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
[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragmen...[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.展开更多
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of re...To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E.Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.9921 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.展开更多
The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vect...The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 (DE3) strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.展开更多
[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activi...[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 (DE3) and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 (DE3). Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.展开更多
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
Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidate...Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated.However,the function of the arsH gene in At.ferrooxidans remains unclear.In order to evaluate the function of the arsH gene,we cloned it and expressed it in Escherichia coli.The protein was purified and its relative molecular mass was determined by SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa.The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.展开更多
Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the E...Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GSll5. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.展开更多
Guided pesticide is an unique compound resulted from the conjugation with carrier (amino acid, protein, sugars, etc) and the active pesticide ingredient. One of the attributes of the guided pesticide is its potentia...Guided pesticide is an unique compound resulted from the conjugation with carrier (amino acid, protein, sugars, etc) and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein (MP) is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein (GFP) and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG (isopropyl-b-dthiogalactopyranoside) at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.展开更多
[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
Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca2+, it undergoes a conformational change, expos...Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca2+, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity. Washing with EDTA chelates the Ca2+ ions from the protein, inducing a conformational change back to the more folded state and eluting the protein from the column. We describe herein the use of a protein expression and purification technique using the calmodulin variant and a short linker for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease. We have shown this approach to be useful in obtaining purified quantities of various small proteins that could not be expressed using other methods, including high enough concentrations of a designed WW domain protein for NMR structural analysis. We have also obtained promising results on the usefulness of this procedure to express and purify zinc finger proteins without the addition of zinc ions or other cofactors.展开更多
Objective To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hlL-2/mGM-CSF). Methods SOE PCR was used to change the linker of the fusion protein for high...Objective To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hlL-2/mGM-CSF). Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coil) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.展开更多
mCLCA3 is a member of calcium activated chloride channel(CACC) family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expres...mCLCA3 is a member of calcium activated chloride channel(CACC) family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.展开更多
Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. T...Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.展开更多
AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion ...AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB (DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 (DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.展开更多
The cDNA containing full encoding region or E1 antigen or HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid PRSETE1 was introduced into the BL21 (DE3) strain or E. coli. The engineering bact...The cDNA containing full encoding region or E1 antigen or HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid PRSETE1 was introduced into the BL21 (DE3) strain or E. coli. The engineering bacteria harhouring the pRSETEI was cultivated in 2YT medium at 37℃.When the Expression of E1 protein was induced by 1 mmol IPTG, the bacteria was killed and the number of living cell was droned down from 107 to 103 cell/mL one hour post induction. suggest that E1 protein is poisoned to E. coli. However, the 26kD polypeptide or E1 fussion still synthesized in appropriate condition. The expression level was about 10% or total protein 4 h after Inducing. Th. E1 protin was purfied by Ni2+-NTAAgarose column chromatography to homogeneous. The purified E1 protein was sensitive and specific in reaction with anti-HCV antibody in sera.展开更多
To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was ampli...To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.展开更多
BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics o...BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil (E. coil) JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION: From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.展开更多
The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and de...The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and development,including seedling photomorphogenesis,shade avoidance,flowering time,and biotic and abiotic stress responses.Previous studies have identified many different BBXs from several plant species,although the BBX family members in maize are largely unknown.Genome-wide identification and comprehensive analysis of maize BBX(ZmBBX)expression and interaction networks would therefore provide valuable information for understanding their functions.In this study,36 maize BBXs in three major clades were identified.The ZmBBXs within a given clade were found to share similar domains,motifs,and genomic structures.Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.The expression levels of ZmBBXs were analyzed in various organs and tissues,and under different abiotic stress conditions.Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation(BiFC)assays.Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions.展开更多
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
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
基金funded by Natural Science Foundation of Jian-gsu Province (BK2009660)Huaiyin Institute of Technology Foundation (35170706)
文摘[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.
文摘To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E.Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.9921 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 (DE3) strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.
文摘[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 (DE3) and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 (DE3). Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
基金Supported by the National Major Science and Technology Projects,No.96-901-01-54.
文摘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 .
基金Project(50621063) supported by the National Natural Science Foundation of ChinaProject(2004CB619201) supported by the National Basic Research Program of China
文摘Four arsenic-resistance genes(arsB,arsC,arsH,arsR) have been discovered in Acidithiobacillus ferrooxidans.Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated.However,the function of the arsH gene in At.ferrooxidans remains unclear.In order to evaluate the function of the arsH gene,we cloned it and expressed it in Escherichia coli.The protein was purified and its relative molecular mass was determined by SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa.The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.
基金Key Project of Science and Technology Department of Wuhan(zz2011-12)Key Project of the Educational Bureau of Wuhan(sz2011-13-10)+1 种基金Project of Science and Technology Department of Hubei(2010CDB04801)The State Key Laboratory program of Viral Genetic Engineering(2010KF10)
文摘Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GSll5. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.
基金supported by the National Natural Science Foundation of China(30571235, 30840058)the Natural Science Fundamental Research Project of Jiangsu Colleges and Universities,China (10KJB210006)High-Level Talented Person Fund of Yangzhou University, China (2009-2010)
文摘Guided pesticide is an unique compound resulted from the conjugation with carrier (amino acid, protein, sugars, etc) and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein (MP) is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein (GFP) and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG (isopropyl-b-dthiogalactopyranoside) at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.
基金Supported by Scientific Research Start-up Fund for Doctors of Liaocheng University(31805)~~
文摘[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.
文摘Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca2+, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity. Washing with EDTA chelates the Ca2+ ions from the protein, inducing a conformational change back to the more folded state and eluting the protein from the column. We describe herein the use of a protein expression and purification technique using the calmodulin variant and a short linker for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease. We have shown this approach to be useful in obtaining purified quantities of various small proteins that could not be expressed using other methods, including high enough concentrations of a designed WW domain protein for NMR structural analysis. We have also obtained promising results on the usefulness of this procedure to express and purify zinc finger proteins without the addition of zinc ions or other cofactors.
基金supported by the National Natural Science Foundation of China (30771952)the National Natural Science Foundation of Guangdong Province (07117783)NSFC and the Research Grants Council of Hong Kong Joint Research Scheme (30418003).
文摘Objective To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hlL-2/mGM-CSF). Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coil) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.
基金Supported by the Distinguished Young Scholars Fund of China(No.30325011)the National Natural Science Foundation of China(Nos.30470405and30670477)+1 种基金Distinguished Young Scholars Fund of Jilin Province,China(No.20030112)Excellent Young Teachers Program of MOE,China.
文摘mCLCA3 is a member of calcium activated chloride channel(CACC) family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.
基金This work was supported by a grant fromthe International Atomic Energy Agency (IAEA) (grantNo: 12510/R1) a grant from the Chinese NationalNatural Science Foundation (grant No: 30400120)
文摘Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.
文摘AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB (DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 (DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.
文摘The cDNA containing full encoding region or E1 antigen or HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid PRSETE1 was introduced into the BL21 (DE3) strain or E. coli. The engineering bacteria harhouring the pRSETEI was cultivated in 2YT medium at 37℃.When the Expression of E1 protein was induced by 1 mmol IPTG, the bacteria was killed and the number of living cell was droned down from 107 to 103 cell/mL one hour post induction. suggest that E1 protein is poisoned to E. coli. However, the 26kD polypeptide or E1 fussion still synthesized in appropriate condition. The expression level was about 10% or total protein 4 h after Inducing. Th. E1 protin was purfied by Ni2+-NTAAgarose column chromatography to homogeneous. The purified E1 protein was sensitive and specific in reaction with anti-HCV antibody in sera.
文摘To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.
基金Supported by:the National Natural Science Foundation of China,No.30600224Supported by:the National Natural Science Foundation of China,No.30700438+2 种基金China's Post-doctoral Science Fund, No.20060390886Hunan Province Natural Science Foundation,No.06JJ30014 Hunan Province Scientific Program,No.2008FJ3138
文摘BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil (E. coil) JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION: From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.
基金financially supported by grants from the Natural Science Foundation of Shandong Province,China(ZR2018LC005 and ZR2019BC107)the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences,China(CXGC2022C02)。
文摘The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and development,including seedling photomorphogenesis,shade avoidance,flowering time,and biotic and abiotic stress responses.Previous studies have identified many different BBXs from several plant species,although the BBX family members in maize are largely unknown.Genome-wide identification and comprehensive analysis of maize BBX(ZmBBX)expression and interaction networks would therefore provide valuable information for understanding their functions.In this study,36 maize BBXs in three major clades were identified.The ZmBBXs within a given clade were found to share similar domains,motifs,and genomic structures.Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.The expression levels of ZmBBXs were analyzed in various organs and tissues,and under different abiotic stress conditions.Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation(BiFC)assays.Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions.
基金Major State BasicResearch (973) Program of China, (G1999053905).
文摘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.