432 results about "Fluorescent protein" patented technology

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

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 belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

The invention discloses a watermelon transgenic screening expression cassette and application thereof, and belongs to the technical field of biology. According to the invention, an expression cassette of a TagRFP gene driven by a d35s promoter is constructed on a pCambia1300 vector, and a recombinant vector is obtained. A recombinant vector is introduced into watermelon cells by utilizing an agrobacterium-mediated genetic transformation technology, and a red fluorescent protein TagRFP gene is integrated into a watermelon genome, so that transgenic watermelons can be screened and identified by taking red fluorescence as an identifier in the whole growth period of watermelon genetic transformation. Compared with a traditional screening method, the screening method has the advantages that the screening process is greatly simplified, and the transgenic watermelon and the non-transgenic watermelon are clearly distinguished, so that the genetic transformation efficiency of the watermelon is remarkably improved.

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 provides a genetically engineered bacterium and a construction method of the genetically engineered bacterium, and the genetically engineered bacterium is characterized in that a green fluorescent protein sfGFP is used for mediating protein glutaminase, PG (protein glutaminase), PGF (protein glutaminase), PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF and PGF; eC 3.5. 1.44) is efficiently secreted and expressed in engineering bacteria, and belongs to the technical field of biological engineering. The method comprises the following steps: an obtained sfGFP gene is derived from an NCBI database (GenBank numbering: CP035486.1), an obtained PG zymogen gene PP (Propeptide-Protein glutaminase) is derived from a Chryseobacterium prion prgA gene (GenBank numbering: AB046594.1) in the NCBI database, a recombinant plasmid pHT01 / sfGFP-PP is introduced into bacillus subtilis WB800N to obtain an engineering bacterium, extracellular secretion expression of PP is realized, the sfGFP-PP is activated by trypsin to obtain PG, and the PG is subjected to enzyme activation to obtain the recombinant bacillus subtilis. The expression quantity and the secretion efficiency of the PG in the bacillus subtilis are greatly improved. The recombinant PG produced by the method can generate 23.5 U / mL enzymatic activity under activation of trypsin, can be used for improving protein functional characteristics, and has relatively high industrial production and application values.

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 provides a probe for cell senescence analysis and a preparation method and application thereof, and belongs to the technical field of cell detection. The invention discloses a probe for cell aging analysis. The probe comprises fusion protein formed by fibronectin RGD and fluorescent protein, the fibronectin RGD is a polypeptide fragment containing an amino acid sequence as shown in SEQ ID NO: 1. The probe is fixed on a solid-phase carrier and then co-incubated with cells to be detected, the condition of senescent cells can be analyzed and judged by detecting the intensity of a fluorescence signal, the purpose of rapid analysis is achieved, the analyzed cells can be recycled and then continuously cultured, the detection cost is low, and the probe is suitable for large-scale popularization and application in the market.

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

The invention discloses a method for high-throughput screening of key genes in a 1, 5-pentamethylene diamine biosynthetic pathway of escherichia coli, and the high-throughput screening is carried out by using a 1, 5-pentamethylene diamine biosensor. The 1, 5-pentamethylene diamine biosensor is a recombinant plasmid obtained by integrating a lysine decarboxylase coding gene cadA and a promoter trc thereof, and a green fluorescent protein (GFP) coding gene and a promoter pyrF thereof into a plasmid skeleton. A fluorescent quantitative flow cytometer is used for screening single-cell thalli with high yield of 1, 5-pentanediamine according to the fluorescence intensity of GFP, key genes can be rapidly screened out from a large number of genes to be screened in a high-throughput mode, and the screening workload and time are greatly saved. According to the application of the key gene in the biosynthetic pathway of the escherichia coli 1, 5-pentanediamine screened by the method disclosed by the invention, the yield of the 1, 5-pentanediamine produced by fermenting the escherichia coli KA30 can be greatly improved, the substrate conversion rate in the fermentation process is improved, the production cost of the 1, 5-pentanediamine is reduced, and the economic benefit is improved.

The present invention relates to a recombinant human elastin protein, and its preparation method and application. The recombinant human elastin protein comprises one or more elastin domains in the amino acid sequence of the full-length human elastin protein as shown in SEQ ID NO.1, as well as one or more of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. The amino acid sequence of the recombinant human elastin protein is shown in SEQ ID NO.2. The nucleotide sequence is a recombinant plasmid, a recombinant vector, or a recombinant bacterium. The preparation method comprises: synthesizing the nucleotide sequence as shown in SEQ ID NO.1, constructing a recombinant protein, inducing expression, centrifuging, purifying, and drying to obtain the recombinant human elastin protein as shown in SEQ ID NO.2. The application is to detect and evaluate the anti-aging, anti-wrinkle, and firming effects of the recombinant human elastin protein, as well as in cosmetics. The preparation method of the present invention is simple, the protein expression level is high, and the cosmetic product has anti-aging, anti-wrinkle, and firming effects.

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 present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

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 invention relates to a reporter responding to phenylalanine embedding and a construction method and application thereof. Specifically, the reporter comprises a first fluorescent protein and a second fluorescent protein, the first fluorescent protein and the second fluorescent protein emit fluorescence of different colors, the emission spectrum of the first fluorescent protein and the emission spectrum of the second fluorescent protein are not overlapped or the overlapping degree is very low, the first fluorescent protein is mBlueBerry2, and the second fluorescent protein is mBlueBerry2. The codon of phenylalanine at the 72nd site of the mBlueBerry2 is mutated into a termination codon TAG. The invention also provides an application of the reporter in screening the aminoacyl tRNA synthetase or an application of the reporter in preparing a preparation or a kit for screening the aminoacyl tRNA synthetase. The invention proves that the reporter can be used for screening the aminoacyl tRNA synthetase which is embedded into phenylalanine in a non-specific manner, and has wide applicability.

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.

A macromolecular interaction regulator screening method, comprising the steps of: providing an engineered cell, the cell containing a target condensate A having dynamic fluidity, the target condensate A containing a first fusion protein formed by a target molecule X, a phase separation scaffold protein Y and a first fluorescent protein F1 module, the cell further co-expressing a second fusion protein, and the second fusion protein being a fusion protein formed by a target molecule B and a second fluorescent protein F2 module; by means of interaction of the target molecule X and the target molecule B, forming a condensate complex A-B; in the presence or absence of a candidate compound, separately performing photobleaching treatment on the cell, and measuring fluorescence signals obtained before and after the photobleaching treatment of the condensate complex A-B in the cell; and performing comparison for the change of the fluorescence signals, and evaluating the impact of the candidate compound on the interaction of the target molecule X and the target molecule B.