111 results about "EGFP protein" patented technology

The invention discloses construction and application of an escherichia coli L-tryptophan biosensor, and belongs to the technical field of bioengineering. The L-tryptophan biosensor provided by the invention comprises a mutated leader peptide fragment tnaC-tnaA, a promoter of the mutated leader peptide fragment tnaC-tnaA, a green fluorescent protein coding gene eGFP and a pTrc99a plasmid skeleton, a recombinant strain containing the L-tryptophan biosensor is cultured, the expression of green fluorescent protein in a culture solution is gradually enhanced along with the increase of the concentration of the L-tryptophan, and the fluorescence value of a unit cell shows better correlation when the concentration of the L-tryptophan is 0-1.5 g / L; therefore, when the biosensor is used for detecting the L-tryptophan in the fermentation liquor, the biosensor has the characteristics of simplicity in operation, sensitive response and high threshold value.

The invention discloses a construction method and application of a probiotic engineering bacterium EcNpAm targeting animal intestinal tracts and improving inflammation. According to the present invention, the heterologous expression of the immunomodulatory protein Amuc1100 is successfully achieved through the gene engineering technology by using the Escherichia coli Niss1917 (EcN) as the host and using the anaerobic inducible plasmid pnirBMisL as the expression vector, and the novel engineering bacterium EcNpAm suitable for the animal intestinal anaerobic environment is constructed; furthermore, the expression efficiency of the target protein is represented by fusion expression of green fluorescent protein (GFP). The engineering bacterium is applied to prevention and treatment of animal intestinal inflammation, and successfully realizes targeted delivery of therapeutic protein to intestinal tracts of mice. Experiments show that EcNpAm can effectively relieve typical symptoms such as weight loss, colon shortening, pathological injury, colitis and the like of mice induced by dextran sodium sulfate (DSS) and protect integrity and functions of an intestinal barrier structure.

The invention provides a single-chain and double-protein-expressed circular RNA construct and a preparation method and application thereof, and the single-chain and double-protein-expressed circular RNA construct comprises 5'and 3 'introns used for realizing RNA self-splicing and cyclization as a first kind of introns; the 5'and 3 'homologous arms are used as pairing sites in RNA molecules; the exon 1 and the exon 2, which are connected after being spliced, can be spliced and connected together under the action of the first type of introns to form a complete and continuous open reading frame; an interval sequence for providing spatial and adjustment sequence structures; the IRES is used for starting translation; a glycine-serine linker as a flexible linker; a green fluorescent protein for gene expression monitoring; a luciferase for producing light by a catalytic chemical reaction. All the advantages of the double-IRES system for generating natural complete protein are reserved, and the core defect of low efficiency can be thoroughly overcome.

This invention provides a method for constructing a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and negative mast cells. First, the Cre gene is knocked into the Mcpt4 gene sequence of C57BL / 6 mice using CRISPR / Cas9 technology. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive mast cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. Using this fluorescently traced mouse model, Mcpt4-positive and negative mast cells can be rapidly, accurately, and effectively sorted, providing a reliable technical basis for exploring the roles and mechanisms of Mcpt4-positive and negative mast cells in immune responses.

The application discloses a use of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof in preparation of a medicine for inhibiting African swine fever virus. The application uses a double reporter virus rASFV-Gluc / EGFP co-expressing green fluorescent protein and Gaussia luciferase to screen 246 natural small molecule compounds, and finds that phenazine-1-carboxylic acid has a significant inhibiting effect on replication of the ASFV; further evaluation of the inhibiting effect finds that the half maximal cytotoxicity concentration of PCA on target cells of primary porcine alveolar macrophages is 470.5 muM, and the half maximal inhibitory concentration of PCA on the ASFV in PAMs is 1.59 muM. According to a dose-dependent inhibiting experiment result, when the concentration of PCA is 25 muM, the inhibiting effect on the ASFV can reach more than 100 times. The application has application prospects in preparation of medicines or preparations against the ASFV.

The present application relates to a gene editing vector of Caulobacter crescentus, a gene editing method thereof and a Caulobacter crescentus editing strain. The gene editing vector of the Caulobacter crescentus comprises the following gene elements: a plasmid backbone, a Pvan promoter, a spCas9M gene, a green fluorescent protein gene GFP, a gRNA, an upstream homologous arm and a downstream homologous arm of a target gene. The gene editing vector of the Caulobacter crescentus adopts a CRISPR / spCas9M-GFP gene editing system, constructs a high-efficiency, simple and scarless genome editing system in the Caulobacter crescentus, and can realize gene knockout and insertion.

This application discloses an embedded whole-cell biosensor, its fabrication method, and its application in machine learning-assisted mercury ion detection, belonging to the field of sensor materials and detection technology. This sensor encapsulates *E. coli* expressing green fluorescent protein (GFP) in a polyacrylamide / hyaluronic acid (PAAM / HA) hydrogel matrix, enabling highly selective and sensitive detection of mercury ions. It effectively eliminates interference from competing metal ions and can be completely regenerated through EDTA-2Na treatment, allowing for repeated use without performance loss. It maintains a stable fluorescence response for up to three weeks, possesses excellent biocompatibility and biodegradability, minimizing the risk of secondary contamination, and facilitates rapid on-site analysis.

A fluorescent protein of the present invention has an amino acid sequence of a green fluorescent protein (GFP) derived from a crystal jelly or of a mutant fluorescent protein of the green fluorescent protein, the amino acid sequence having an amino acid residue (alanine residue) substituted with a phenylalanine residue, the amino acid residue corresponding to position 206 with the amino acid sequence of the GFP used as a reference sequence.

The application discloses a lithium acetate-polyethylene glycol mediated protoplast lipid nanoparticle delivery method and belongs to the technical field of biology. 2+ )concentration to improve the PEG formula, successfully delivers the lipid nanoparticle (LNP) to the protoplast of oil palm, coconut and other palm crops, the mRNA of the nanoparticle is wrapped with enhanced green fluorescent protein (eGFP), and the fluorescent protein can be directly expressed in the protoplast with the mRNA as a template without a promoter. After the LNP is delivered to the protoplast of the palm crops by the method, green fluorescent signals are detected by laser confocal microscopy. The method can be used for directly delivering target gene mRNA to the protoplast of the palm crops, and can also deliver CRISPR / Cas system protein and the like for gene editing technology research.

Fluorescent proteins and split-fluorescent proteins (SFPs) including split-green fluorescent proteins from Corynactis californica are provided. Nucleic acid molecules encoding the fluorescent proteins and SFPs, vectors, and kits are also provided. Methods of using the fluorescent proteins and SFPs, such as detecting a protein of interest or protein-protein interactions are also provided.

The invention belongs to the technical field of gene engineering, and particularly relates to a construction method and application of a recombinant vector based on bCoro1a gene 3 'UTR editing. The recombinant vector consists of an upstream homologous arm of a targeting site, 3 'UTR inserted into a target sequence, 200bp of a nucleotide tailing signal of bCoro1a, a selection marker and a downstream homologous arm of the targeting site; the targeting site is the 29 or 70 site of the 3 'UTR of the bCoro1a gene; the sequence of the 3 'UTR of the bCoro1a gene is as shown in SEQ ID NO. 48; the selection marker comprises enhanced green fluorescent protein and puromycin, the bCoro1a 3 'UTR edited gene expression regulation and control system is based on bCoro1a 3' UTR edited gene expression regulation and control system, and the expression level of bCoronin-1A is reduced in a targeted manner by inserting a specific bta-miR-27b target sequence into 3 'UTR, so that M.tb immune escape is inhibited, and the M.tb infection resistance of a host is improved.

The invention discloses a surface modification method of a cycloolefin copolymer membrane material, and belongs to the technical field of high polymer materials. The surface modification method of the cycloolefin copolymer membrane material comprises the following steps: (1) putting a cycloolefin copolymer membrane into an acid mixed solution, and carrying out acid treatment; after acid treatment, filtering, washing and drying to prepare an acid-treated cycloolefin copolymer membrane; (2) putting the cycloolefin copolymer membrane subjected to acid treatment into a solvent, adding an amination reagent under a stirring condition, and heating to react; and after the reaction is finished, washing and drying to obtain the surface-modified cycloolefin copolymer film. The surface-modified cycloolefin copolymer membrane prepared by the invention has good hydrophilic performance, and the contact angle can be as low as 69 degrees; the mechanical property is slightly reduced, but the use is not influenced; the amino content is higher; the protein FITC-BSA with green fluorescence can be better adsorbed; therefore, subsequent development and research of biological materials are facilitated.

The invention discloses a fusion protein and a mycobacterium detection method based on the fusion protein. The method comprises the following steps: constructing a fusion protein Gp16-EGFP by combining a tail silk protein gene gp16 of a bacteriophage TM4 and a green fluorescent protein gene egfp fragment; the nucleotide sequence of the tail silk protein gene gp16 is as shown in SEQ ID NO.1, and the amino acid sequence of the tail silk protein gene gp16 is as shown in SEQ ID NO.2; the nucleotide sequence of the fluorescent protein gene egfp is as shown in SEQ ID NO.3, and the amino acid sequence of the fluorescent protein gene egfp is as shown in SEQ ID NO.4; in the invention, by amplifying fragments of a tail silk protein gene gp16 and a green fluorescent protein gene gfp of a bacteriophage TM4, cloning the two fragments to a pET28a plasmid and transforming the pET28a plasmid into an escherichia coli expression strain BL21, the obtained recombinant strain is obviously green and also has green fluorescence under a fluorescence microscope, and the fusion protein Gp16-EGFP with higher purity is obtained by expression and purification. The fusion protein Gp16-EGFP can be specifically combined with the surface of the mycobacterium to play a marking role, so that the detection of the mycobacterium is realized.

The invention discloses a green fluorescence report system for screening a Listeria monocytogenes quorum sensing inhibitor as well as a construction method and application of the green fluorescence report system. The report system is constructed through the following steps: synthesizing a DNA fragment according to a listeria monocytogenes AGR system pII promoter sequence, connecting the DNA fragment with a green fluorescent protein pEGFP plasmid, amplifying an EGFP-pII target fragment through PCR, and connecting the target fragment to a pKSV7 shuttle vector plasmid through homologous recombination to obtain a pKSV7-EGFP-pII plasmid, and then transferring the gene into a competent cell of listeria monocytogenes ATCC BAA-679, and screening to obtain a report system. The report system disclosed by the invention can be used for intuitively evaluating the expression condition of the quorum sensing system of the listeria monocytogenes and screening out the quorum sensing inhibitor.

The invention relates to an efficient preparation and activation method of natural protein glutaminase, and belongs to the technical field of biology. The invention provides a method for preparing and activating natural protein glutaminase, which comprises the following steps of: expressing fusion protein consisting of super-folded green fluorescent protein, a protein glutaminase propeptide region, intein and a protein glutaminase mature region in bacillus subtilis; extracellular expression of fusion protein is guided by super-folded green fluorescent protein, the fusion protein is spontaneously cut by intein Mxe GyrA, activated protein glutaminase is obtained, the sequence of the activated protein glutaminase is completely consistent with that of natural protein glutaminase, the highest enzyme activity reaches 52.3 U / mL, and the activity of the protein glutaminase is remarkably improved. The problem that the mature enzyme sequence is changed due to poor protease activation specificity in the prior art is solved, a brand new thought and tool are provided for efficient preparation and activation of protein glutaminase, and the method has wide application prospects.

The invention relates to an improved embryo rescue technology for cucumber haploid breeding, and aims to solve the problem of low embryo rescue operation efficiency in a technology of inducing haploid by radiation pollen. By using a male parent material carrying a green fluorescent protein (GFP) marker, the haploid screening process in the seedling stage is simplified, and efficient and simple large-scale haploid cultivation is realized in combination with an optimized rapid embryo rescue method. The method comprises the specific steps of radiation pollen preparation, pollination and subsequent embryo rescue processes, particularly emphasizes the technical key points of disinfection, seed taking, cleaning and inoculated culture, and ensures that the operation speed and the success rate are improved while the pollution risk is strictly controlled. Compared with a traditional method, the method has the advantages that the obtaining efficiency of haploid cucumber plants is remarkably improved, the method is suitable for large-scale haploid breeding of cucurbitaceae crops, breeding of new cucumber varieties is accelerated, and agricultural yield increase and income increase of farmers are promoted.

The application discloses a recombinant strain and a preparation method thereof and application of the recombinant strain in preparation of a vaccine for Streptococcus agalactiae of tilapia, and the recombinant strain is successfully constructed by the following steps: designing an ORF4 primer, amplifying an ORF4 gene fragment from a DNA template of Streptococcus agalactiae, editing the ORF4 gene fragment into Escherichia coli by means of a CRISPR / Cas9 gene editing technology, and connecting a green fluorescent protein (GFP). The recombinant strain is subjected to immunoprotection research on the Streptococcus agalactiae of tilapia, and it is shown that the recombinant strain has a good protection effect, and can provide a solid theoretical basis and technical support for application and development of the vaccine for Streptococcus agalactiae of tilapia in the field of aquaculture.

The present disclosure provides compositions and methods for Agrobacterium infiltration of plant tissues, for example fruit, vegetable or leaf tissues, and the use of such plant tissues for production of high-value compounds, for example mogroside compounds, in particular mogroside V, betalain, lactoferrin, or fluorescent proteins, such as green fluorescent protein (GFP), and the high-value compounds so produced.

The invention discloses a method for constructing a genetic transformation system of cymbidium kanran, which belongs to the technical field of plant genetic engineering, and comprises the following steps of: carrying out monoclonal propagation on rhizomes after germination of cymbidium kanran seeds to obtain vegetative propagules with stable characters, taking the vegetative propagules as transformation receptors, taking GFP (Green Fluorescent Protein) as marker genes, and constructing the genetic transformation system of cymbidium kanran. The method comprises the following steps: adding agrobacterium EHA105 into a YEB liquid culture medium containing 50 mg / L of kanamycin and 25 mg / L of rifampicin as a dip dyeing solution; placing the injured transformation receptor in dip dyeing for transformation, and then carrying out regeneration culture. The invention relates to a method for carrying out genetic transformation on cymbidium kanran by utilizing the innovative receptor and the screening culture medium, and the method can realize efficient and stable genetic transformation without genotype dependence and can solve the problem that the genetic transformation of cymbidium kanran varieties is difficult to carry out. According to the invention, an efficient genetic transformation and regeneration system of cymbidium kanran is established for the first time, and a foundation is laid for verification of related functional genes of cymbidium kanran and further development of biological research of cymbidium transgenic breeding and the like.

The invention discloses an in-vitro transcription system of theileria annulata transformed cells and a construction method of the in-vitro transcription system, the in-vitro transcription system comprises IVT-EGFP-mRNA, and the structure of the IVT-EGFP-mRNA is 5 'UTR-Cl-Kozak-EGFP-Pacl-3' UTR-polyA (120). According to the invention, mRNA for coding the EGFP gene is synthesized through in-vitro transcription, through capping, purification and agarose gel electrophoresis analysis, the expression condition of the target gene in TaNM1 cells is finally verified through an in-vitro transfection test and an indirect immunofluorescence technology, and the result shows that the in-vitro transcription system transfected and coded with the EGFP successfully expresses the EGFP, and the expression condition of the target gene in the TaNM1 cells is verified. The invention shows that a system for transfecting the EGFP gene into the theileria annulata transformed cell TaNM1 through an in-vitro transcription method is successfully established, and an important reference value is provided for design and research and development of theileria annulata RNA vaccines.

This invention belongs to the field of genetic engineering technology, specifically disclosing a method for preparing and applying gene-modified homozygous cells of chicken progenitor cells (PGCs). Using a dual-fluorescent marker gene, two donor plasmids carrying green fluorescent protein (EGFP) and red fluorescent protein (mCherry), respectively, are transfected into PGCs. Cells simultaneously expressing red and green fluorescence are obtained using flow cytometry, thus forming homozygous cell lines. Then, the dual-fluorescent marker gene is deleted using the Cre-LoxP system combined with electroporation, and residual LoxP fragments are deleted using ssODN as a donor template, achieving traceless gene editing in recipient chicken embryos. This invention does not introduce any exogenous genes, increasing the safety of gene-modified chickens and is of great significance for eliminating the biosafety risks associated with gene-edited chickens.

The invention discloses a virus aerosol experiment system based on pseudovirus. The virus aerosol experiment system comprises the following steps: preparing a lentiviral vector for expressing green fluorescent protein; adding the lentiviral vector expressing the green fluorescent protein into a PBS solution to obtain a virus aerosol generation solution, adding the virus aerosol generation solution into an aerosol generator, taking a DMEM culture medium containing 1% of fetal calf serum as an aerosol collection solution, putting the aerosol collection solution into a liquid impact type aerosol collector for treatment, culturing HEK293T cells, inoculating the aerosol collection solution with the HEK293T cells, and culturing the HEK293T cells to obtain the lentiviral vector expressing the green fluorescent protein. Culturing the inoculated cells for 72 hours; and after culture is completed, measuring the proportion of cells expressing fluorescence, and calculating the number of active virus particles in the virus aerosol. According to the invention, a set of virus aerosol experiment system is established, the system adopts a pseudovirus expressing fluorescent protein as a model virus to carry out a virus aerosol active particle detection experiment, and the system can be applied to a method and a technology for evaluating detection and disinfection of virus aerosol active particles in air.

The present application relates to an oil palm UBQ promoter and application thereof, and belongs to the technical field of biology.The nucleic acid sequence of the promoter is shown as SEQ ID NO.1, the present application firstly aligns and intercepts a sequence of 2000bp upstream of a start codon in an oil palm genome, and synthesizes an oil palm RNA polymerase II type promoter, i.e., an oil palm endogenous ubiquitin promoter EgUBQ.The promoter has transcription activity, and can drive expression of downstream green fluorescent protein NeonGreen.The present application provides an oil palm endogenous UBQ promoter for genetic transformation and gene editing of oil palm.

The invention discloses a cell strain capable of stably expressing a multi-protein tag as well as a construction method and application of the cell strain, and belongs to the technical field of biology. The cell strain is transfected by a lentiviral vector, a fusion expression vector containing P2A self-cleavage polypeptide and a plurality of protein tag coding sequences is integrated into a host cell, and a stable expression cell strain and a fusion protein expressing a plurality of protein tags including green fluorescent protein are obtained through antibiotic screening. The cell strain can be used for antibody specificity verification, protein interaction research and multi-label system standardization experiment platforms, and has the advantages of stable expression, simple operation, reliable result and the like.

The application provides a construction and application of a recombinant pseudorabies virus strain stably and efficiently expressing green fluorescent protein, in the research, a green fluorescent protein gene (EGFP) is inserted into different positions of a double gene deletion vaccine strain (JS-2012-△gE / gI) of a pseudorabies virus mutant strain by using a homologous recombination method. Finally, it is proved that the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP has good genetic stability, and the EGFP gene can also be stably and efficiently expressed, and the growth rate on cells is also comparable to that of the parent virus, so that the VP1 / 2 gene is the best exogenous gene insertion site. Subsequently, the classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be replaced or inserted into the EGFP expression frame in a single or combined manner to construct different types of multi-vaccines, and the multi-vaccines have extremely high research value and platform significance.

This invention provides a research model and construction method for calcium ion imaging of ASH neurons in nematodes, belonging to the field of biological model construction technology. The construction method includes the following steps: nematode culture; construction of a transgenic probe plasmid: obtaining the ASH neuron-specific promoter sra-6 using PCR technology, and connecting HindIII and BamHI restriction sites to both ends of the promoter; amplifying YC3.60 using PCR technology; then inserting the promoter sequence into the pPD95.75 plasmid, and then inserting YC3.60 after the promoter. Transgenic microinjection of nematodes is used, followed by screening to obtain nematodes with ASH neurons carrying green fluorescent protein. In this application, YC3.60 is transferred into nematodes via transgenic means. Using a promoter specifically expressed in ASH neurons to link the gene of this fluorescent probe protein into the nematode, the fluorescent probe can be specifically expressed in the neuron. The constructed nematode model can be used for neuronal structure and functional imaging studies, as well as neuropharmacological efficacy analysis.

A derivative of insulin aspart and a method for preparing the same, the derivative comprising a fusion protein of a green fluorescent protein folding unit and an insulin aspart precursor or an active fragment thereof. The fusion protein is expressed in a significantly increased amount, the insulin aspart precursor protein in the fusion protein is folded correctly and has biological activity. Furthermore, the green fluorescent protein folding unit in the fusion protein can be digested by a protease into small fragments, and has a large difference in molecular weight compared to the target protein, and is easy to separate. A method for preparing insulin aspart and an intermediate using the fusion protein is also provided.

The present invention discloses a mercury response reporting vector and a visual monitoring and early warning microbial sensor cell and its application. p2_244 The expression level is finely regulated and high-intensity ribosome binding site RBS is used GroEL A double-copy fluorescent protein gene is used as a reporter element, with a wide host vector as the backbone; the constitutive promoter sequence is shown in any one of SEQ ID NO. 4-9; the RBS GroEL The sequence is shown in SEQ ID NO.10; the green fluorescent protein gene sequence after codon optimization is shown in SEQ ID NO.11. Microbial sensor cells have lower Hg 2+ ELISA detection limit and Hg 2+ The visual detection limit provides a simple, feasible, portable and efficient solution for the rapid detection and visual early warning of environmental heavy metal mercury pollution.

The invention discloses an enhanced green fluorescent protein mutant, and the amino acid sequence of the enhanced green fluorescent protein mutant has the following mutation sites: S30R, Y39N, N105T, I171V, A206V and L220F compared with the amino acid sequence of green fluorescent protein roGFP1 or roGFP2. The invention discloses a fluorescent probe. Raw materials for preparation comprise an enhanced green fluorescent protein mutant. The invention also discloses a preparation method of the enhanced green fluorescent protein mutant and application of the enhanced green fluorescent protein mutant in an oxidation sensor. Compared with green fluorescent protein, the enhanced green fluorescent protein mutant disclosed by the invention has the advantages that the fluorescence intensity is enhanced, the response range is expanded, and the enhanced green fluorescent protein mutant can be used as a strong and high-sensitivity fluorescent indicator for monitoring the redox potential in cells.

The invention discloses a fungus expression vector containing a green fluorescent protein reporter gene as well as a construction method and application of the fungus expression vector, and belongs to the technical field of edible fungus genetic engineering. On the basis of flammulina velutipes strain 0747 transcriptome data, a promoter sequence of a gene hyd9 efficiently expressed in hyphae is obtained through screening and cloning, and the promoter sequence has conserved structures such as a TATA box and a CAAT box. The expression activity of the promoter in hyphae is about 60 times that of an Ffgpd promoter. By constructing a hyd9-driven egfp expression vector and converting flammulina velutipes, the result shows that a transformant presents strong green fluorescence, the egfp transcriptional level is significantly higher than that of a Glgpd and Ffgpd promoter control group, a fluorescence signal is weakened in primordium and sporocarp but still can be detected, the mode is consistent with an endogenous hyd9'hypha high expression-sporocarp low expression 'mode, and the expression of the endogenous hyd9-sporocarp low expression is consistent with that of the endogenous hyd9. The promoter is proved to be capable of driving the expression of exogenous genes in a space-time specific manner.