87 results about "EGFP protein" patented technology

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.

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 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 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 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.

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 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.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The application relates to the technical field of biomedical engineering, and particularly discloses a method for extracting binary condensate immune signals, which comprises the following steps: screening disease-related high-expression genes by using a disease model, a normal model and a drug treatment model; screening important protein molecules based on the high-expression genes by using a nucleic acid-protein affinity analysis method; constructing a fusion carrier based on cytoskeleton proteins, important protein molecules and green fluorescent protein tracing; and identifying risk genes and possible downstream proteins associated with condensates by spatial omics imaging based on the fusion carrier of the disease model, the normal model and the drug treatment model, and quantifying the condensation process of the condensates. The application reduces the influence of problems such as condensate state destruction and spatial distribution information loss, and accurately realizes in-situ spatial omics imaging and analysis of an immune microenvironment evolution process of a complex disease.

The invention belongs to the technical field of animal genetic engineering and biological breeding, and particularly relates to a recombinant plasmid for co-expressing human antibacterial peptide and human interferon and application of the recombinant plasmid in the aspect of improving the disease resistance of aquarium fish. The recombinant plasmid is a plasmid which contains a human antibacterial peptide gene hLL-37 and a human interferon gene hIFNA1 and has a Tol2 transposon, a goldfish pan-expression promoter gActb1, a green fluorescent protein gene EGFP and a polyA sequence, and the nucleotide sequence of the recombinant plasmid is as shown in SEQ ID NO. 1. The recombinant plasmid and the Tol2 transposase mRNA are jointly introduced into the aquarium fish, so that the antiviral capability and the antibacterial capability of the aquarium fish can be improved.

The invention discloses an infectious pseudo-ginseng virus A cloning vector, the nucleotide sequence of the infectious pseudo-ginseng virus A cloning vector is shown as SEQ ID NO: 1, the infectious pseudo-ginseng virus A cloning vector belongs to the field of plant virology and genetic engineering, and the infectious pseudo-ginseng virus A cloning vector is obtained by cloning CP gene and RdRp gene of pseudo-ginseng virus A into a pCB301 vector; on the basis of the vector, a PnVA infectious cloning vector pPnVA-GFP carrying a green fluorescent protein GFP is constructed; a PnVA infectious cloning vector is transferred into an agrobacterium GV3101 strain through a freeze-thaw method, the agrobacterium GV3101 strain is transferred into nicotiana benthamiana and pseudo-ginseng through an injection method for expression, and results show that pCB301-PnVA and pPnVA-GFP can complete PnVA gene replication in pseudo-ginseng and tobacco and trigger typical virus disease symptoms.

The application discloses a shark-derived synthetic nanobody targeting green fluorescent protein and an application thereof. The nucleotide sequence of the nanobody is as shown in SEQ ID NO:1-4, and the amino acid sequence is as shown in SEQ ID NO:5-8. The preparation method comprises the following steps: screening a green fluorescent protein specific nanobody sequence from a shark-derived synthetic antibody library, performing eukaryotic expression vector construction, then performing transient transfection in HEK 293F cells for expression, and finally obtaining the shark-derived synthetic nanobody targeting the green fluorescent protein. The shark-derived synthetic nanobody is used for affinity purification of the green fluorescent protein. The nanobody of the application is derived from Chlamydoselachus anguineus, has small molecular weight, strong stability, strong tissue penetration ability and can recognize hidden antigen epitopes, and can be used in the fields of affinity purification, immunological detection, molecular imaging, immunosensing probes and the like of the green fluorescent protein.

The invention relates to the field of biomedical engineering, and discloses a human-derived glioblastoma GBM invasion organ-like model as well as a construction method and application of the human-derived glioblastoma GBM invasion organ-like model. The model is a fused organ BGO-CC formed by performing physical contact co-culture on a dorsal forebrain organ BO for expressing green fluorescent protein GFP and a glioblastoma organ GO from a patient. The invention can deeply reveal the recurrence and invasion mechanism of GBM, and provides an efficient and reliable platform for screening anti-invasion drugs.

The invention provides application of organic peroxide resistant protein Ohr in inducing phase separation of protein in plant cells. The invention proves that the organic peroxide resistant protein Ohr is coagulated in plant cells in nicotiana benthamiana and can cause the cell death of nicotiana benthamiana, and the coagulation of the organic peroxide resistant protein is generated through phase separation and depends on two internal disordered regions IDR2 and IDR3 of a C segment of the organic peroxide resistant protein, so that the cell death of the nicotiana benthamiana can be caused. The organic peroxide resistant protein without the N-segment IDR1 does not cause cell death, but can cause phase separation of green fluorescent protein. A plant regulates and controls many physiological and biochemical reactions through protein phase separation, it is proved that the organic peroxide resistant protein derived from microorganisms can trigger phase separation in the plant, it is proved that the N-segment truncated mutant of the organic peroxide resistant protein can serve as tag protein to promote phase separation of other proteins, plant death is not caused, and the application is wide in application prospect. The method is suitable for researching the functional requirements of protein phase separation, and has wide application and market prospects.