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Induced Expression and Optimal Expression Conditions of Recombinant Alpha-bungarotoxin Gene Fusion Protein
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作者 贾艳 陈一飞 +3 位作者 胡延春 谢光洪 张乃生 柳增善 《Agricultural Science & Technology》 CAS 2013年第5期716-721,共6页
[Objective] This study aimed to obtain recombinant alpha-bungarotoxin (a-BG-0 gene fusion protein with biological activity and investiagte its fusion expression. [Method] The plasmid pGEX-a-BGT was transformed into E... [Objective] This study aimed to obtain recombinant alpha-bungarotoxin (a-BG-0 gene fusion protein with biological activity and investiagte its fusion expression. [Method] The plasmid pGEX-a-BGT was transformed into E coil BL21 (DE3) and BL21 (DE3) plysS host bacteria to identify the optimal engineering strain. Fusion expression of the optimal engineering strain was induced, in order to optimize the induced expression conditions of the soluble fusion protein. [Result] JP-a-BGT was identified as the optimal engineering strain, which could express fusion protein after induced by IPTG. The optimal induced expression conditions of the soluble fusion protein were investigatect JP-a-BGT was incubated at 37 ℃ for 2.5 h and induced with 0.50 mmol4. IPTG for 4 h at 22 ℃, and the expression level of the soluble fusion protein reached 18.42%. [Conclusion] This study laid a solid foundation for the subsequent purification of fusion proteins and the separation and purification of a-BGT. 展开更多
关键词 recombinant alpha-bungarotoxin fusion protein expression condition
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Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells
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作者 WEI HE FANG LIU +3 位作者 LING BO LIU MIN ZHANG ZHONG BO HU PING ZOU 《Journal of Microbiology and Immunology》 2005年第4期293-300,共8页
To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukem... To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNMB7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTr colorimetry and ELISA assay. The experimental resuits showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7 % up to 84.6 %. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumorspecific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo. 展开更多
关键词 gene fusion recombinant fusion proteins Immune escape Immunotherapy
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Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation
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作者 宋自芳 《外科研究与新技术》 2005年第3期171-172,共2页
To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites ... To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs. 展开更多
关键词 Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation
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Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap/GFP Fusion Protein in Mammal Cells
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作者 SONGYi-shu SONGZhi-yu +4 位作者 LIHong-jun WuYin BAOYong-li TANDa-peng LIYu-xin 《Chemical Research in Chinese Universities》 SCIE CAS CSCD 2005年第3期298-300,共3页
s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of t... s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B_~16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. ~s-Lap/GFP fusion protein can be expressed in CHO and B_~16 cells with a high rate expression in the nuclei. 展开更多
关键词 s-Lap gene fusion protein Mammal cell expression LOCALIZATION
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Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti 被引量:2
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作者 李文学 李海峰 金清洙 《Agricultural Science & Technology》 CAS 2010年第5期96-100,共5页
[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surf... [Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti. 展开更多
关键词 Theileria sergenti P23 major surface protein gene Prokaryotic expression
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Prokaryotical expression of structural and non-structural proteins of hepatitis G virus 被引量:4
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作者 Ning-Shao Xia~1 Hai-Jie Yang~1 Jun Zhang~1 Chang-Qing Lin~1 Ying-Bin Wang~1 Juan Wang~1 Mei-Yun Zhan~2 MH Ng~3 1 Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering,Xiamen University,Xiamen 361005,Fujian Province,China2 Institute of Virology,Chinese Academy of Preventive Medicine Beijing 100052,China3 Department of Microbiology,Hoog Kong University,Hongkong,China 《World Journal of Gastroenterology》 SCIE CAS CSCD 2001年第5期642-646,共5页
AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragm... AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis. 展开更多
关键词 Blotting Western Enzyme-Linked Immunosorbent Assay Epitope Mapping Escherichia coli GB virus C PURIFICATION gene expression Regulation Viral Humans Plasmids recombinant proteins Viral Envelope proteins Viral Nonstructural proteins
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Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori 被引量:5
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作者 Chao Wu~1 Quan Ming Zou~1 Hong Guo~2 Xiao Peng Yuan~1 Wei Jun Zhang~1 Dong Shui Lu~1 Xu Hu Mao~1 ~1Department of Clinical Microbiology,Third Military Medical University,Chongqing 400038,China ~2Department of Gastroenterology,Xinqiao Hospital,Third Military Medical University,Chongqing 40003?,ChinaDr.Chao Wu graduated from Third Military Medical University as a postgraduate in 2000,now a lecturer,specialized in diagnosis,prevention and therapy of Helicobacter pylori infection,having 6 papers published. 《World Journal of Gastroenterology》 SCIE CAS CSCD 2001年第3期389-393,共5页
INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat ... INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens . 展开更多
关键词 UREASE PURIFICATION Animals Antibodies gene expression Regulation Bacterial Helicobacter pylori MICE Mice Inbred BALB C Plasmids recombinant proteins PURIFICATION Research Support Non-U.S. Gov't
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Contruction of the Genetic Engineering Strain Expressed Nontoxic ST_1-LT_B Fusion Protein Against Enterotoxigenic Eschenichia coli 被引量:1
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作者 BAIJia-ning SUNYi-min BIANYan-qing ZHAOBao-hua 《Agricultural Sciences in China》 CAS CSCD 2004年第7期535-540,共6页
Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-... Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain. 展开更多
关键词 Thermostable enterotoxinⅠgene Thermolabile enterotoxin B subunit gene fusion gene fusion protein gene expression
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Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis 被引量:1
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作者 周建华 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 1999年第4期253-256,共4页
Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118... Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku. 展开更多
关键词 B. Subtilis diphtheria toxin Western blot recombinant protein gene expression MUTATION
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The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria 被引量:1
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作者 GaoCF KongXT 《Cell Research》 SCIE CAS CSCD 2001年第2期95-100,共6页
In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-39... In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies. 展开更多
关键词 Amino Acid Sequence Animals Base Sequence EPITOPES Escherichia coli gene expression Regulation Bacterial genetic Vectors Molecular Sequence Data Plasmids protein Structure Tertiary Rats recombinant proteins Research Support Non-U.S. Gov't Transformation genetic Transforming Growth Factor beta
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Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli
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作者 廖芳 宋启发 万沐芬 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2004年第5期417-420,共4页
In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot... In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed. 展开更多
关键词 Neisseria gonorrhoeae porin B porin A prokaryotic expression plasmid recombinant fusion protein
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Expression of a Carrot Antifreeze Protein Gene in Escherichia coli
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作者 Ma Xinyu Shen Xin Lu Cunfu 《Forestry Studies in China》 CAS 2003年第4期22-25,共4页
The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA... The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h. 展开更多
关键词 antifreeze protein (AFP) fusion protein induced expression polymerase chain reaction antifreeze protein gene
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Expression of GST-IL-1 fusion gene
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作者 陈梅红 王字玲 +3 位作者 邓健蓓 赵忠良 陈南春 苏成芝 《Journal of Medical Colleges of PLA(China)》 CAS 1996年第2期79-83,共5页
Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ... Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene. 展开更多
关键词 gene expression fusion protein INTERLEUKIN-1 GLUTATHIONE-S-TRANSFERASE
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Expression and purification of recombinant human hemangiopoietin in Escherichia coli
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作者 Ren Qian Ma Fengxia Chen Zhong Lu Shihong Han Zhibo Liu Yongjun Xu Bin Zhang Xiangyu Han Zhongchao 《Journal of Medical Colleges of PLA(China)》 CAS 2008年第3期148-153,共6页
Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into pl... Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application. 展开更多
关键词 ENTEROKINASE Escherichia coli fusion protein Hemangiopoietin PURIFICATION recombinant protein expression
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Expression of mature peptide of human bone morphogenetic protein-2 in Escherichia coli 被引量:1
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作者 蒲勤 陈苏民 陈南春 《Journal of Medical Colleges of PLA(China)》 CAS 1998年第1期40-42,共3页
To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expressio... To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity. 展开更多
关键词 HUMAN BONE morphogenetic protein-2 recombinant DNA gene expression
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Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells 被引量:16
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作者 GuoBC XuYU 《Cell Research》 SCIE CAS CSCD 2001年第2期101-106,共6页
Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role... Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell. 展开更多
关键词 Apoptosis Apoptosis Regulatory proteins CARCINOGENS gene expression Regulation Humans INTERLEUKIN-2 Jurkat Cells LIPOPOLYSACCHARIDES Membrane Glycoproteins protein Kinase C Proto-Oncogene proteins c-bcl-2 recombinant proteins Research Support Non-U.S. Gov't Tetradecanoylphorbol Acetate TRANSFECTION Tumor Necrosis Factor-alpha
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Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression 被引量:3
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作者 LI Jun Lü Chang-rong DOU Lin DOU Zhong-ying 《Agricultural Sciences in China》 CAS CSCD 2007年第4期487-492,共6页
The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene... The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction (PCR) from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21(DE3) for expression. After induction by isopropyl-β-D-thiogalactoside (IPTG) at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody. 展开更多
关键词 Nanog gene prokaryotic expression glutathione-S-transferase (GST) fusion protein MOUSE
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Expression, purification and bioactivity of human augmenter of liver regeneration 被引量:2
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作者 Yang-De Zhang Jian Zhou +4 位作者 Jin-Feng Zhao Jian Peng Xiao-Dong Liu Xin-Sheng Liu Ze-Ming Jia 《World Journal of Gastroenterology》 SCIE CAS CSCD 2006年第27期4401-4405,共5页
AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. METHODS: hALRcDNA clone was obtained from plasmid pGEM-T... AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. METHODS: hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS: The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P 〈 0.01).CONCLUSION: Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application. 展开更多
关键词 Human augmenter of liver regeneration gene recombination expression PURIFICATION fusion protein TRANSFORMATION Biological activity
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A preliminary study on the cloning and expression of human papiilomavirus type 18 E6 gene in Escherichia coli
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作者 文维延 徐钤 《Journal of Medical Colleges of PLA(China)》 CAS 1991年第1期15-20,共6页
We have cloned the E6 gene of human papillomavirus type 18 into anexpression plasmid pBD2.One of the recombinant plasmids (named pDV11) wasidentified by DNA analysis and protein product analysis.It could express a new... We have cloned the E6 gene of human papillomavirus type 18 into anexpression plasmid pBD2.One of the recombinant plasmids (named pDV11) wasidentified by DNA analysis and protein product analysis.It could express a newprotein whose molecular weight correspods well with the expected one.Afterpurification,the expressed protein showed a positive result in countercurrentimmuno-electrophoresis with anti-β-gal serum and was proved to be the expectedβ-gal/E6 fusion protein.The physical map of pDV11 was also prepared. 展开更多
关键词 HUMAN PAPILLOMAVIRUS TYPE 18 recombinant and expression LAC promoter expression PLASMID fusion protein
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Construction and Expression of Prokaryotic Expression Vector of MPT-64 Gene
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作者 Long PENG Linbo ZHANG 《Agricultural Biotechnology》 CAS 2014年第3期11-13,17,共4页
[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the ... [Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines. 展开更多
关键词 Mycobacterium tuberculosis Protective antigen genes Secreted protein MPT64 Prokaryotic expression fusion protein
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