ABSTRACT
Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Subject(s)
Animals , Foot-and-Mouth Disease Virus/genetics , Capsid Proteins , Viral Proteins/metabolism , Foot-and-Mouth Disease/prevention & control , Tetracyclines/metabolism , Viral Vaccines , Antibodies, Viral , Mammals/metabolismABSTRACT
Objective@#An innovative technique was established to rapidly construct various cell lines that could be induced to express multiple influenza A virus (IAV) proteins.@*Method@#The HA protein genes of multiple IAVs were cloned into the Cumate-induced expression system which was positioned between two PiggyBac transposon sites. These HA plasmids were transfected into the HEK293A cell line with the PiggyBac transposase plasmids. The transfected cells were screened with puromycin, and after that the corresponding virus proteins were induced with Cumate.@*Results@#The results of flow cytometry and Western blotting showed that the virus proteins were expressed in most of the cells in corresponding lines after the induction of Cumate.@*Conclusion@#Cell lines which were inducible to express IVA HA protein can be efficiently constructed by using the PiggyBac transposon system.
ABSTRACT
Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from().(GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by thecDNA was expressed and purified as a recombinant protein from() and showed SMT activity. The expression ofwas highly up-regulated incell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis ofand will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of
ABSTRACT
Objective: To construct a mifepristone(an oral nontoxic chemical)-inducible eukaryotic expression vector and to evaluate its regulatory effect in vitro using luciferase reporter gene. Methods: Vector pDC-RULUC, which contains firefly luciferase reporter gene, promoter and mifepristone-inducible system, was constructed by molecular biological methods. A 1.2 kb insulator was inserted to reduce the interference between two transcription units. The vector was verified by PCR, restriction enzyme digestion, and sequencing. pDC-RULUC was used to transfect SW620 cells using Lipofectamine2000. Cells transfected with pGL3-Control and pGL3-Basic were used as positive and negative controls, respectively. Cotransfectant with pRL-TK renilla luciferase reporter vector was used as internal control. Cells of experimental group were incubated for 48 h in presence of different concentrations of mifepristone after transfection and were harvested for luciferase assay by using the Dual-Luciferase Reporter Assay System. Half of the wells were replaced with fresh medium and were measured after another 48 h. Results: The recombined plasmid vector was identified by digestion with different enzyme restrictions, PCR and sequencing analysis. The relative activity increased with the increase of mifepristone concentration. When the concentration of mifepristone reached 1 × 10-6 mol/L, the relative activity increased to approximately 50 folds of the original. No significant luciferase activity was detected when the mifepristone was removed. Conclusion: We have successfully established mifepristone-regulated eukaryotic expression vector, which can be used for controllable gene expression in vitro, providing a way for gene regulation and gene therapy.
ABSTRACT
Objective To develop mycobacterial inducible expression vectors which permit to overexpress Mycobacterium tuberculosis (Mtb) immunodominant antigen, and to analyze its immunogenicity after purification by affinity chromatography. Methods The regulatory region of M. smegmatis (Ms) acet-amidase(pACE) was obtained by PCR amplification, and was used as promoter to construct the mycobacteri-al inducible expression vectors, pMF series. The coding gene of Mtb chimeric antigen Ag856A2 which is a recombinant Ag85A with 2 copies of ESAT-6 inserted in its Acc Ⅰ site and showed excellent immunogenicity in the animal experiments we described previously, was cloned into the pMF vector series, and was induced to express by the addition of acetamide. The recombinant protein expressed in the Ms was purified by the Ni~(2+)-NTA affinity chromatography, the resulted homologous recombinant antigen was added into the spleen cells separated from BCG vaccinated mice, and the immunogenicity was analyzed by the IFN-γ ELISPOT as-say. Results The mycobacterial inducible expression vectors, pMF series was constructed successfully, target antigen could be. induced to express in the Ms by the addition of 0.02% acetamide, and could be puri-fied by the Ni~(2+) -NTA affinity chromatography due to the addition of 6×His tag in the vector pMF406. Fur-thermore, the mycobactefial homologous antigen could induce more IFN-γ secretion than the heterogonous one. Conclusion The mycobacterial inducible expression system based on the regulatory region of Ms acet-amidase as promoter could permit the Mtb target antigen of interest overexpression and purification, and the immunogenicity of the homologous antigen from Ms is better than that of be expressed from E. coli, which may be more potential for immunological detection of tuberculosis.
ABSTRACT
The promoter and signal peptide sequence of sacB gene (sacR gene) has been amplified by PCR.An inducible expression and secretion vector pHP13SN has been constructed with this amplified sequence,which was ligated with the pro-peptide and mature peptide of neutral protease gene on the vector pHP13.Transforming Bacillus subtilis DB104 with the vector pHP13SN, and the recombinant strain DB104(pHP13SN) can be got.The neutral protease gene has been expressed by the inducement of sucrose and the regulation of sacR,and the production has been secreted with bioactivity.
ABSTRACT
RIG-Ⅰ, the abbreviation of retinoic acid inducible gene-Ⅰ, can be induced to express in many type cells by various stimuli. Recently, it was identified as an intracellular regulator for RNA virus-induced antiviral response in innate immunity. Its discovery, expression induction, structure, research status of its function, homologous proteins and functional mechanism etc. were summarized, and its further research pulses are also prospected meanwhile.
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Objective:To construct eukaryotic inducible expression plasmids pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 using Tet-On gene expression system and co-transfect them into Chinese hamster ovary(CHO) cells,so as to achieve the inducible expression of Mac-1-FP.Methods: The eukaryotic expression vectors pTRE-Tight-CFP-CN11b and pTRE-Tight-YFP-CD18 were constructed by recombinant DNA technique.The 2 vectors were co-transfected into CHO cells using liposome to fuse CD11b and CD18: the 2 subunits of Mac-1. Fluorescence microscope was used to observe the cyan fluorescence and the yellow fluorescence of Mac-1-FP.The influence of different levels of Dox(0,0.01, 0.1,0.5,1,2 ?g/ml) on expression levels of CD11b and CD18 in CHO cells was analyzed by RT-PCR and fluorescence intensity analysis.The adhesive rate of CHO-Mac-1-FP cells with ligand ICAM-1 was analyzed before and after PMA(1 ?g/ml) stimulation.Results: The recombinant plasmids of pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 were successfully constructed.The cyan and yellow fluorescences were observed in co-transfected CHO cells under fluorescence microscope.The fluorescence intensity of the cells was increased with the increase of Dox concentration.RT-PCR analysis demonstrated that the CD11b and CD18 mRNA increased with the increase of Dox concentration.The adhesive rate of CHO-Mac-1-FP cells with ICAM-1 was increased after PMA stimulation(peaked at 2 h and 4 h after stimulation and decreased afterwards).Conclusion: This study achieves the inducible expression of Mac-1-FP in CHO cells.And Mac-1-FP,like widetype Mac-1,exhibits adhesive activity with ligand ICAM-1,which lays a foundation for studying the consisting dimmer,clustering,conformation and affinity of the ligands of Mac-1 using single molecule spectroscopy and fluorescence resonance energy transfer technique in living cells.
ABSTRACT
The regulatory region and the signal peptide sequence of the sacB gene has been amplified by PCR using Bacillus subtilis chromosomal DNA as template, and an inducible secretion vector has been developed based on this sequence, which was ligated with Bacillus subtilis alkaline proteinase gene. Transform Bacillus subtilis DB403 with this vector, and the expression of the inserted Bacillus subtilis alkaline proteinase gene can be induced by addition of sucrose into the medium.
ABSTRACT
Objective:To construct a mifepristone(an oral nontoxic chemical)-indueible eukaryotic expression vector and to evaluate its regulatory effect in vitro using luciferase reporter gene.Methods:Vector pDC-RULUC,which contains firefly lucif- erase reporter gene,promoter and mifepristone-inducible system,was constructed by molecular biological methods.A 1.2 kb in sulator was inserted to reduce the interference between two transcription units.The vector was verified by PCR,restriction en- zyme digestion,and sequencing,pDC-RULUC was used to transfect SW620 cells using Lipofectamine2000.Cells transfected with pGL3-Control and pGL3-Basic were used as positive and negative controls,respectively.Cotransfectant with pRL-TK renilla lu- ciferase reporter vector was used as internal control.Cells of experimental group were incubated for 48 h in presence of different concentrations of mifepristone after transfection and were harvested for luciferase assay by using the Dual-Luciferase Reporter Assay System.Half of the wells were replaced with fresh medium and were measured after another 48 h.Results:The recom- bined plasmid vector was identified by digestion with different enzyme restrictions,PCR and sequencing analysis.The relative activity increased with the increase of mifepristone concentration.When the concentration of mifepristone reached 1?10~(-6)mol/ L,the relative activity increased to approximately 50 folds of the original.No significant luciferase activity was detected when the mifepristone was removed.Conclusion:We have successfully established mifepristone-regulated eukaryotic expression vector, which can be used for controllable gene expression in vitro,providing a way for gene regulation and gene therapy.