334 results about "Fluorescent protein" patented technology

The invention provides a fluorescent protein selection marker staygold-ynaMr gene and application thereof, and belongs to the technical field of biological engineering. The method comprises the following steps: replacing codons of L-isoleucine in nucleotide sequences of a fluorescent protein gene staygold and a gene ynaM by using a rare codon ATA to respectively obtain the fluorescent protein gene staygold after codon replacement and the gene ynaMr after codon replacement, and connecting the two segments of genes after codon replacement by using a flexible protein peptide, and the fluorescent protein selection marker staygold-ynaMr gene is obtained. Experiments prove that the gene can obviously improve the screening efficiency of L-isoleucine high-yield strains.

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 an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter gene is shown as SEQ ID NO.1. The oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, is obtained by cloning in an oil palm genome for the first time, and the promoter gene has high transcriptional activity and can drive downstream fluorescent protein mNeonGreen expression. The candidate oil palm endogenous U6 promoter gene can be provided for subsequently establishing a high-efficiency oil palm gene editing technology system based on a CRISPR / cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) system.

The invention discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for targeted knockout of Pten and Trp53 genes, and the recombinant plasmid comprises an EPI vector system. The recombinant plasmid takes an ori element as a replication start site, and sequentially comprises an sgRNA sequence of a targeted Trp53 gene and Pten controlled by double U6 promoters, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, an orip element and an EBNA1 protein expression element. The invention also discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and the established ovarian cancer cell model. By adopting the method to construct the ovarian cancer cell model, the period from cell editing to animal tumor formation is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.

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 discloses a rapid degreasing method for a biological tissue sample, which comprises the following steps: sequentially carrying out fixing, tissue dehydration, azeotropic degreasing, tissue rehydration and other treatments on the sample, and the degreasing solution is composed of alcohols and hydrocarbons according to a mass ratio of 1: 1-1: 2. According to the rapid degreasing method provided by the invention, the lipid in the tissue is efficiently extracted through mixed azeotropy of the alcohol and hydrocarbon organic reagents, so that the tissue is transparent and transparent; the problem of poor degreasing in tissues is solved by utilizing the high permeability characteristic of the micromolecular organic reagent; the dissolving capacity to phospholipid and cholesterol is improved by mixing organic reagents; through azeotropy, the degreasing efficiency is improved, the degreasing time is shortened, the internal permeability effect of the tissue is improved, and internal and external uniform permeability of the tissue is realized; by adjusting the components of the organic reagent, boiling rapid degreasing at low temperature is realized, and the retention of a fluorescence signal is improved; the tissue treated by the method is transparent and small in deformation, a fluorescent protein signal can be maintained, and the immunofluorescence staining effect can be improved.

The invention provides a recombinant lentivirus capable of indicating bovine spermatogonial stem cell differentiation, a spermatogonial stem cell line and application, and belongs to the technical field of cell biology. The invention provides a method for constructing a spermatogonial stem cell line for indicating bovine sperm differentiation on the basis of a lentivirus transduction technology, and a visual detection system for differentiating spermatogonial stem cells into spermatoblasts is constructed by regulating expression of mCherry protein by utilizing a PRM1 promoter. According to the method, the expression of PRM1 is indicated by fluorescent protein mCherry, so that the differentiation process of the bovine spermatogonial stem cells is indicated, and a good tool is provided for the induction of differentiation of the bovine spermatogonial stem cells to sperms, the research of a development mechanism and the optimization of a differentiation system of the bovine spermatogonial stem cells. The method is a new way for solving the neck clamping problem that good-variety bovine sperms are difficult to obtain in scientific research and production, can also be used for optimization of a spermatogonial stem cell differentiation system, sorting and purification of sperms, even downstream research of drug screening and the like, and has a good application prospect.

The invention discloses a stably transfected cell strain for evaluating fibroblast autophagy flux and a construction method of the stably transfected cell strain. The construction method comprises the following step: infecting fibroblasts by using HBAD (Hepatitis B Antigen)-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus. The construction method can be combined with a super-resolution microscope to dynamically observe the autophagy intensity change in the primary fibroblasts through an imaging method.

Protease activity can be assessed using a microfluidic system that comprises a microfluidic device and solid support, wherein the solid support comprises a protease fusion protein bound to a first surface of the solid support; wherein the protease fusion protein comprises a protease domain, a protease activation sequence, and a fluorescent protein tag, wherein the fluorescent protein tag is bound to the protease domain and to the first surface of the solid support. Protease activity can be assessed in a microfluidic system by reacting an activating protease with a protease fusion protein; wherein the protease fusion protein comprises a protease domain and a protease activation sequence; wherein the activating protease cleaves the protease activation sequence, thereby activating the protease domain in the protease fusion protein to produce an activated protease fusion protein; reacting the activated protease fusion protein with a detectable substrate, thereby generating a detectable signal; and detecting the detectable signal, thereby detecting protease activity in the microfluidic system.

The application relates to establishment and application of an isobutyraldehyde biosensor and belongs to the technical field of bioengineering. yqhD The RFP report system responding to isobutyraldehyde is established, the yqhC gene is rationally designed, a mutant library is constructed through random mutation by using error-prone PCR, isobutyraldehyde is used as a substrate, and through screening of response concentrations and response thresholds, a YqhC mutant without background fluorescence response, with a wider substrate detection range (0-5 g / L) and higher response intensity (24 times) is obtained, and the YqhC mutant is named YqhCm9. The isobutyraldehyde response element (YqhCm9) and the fluorescent protein report element (RFP) are used to construct a high-efficiency and specific isobutyraldehyde biosensor, real-time monitoring of an isobutyraldehyde production process and screening of an isobutyraldehyde production strain can be carried out.

The invention provides a recombinant herpes simplex virus and a herpes simplex virus modification method. The herpes simplex virus modification method comprises the step of replacing an ICP4 protein coding gene in a herpes simplex virus genome containing an infected cell protein 4 (ICP4) gene with an hTERTp-fluorescent protein expression cassette. The expression cassette comprises an hTERTp promoter and a fluorescent protein coding sequence controlled by the hTERTp promoter, and the transcription direction is opposite to that of an ICP4 promoter in a genome. The hTERTp-fluorescent protein expression cassette in the recombinant virus obtained by the method disclosed by the invention can be normally expressed in response to telomerase activity. Therefore, the recombinant virus is capable of expressing a fluorescent protein, such as mBaoJin, in a cell having human telomerase activity. Cells infected by the virus can be identified through fluorescence signals, and the higher the telomerase activity is, the stronger the fluorescence intensity is. The virus has wide application value in research of tumor action mechanisms and stem cell action mechanisms, health assessment, screening of tumor drugs, research and development of diagnostic reagents and establishment of animal models.

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 provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

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 invention discloses a monoclonal cell strain screening method based on fluorescence intensity, which comprises the following steps: determining a cell line, and stably transfecting fluorescent protein to cells of the cell line; a microwell plate is selected, a datum hole is set, the cells stably transfected with the fluorescent protein are separated and put into the microwell plate, a plurality of cells are put into the datum hole, and only one cell is put into the other holes; screening out single-cell pores with only one cell from the rest of pores except the reference pore; screening out clone group holes capable of growing clone groups; respectively digesting cells in the cloning group pores and then transferring the cells into new pores for culturing; screening out fluorescent screening holes; splitting proliferation holes are screened out; and determining the cells in the division proliferation hole as the screened monoclonal cell strain. According to the monoclonal cell strain screening method provided by the invention, the monoclonal cell strain which is high in fluorescence intensity and can proliferate at a normal speed can be screened, compared with fluorescence microscope observation and imaging, the method is convenient and rapid, and errors possibly generated by comparing the fluorescence intensity with human eyes are reduced.

The present invention relates to a method for sanitising, disinfecting or sterilising microbially contaminated water-containing fluids, comprising treating the microbially contaminated water-containing fluid with an oxidising agent, wherein a fluorescence measurement is carried out on the water-containing fluid before, after and / or during treatment of the microbially contaminated water-containing fluid with the oxidising agent. The fluorescence measurement comprises irradiation with an excitation wavelength characteristic of a fluorescent protein and / or peptide building block and measurement of the corresponding fluorescence signal, and the method is characterised in that treatment with the oxidising agent proceeds in such a way that addition of the oxidising agent is controlled as a function of the fluorescence signal. The invention further relates to a corresponding device for sanitising, disinfecting or sterilising microbially contaminated water-containing fluid.

The invention relates to the field of medicine. In particular, the present invention relates to a protein-based biosensor comprising a substrate binding protein that binds to an MRI contrast agent by a chelating agent. More specifically, the present invention relates to a glutamic acid biosensor comprising a glutamic acid binding protein bound to an MRI contrast agent, characterized in that the glutamic acid binding protein comprises SEQ ID NO: 10, preferably SEQ ID NO: 5, and a sequence encoding a fluorescent protein.

The present application relates to a kind of high efficiency response vanillin biosensor, belong to the field of bioengineering technology.The present application utilizes aldehyde response transcription factor YqhC of escherichia coli ( Escherichia coli ) origin and the promoter element of the regulated P yqhD Establishment of RFP report system of vanillin response, to yqhC Gene semi-rational design is carried out, and mutant library is constructed by saturation mutation, with vanillin as substrate, by screening response concentration and response threshold, obtain no background fluorescence response, with wider substrate detection range (0-10 mM), and response intensity is higher (167 times) YqhC mutant, it is named YqhC-V18.Further, the biosensor containing vanillin response element (YqhC-V18) and fluorescent protein report element (RFP) is obtained, which can be applied to detect vanillin-containing sample, or screen vanillin production strain, or regulate vanillin production process.

The invention belongs to the technical field of GPCR drug research and development, and discloses a ratio type cAMP fluorescent probe RaCamp and a construction method of a transgenic mouse, the probe RaCamp is formed by series fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein through a flexible linker, and the preparation method of the probe RaCamp comprises the following steps: S1, obtaining an mCherry fragment; s2, a G-Flamp2 linear carrier is obtained; s3, homologous recombination; s4, converting and screening; the construction method of the transgenic mouse comprises the following steps: T1, designing a carrier; t2, microinjection of fertilized eggs; t3, embryo transplantation and reproduction; the invention has the beneficial effects that mCherry is added as an internal reference protein on the premise of keeping the sensitivity of the original G-Flamp2 probe, and the selected connecting peptide (GGGGS) 2 enables the expression quantity of the two proteins in cells to be accurately controlled to be 1: 1, so that the problem of background interference in cell observation of in-vivo imaging is well solved. And the sequence is inserted into a Rosa26-LSL box, so that the construction of a Rosa26-LSL-RaCamp mouse is realized.

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.

The invention discloses a method for bifluorescence quantitative detection of epithelial cells of a lacrimal gland organ and application. The method comprises the following steps: providing a mouse lacrimal gland organ; a recombinant adeno-associated virus combination is added to the organoid, the combination comprising: a first AAV vector comprising a promoter specifically driving the expression of alveolar epithelial cells and a first fluorescent protein coding sequence; the second AAV vector contains a promoter for specifically driving expression of myoepithelial cells and a second fluorescent protein coding sequence; according to the method, the two cells can be visually distinguished, and by calculating the fluorescence area or the number of the cells, the alveolar / muscular epithelial cell proportion is established as a damage index, so that high-throughput drug screening is realized.

The invention discloses a synchronous imaging method for a multicellular structure in a multicellular type. The synchronous imaging method comprises the following steps: S1, marking the cellular structure in the multicellular type by using fluorescent protein; s2, forming a spectrum-spatial feature joint coding set according to the spectrum and spatial features; s3, performing spectral unmixing on the spectral image to obtain an abundance image of each fluorescent component; s4, performing cell segmentation and cell structure positioning, and extracting spatial features; and S5, carrying out joint decoding on the spectral features and the spatial features to distinguish different cell types, and carrying out synchronous imaging on the multi-cell structure. The method has the beneficial effects that different cell types are marked by using corresponding fluorescent proteins, and a spectrum-space coupling coding strategy is formed in combination with spatial position characteristics, so that simultaneous observation and dynamic analysis of different cell types and a plurality of internal structures in the same imaging view field are realized; and the limitation that traditional spectral imaging can only work in a single cell type is broken through.

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.

A monomeric Scarlet Red Fluorescent protein (mScarlet RFP) includes at least amino acid substitutions V196I and / or G220A when compared to the amino acid sequence of synthetically designed mScarlet RFP. The V196I and / or G220A variants may include one or more further amino acid substitutions.

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 bioengineering, and discloses a plasma membrane localized green fluorescence overexpression vector as well as a construction method and application thereof. The cytoplasmic membrane localized green fluorescence overexpression vector contains an expression vector skeleton and an AtCBL1 gene connected to the expression vector skeleton, wherein the nucleotide sequence of the AtCBL1 gene is shown as SEQIDNO: 1, and the expression vector skeleton also carries a fluorescent protein gene. The construction method of the vector comprises the following steps: S1, carrying out enzyme digestion and recovery on an AtCBL1 gene sequence with a nucleotide sequence as shown in SEQIDNO: 1 obtained by amplification; s2, performing same enzyme digestion on the expression vector carrying the fluorescent protein gene, and recovering; and S3, connecting the AtCBL1 gene subjected to enzyme digestion with the expression vector to form a recombinant plasmid, converting a connection product into an escherichia coli competent cell, and extracting the recombinant plasmid from positive clone to obtain the plasma membrane localized green fluorescence overexpression vector.

The invention relates to the fields of molecular biology and virology, and provides a method for preparing a specific MHC haplotype-derived cell-binding virus. The method comprises the following steps: firstly, obtaining chick embryos of different haplotypes, separating CEF cells, transiently expressing exogenous fluorescent protein through electrotransfection, then carrying out virus inoculation, carrying out flow sorting to obtain a fluorescent positive cell population carrying MDV, and carrying out quantitative analysis on the cell population to obtain the specific MHC haplotype chicken Marek's Disease Virus (MDV), thereby obtaining the specific MHC haplotype chicken Marek's Disease Virus Marek's Disease Virus Marek's Disease Virus Marek's Disease Virus. And carrying out in-vitro propagation to obtain the purified MDV with a specific haplotype source. The purified virus can provide a test material for related research of haplotype on virus infection, and can be widely applied to in-vitro purification of different cell binding viruses and related research of specific MHC molecule presenting virus peptides. The method disclosed by the invention is beneficial to carrying out related research aiming at the host immune response of the virus and analyzing the hereditary basis of haplotype to MDV differential resistance, and is expected to be applied to preparation of vaccine virus.

The invention belongs to the technical field of transgenic and biomedical research tools, and particularly relates to a method for constructing a transgenic fish system based on a Tol2 transposon system, which comprises the following steps: constructing a myo6b-lhfpl5b-DsRed plasmid by using myo6b as a promoter, lhfpl5b as a target gene and DsRed as a red fluorescent protein tag through a microinjection method, and constructing a Tol2 transposon system-based transgenic fish system. Co-injecting the plasmid containing the Tol2 transposase mRNA and the Tol2 transposase mRNA into a fertilized egg of an AB zebra fish strain, and screening generation by generation to obtain a transgenic fish strain with stable inheritance MET channel gene lhfpl5b marked hair cells. The spatio-temporal dynamic visualization of the living whole fish to the lhfpl5b is realized, the deafness-causing molecular pathway is clarified, and an in-vivo platform is provided for drug screening and intervention strategies.

Provided are nucleic acids encoding kinase-modulated bioluminescent indicator (KiMBI) polypeptides. In certain embodiments, a nuclei acid of the present disclosure encodes a KiMBI polypeptide comprising a first bioluminescent enzyme fragment, a phospho-binding domain, a 5 second bioluminescent enzyme fragment capable of forming an active bioluminescent enzyme with the first fragment via enzyme fragment complementation, and a kinase substrate bound by the phospho-binding domain when phosphorylated. KiMBI polypeptide may be fused to one or more fluorescent proteins, e.g., which exhibit resonance energy transfer (RET). Also provided are KiMBI polypeptides encoded by the nucleic acids of the present disclosure. Cells that express 10 a KiMBI polypeptide are also provided, as are non-human animals comprising such cells. Also provided are methods of assessing activity of a kinase of interest in a non-huma animal, and methods of assessing a test agent for the ability to inhibit a kinase of interest in a non-human animal.