,91,共6页
[Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with... [Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with pC1-neo-hTERT and pEGFP-N1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-hTERT into rat fetal neural stem cells(NSCs),the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells,and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis.[Result]The eukaryotic expression vector pEGFP-N1-hTERT had correct structure and could express in eukaryotic cells.[Conclusion]This study laid a foundation for the establishment of immortalized NSCs line in rats. 展开更多
关键词 GFP HTERT eukaryotic expression vector CONSTRUCTION IDENTIFICATION
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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 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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Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells
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作者 易继林 田耕 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2003年第4期392-395,共4页
To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was a... To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed. 展开更多
关键词 gene cloning α fetoprotein gene eukaryotic expression vector CHO cells
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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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Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1
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作者 Di-Nan HUANG Ying-Hua JIANG Hou GAN(Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang 524023, China) 《生物医学工程学杂志》 EI CAS CSCD 北大核心 2005年第S1期127-128,共2页
关键词 SIRNA HELA Construction and expression of eukaryotic expression vector and Plasmid expressing siRNA of Human Protection of Telomeres 1
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Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1
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作者 Wenhua Xiong Anmin Chen Fengjing Guo Tao Huang 《The Chinese-German Journal of Clinical Oncology》 CAS 2006年第5期358-361,共4页
Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPinl) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPinl cDNA was amplified by RT-PCR. The ... Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPinl) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPinl cDNA was amplified by RT-PCR. The same time the sense and antisense hPinl genes were formed by binding BamH Ⅰ and Hind Ⅲ in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamHⅠ and Hind Ⅲ. Results: The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH Ⅰ and Hind Ⅲ, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion: Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed. 展开更多
关键词 PIN1 ISOMERASE antisense gene eukaryotic expressing vector
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The Generation of Eukaryotic Expression Vectors of shRNA Specific for Stat6
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作者 Ming-Sheng ZHANG Yun-Feng ZHOU~Δ Zhi-Guo LUO Jian-Ping WU Wen Jie ZHANG(Department of Radio-Chemotherapy, Zhongnan Hospital, Cancer Research Center, 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73-74,共2页
关键词 SHRNA RNAI The Generation of eukaryotic expression vectors of shRNA Specific for Stat6
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Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector
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作者 黄洪超 《外科研究与新技术》 2011年第2期91-91,共1页
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
关键词 PCR GFP Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector GENE
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Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes
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作者 冯勇 郑东 +1 位作者 杨述华 李进 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2007年第1期27-30,共4页
The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF)... The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid plRES2-EGFP to construct eukaryotic expression plasmid plRES2-EGFP-bFGF. The plRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the plRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that plRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments. 展开更多
关键词 BFGF eukaryotic expression vector TENOCYTES REPAIRS
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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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Construction of Smac gene-carrying and human uroplakin Ib promoter-Regulated Genetic Vector and its Expression
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作者 Zhongxing Zhang Fuqing Zeng Guiyi Liao Xianghui Yue 《Journal of Nanjing Medical University》 2006年第5期275-278,共4页
Objective: To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib (UpIb) promoter. Methods: For the directionality of Smac expression in the transitional cel... Objective: To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib (UpIb) promoter. Methods: For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results: The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion: The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy. 展开更多
关键词 SMAC Uroplakin Ib PROMOTER eukaryotic expression vector
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Construction of a HERG mutant L539fs/47-*558W pEGFP vector and the expression of the fusion protein in HEK293 cells
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作者 ZHANG Junbo Lü Ying +8 位作者 ZHANG Aifeng SUN Chaofeng HAN Wenqi LI Guoliang GAO Jie HUO Jianhua PAN Junqiang ZHOU Xin NIU Xiaolin 《Journal of Medical Colleges of PLA(China)》 CAS 2013年第4期193-205,共13页
Objective: To construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs/47-558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct... Objective: To construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs/47-558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 (HEK293) cells. Methods: The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 1 and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results: The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558Wand the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery pEGFP-C2-L539fs/47*-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion: pEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-*558W 展开更多
关键词 Human ether-a-go-go-related gene MUTATION eukaryotic expression vector PEGFP-C2
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A Plasmid vector encoding functional human keratinocyte growth factor gene in vitro—Functional human KGF gene expression in vitro
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作者 Lin Qiu Chunbao Guo 《Journal of Biophysical Chemistry》 2010年第1期64-71,共8页
In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lu... In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords: 展开更多
关键词 HUMAN Embryo Lung FIBROBLAST Gene CLONE Reverse Transcriptage POLYMERASE Chain Reaction eukaryotic expression vector
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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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Stable Expression of Antibacterial Peptide CecropinB in Dairy Goat Mammary Gland Epithelial Cells
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作者 TONG Huili YIN Deyun ZHANG Li GAO Xuejun 《Journal of Northeast Agricultural University(English Edition)》 CAS 2010年第1期53-56,共4页
The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the go... The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB. 展开更多
关键词 CecropinB eukaryotic expression vector goat mammary epithelial cell line stable transfection
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Construction and Identification of a Human Liver Specific MicroRNA Eukaryotic Expression Vector 被引量:18
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作者 Shanshan Chen Ming Ni +3 位作者 Bing Yu Tingting Lv Mengji Lu Feili Gong 《Cellular & Molecular Immunology》 SCIE CAS CSCD 2007年第6期473-477,共5页
MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by us... MiR-122 is one of the non-coding RNAs which showed its effects on the lipo-metablism, virus infection and HCC forming through regulation of liver gene expression. Its eukaryotic expression vector was constructed by using pSuper which was widely applied in the siRNA expression. The precursor of human miR-122 gene was amplified by polymerase chain reaction (PCR) from the human genomic DNA. The positive clones were screened by PCR and restriction enzyme digestion. The new expression vector of miR-122 was named pHsa-m122. PHsa-m122 and its controls were transfected to HepG2 cells. The miR-122 expression activity was evaluated by GFP122i sensor reporter plasmid through fluorescence detection and Western blot. It was shown that the fluorescence intensity of GFP122si and pHsa-m122 co-transfection group was weaker than that of the controls, so the functional activity of expressed miR-122 was detected. When HepG2 cells were co-transfected with HBV1.3 and pHsa-m122 plasmids, the results showed miR-122 may down-regulate the gene expression of HBV. The human liver specific microRNA eukaryotic expression vector of miR-122 was constructed successfully, which may facilitate further study of its function in the development of liver virus infection diseases and HCC. Cellular & Molecular Immunology. 展开更多
关键词 MIRNA MIR-122 eukaryotic expression vector PSUPER
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猪催乳素的真核表达与生物活性验证 被引量:1
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作者 谢社风 韩贝贝 +5 位作者 高凤磊 马莹 李莉 张守全 邹娴 卫恒习 《华南农业大学学报》 CSCD 北大核心 2024年第2期179-189,共11页
【目的】催乳素(Prolactin,PRL)具有广泛的生理调节作用,但其多效性机制仍不清楚。为了更好地研究猪PRL的多效性,本研究制备猪源PRL真核重组蛋白并验证其生物活性。【方法】利用分子克隆技术将猪PRL基因克隆到慢病毒表达载体pCDH-CMV-MC... 【目的】催乳素(Prolactin,PRL)具有广泛的生理调节作用,但其多效性机制仍不清楚。为了更好地研究猪PRL的多效性,本研究制备猪源PRL真核重组蛋白并验证其生物活性。【方法】利用分子克隆技术将猪PRL基因克隆到慢病毒表达载体pCDH-CMV-MCS-EF1-GFP+Puro中,经慢病毒包装获得携带猪PRL基因的PRL-慢病毒;用浓缩的PRL-慢病毒感染CHO-K1细胞,经嘌呤霉素筛选后,获得能够分泌PRL重组蛋白的阳性细胞系CHO-K1-PRL;利用镍柱亲和层析法对重组蛋白进行纯化并进行LC-MS/MS质谱鉴定,利用HC11细胞体外培养体系验证PRL重组蛋白的生物活性。【结果】成功构建了携带猪PRL基因的pCDH-CMV-6His-PRL-6HisEF1-GFP+Puro慢病毒表达载体;包装及浓缩后的PRL-慢病毒滴度为9.9×10^(8) TU/mL,其感染的CHO-K1细胞经嘌呤霉素筛选后得到阳性细胞系CHO-K1-PRL;从CHO-K1-PRL细胞培养液中成功纯化出重组蛋白,质量浓度为50μg/mL,LC-MS/MS质谱分析的覆盖率达94%,鉴定为猪PRL重组蛋白;重组PRL具有促进HC11细胞增殖及酪蛋白表达的生物活性。【结论】构建的细胞系CHO-K1-PRL可稳定表达具有生物活性的猪重组PRL,为猪PRL功能的研究和生产应用奠定了基础。 展开更多
关键词 催乳素 CHO-K1细胞 真核表达 慢病毒载体 重组蛋白
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多星韭AwCHI 1的克隆与分析及真核表达载体的构建 被引量:1
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作者 赵鑫 张艳 +3 位作者 肖松 黄菊 陈瑶 孙威 《贵州师范大学学报(自然科学版)》 CAS 北大核心 2024年第2期118-126,共9页
查尔酮异构酶(Chalcone isomerase,CHI)是类黄酮物质合成途径中的关键酶,能催化柚皮素查尔酮生成柚皮素,进而转化生成各种类型的黄酮类化合物。根据多星韭(Allium wullichii)转录组测序结果,运用PCR技术克隆获取多星韭查尔酮异构酶的编... 查尔酮异构酶(Chalcone isomerase,CHI)是类黄酮物质合成途径中的关键酶,能催化柚皮素查尔酮生成柚皮素,进而转化生成各种类型的黄酮类化合物。根据多星韭(Allium wullichii)转录组测序结果,运用PCR技术克隆获取多星韭查尔酮异构酶的编码基因(AwCHI 1),对基因序列进行分析,同时与真核表达载体pBI121进行连接,将其转入农杆菌GV3101感受态细胞中,完成农杆菌的转化。研究结果不仅为该基因的功能解析研究奠定了基础,也为多星韭类黄酮代谢途径的研究提供了重要基因资源。 展开更多
关键词 类黄酮 查尔酮异构酶 多星韭 真核表达载体
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