156 results about "EGFP protein" patented technology

The invention provides an application of an AC2 gene and the like in improving the expression quantity of GFP (Green Fluorescent Protein) and / or inhibiting degradation of an mRNA (Messenger Ribonucleic Acid) attenuated substrate taking the GFP as a reporter gene. Researches find that AC2 has an mRNA decay inhibition function, can inhibit degradation of a nonsense-mediated mRNA decay substrate taking GFP as a reporter gene and degradation of an endogenous nonsense-mediated mRNA decay substrate of arabidopsis thaliana, can improve the expression quantity of mRNA of AT1G0106020, RPS6, SMG7, AT5G35490, AT5G64430, AT1G36730 and the like, can reduce the expression quantity of AT1G72450 and AT2G400, has interaction with Dcp1 and Dcp2, and can be used for inhibiting the expression quantity of mRNA of AT1G0106020, RPS6, SMG7, AT5G35490, AT5G6430, AT1G36730 and the like. The growth ability of the yeast in an auxotroph culture medium can be improved, the expression level of Dcp1 can be reduced, and a research result lays a foundation for clarification of AC2 functions.

The invention relates to a gene editing vector of agrobacterium, a gene editing method of the gene editing vector and an agrobacterium editing strain. The agrobacterium gene editing vector comprises the following gene elements: a plasmid skeleton, a Pvan promoter, a spCas9 gene, a green fluorescent protein gene GFP, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. According to the agrobacterium gene editing vector, a CRISPR / spCas9-GFP gene editing system is adopted, an efficient, simple, convenient and traceless genome editing system is constructed in agrobacterium, and gene knockout and insertion can be achieved.

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 a PDCoV JS2021-LX strain NS6 gene deletion recombinant virus and a construction method thereof, on the basis of PDCoV JS2021-LX strain infectious clone successfully constructed by a BAC system, the upstream and downstream of the NS6 gene are cut by adopting a CRISPR / Cas9 technology, then the original NS6 gene segment is replaced by green fluorescent protein EGFP through a homologous recombination mode, an NS6 gene deletion recombinant plasmid is obtained, and the NS6 gene deletion recombinant virus is constructed. And after the LLC-PK1 cell is transfected, the recombinant virus without the NS6 protein is rescued. The recombinant virus with the PDCoV NS6 gene deleted is successfully rescued, the virus replication level of the recombinant virus and the virus is rescued through in-vivo and in-vitro evaluation, and pig experiment results show that the pathogenicity of the recombinant virus is reduced, and the recombinant virus has the potential of serving as a candidate strain of an attenuated live vaccine.

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 invention discloses a detection method of mycobacteria. The detection method comprises the following steps: S1, constructing a fusion protein gp23RD-GFP; s2, a target protein is obtained from the fusion protein gp23RD-GFP; s3, mixing the target protein with the mycobacterium culture, and incubating; s4, preparing a microscopic examination sample; s5, carrying out microscopic examination by using a fluorescence microscope; according to the method, a functional truncated body of phage TM4 receptor binding protein gp23 is fused with a green fluorescent protein to construct a fusion protein gp23RD-GFP, the fusion protein gp23RD-GFP is purified to obtain a target protein, the target protein and a mycobacterium culture are mixed and incubated, and finally the incubated mycobacterium culture is subjected to microscopic examination sample preparation. The detection mode is simple and rapid, and support can be provided for the joint development of the subsequent mycobacterium detection 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.

The present invention discloses a method for rapidly preparing and on-line characterizing an amino-silane coating in an aqueous phase system. Under negative pressure conditions, an amino-silane buffer solution is introduced into a fused silica capillary. The process of forming Si-O-Si bonds by the bonding of Si-OR in the amino-silane molecule and Si-OH on the surface of the microchannel is utilized to achieve the preparation and real-time characterization of the amino-silane coating: the formation of the amino-silane bonded coating is characterized in real time by the change of the surface electrical signal with time during the formation of the Si-O-Si bond. The stability of the amino-silane bonded coating in the aqueous phase system is characterized in real time by the change of the chemical bond caused by the hydrolysis and cleavage of the Si-O-Si bond in the pure buffer system; the content of amino groups on the capillary surface is indirectly characterized by the change of the fluorescence intensity of the green fluorescent protein bonded and fixed by the amino-silane coating.

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 recombinant humanized collagen type III alpha-1 (rhCol III a1), and an expression vector and use thereof are provided, relating to the technical field of bioengineering. A sequence on positions 154 to 1,221 of a human type III collagen α chain is ligated to an optimized vector pcDNA3.1, a resulting optimized expression vector is transfected into a human embryonic kidney cell Expi293F, and then rhCol III a1 is secreted to form a fusion protein with a soluble green fluorescent protein (GFP). The expressed rhCol III a1 that forms the fusion protein with the GFP shows an activity of promoting the migration of BALB / c 3T3 cells, thus exhibiting a great application potential in the field of skin care.

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 present invention discloses the construction and application of a recombinant Gluconobacter oxydans, belonging to the fields of genetic engineering and whole-cell catalysis. The present invention heterologously expresses an epoxide hydrolase from Sphingomonas in Gluconobacter oxydans, and simultaneously utilizes the alcohol and aldehyde dehydrogenases bound to the membrane of Gluconobacter oxydans itself to synthesize R-mandelic acid in a one-step process using styrene oxide as a substrate. The present invention combines the transcriptome data of Gluconobacter oxydans, screens 7 strong promoters using green fluorescent protein as a reporter gene, and determines the strongest promoter P for expressing the epoxide hydrolase gene. 12780 , ultimately significantly increasing the yield of R-mandelic acid.

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 provides a 293T-CymR-1-2-F6 cell strain as well as a construction method and application of the 293T-CymR-1-2-F6 cell strain. The functions of the 293T-CymR-2-1-F6 are verified through tests, it is proved that a monoclonal cell strain of the 293T-CymR-2-1-F6 can remarkably inhibit the expression efficiency of a CMV promoter after a CuO sequence is inserted, and compared with a cell transfected with normal HEK293T cells, the expression quantity of green fluorescent protein transfected with the 293T-CymR-1-2-F6 cells is reduced by 10 times.

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 invention discloses an SFTSV pseudovirus as well as a preparation method and application thereof. Based on the characteristics of high pathogenicity and transmissibility of the SFTSV, the virus infection animal experiment needs to be operated in a laboratory at the biosafety third level or above, so that the research and development of vaccines and therapeutic drugs are limited to a great extent. A novel SFTSV pseudovirus is obtained by deleting a nucleoprotein coding gene in an S gene of the SFTSV virus based on a reverse genetics technology, and the pseudovirus lacks a complete virus genome and replication ability and can be researched in a low-biosecurity-level laboratory; the pseudovirus system carries luciferase or a green fluorescent protein reporter gene, the virus load is quantified through the expression of the reporter gene, and the pseudovirus system can be used for evaluating the inhibition effect of a small-molecule inhibitor or a natural product on virus invasion; the pseudovirus can be used for simulating a natural infection process and evaluating a neutralizing antibody level induced by a vaccine. The invention provides a new development platform for discovery of anti-SFTSV drugs.

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.