44 results about "Gfp fusion" patented technology

An improved BRET assay, wherein the BRET signal is enhanced and / or prolonged. The improved BRET assay comprises the steps of i) adding a substrate to a cell comprising GPCR-Rluc fusion protein and a β-arrestin-GFP fusion protein, wherein the (β-arrestin is mutated, ii) adding a ligand to obtain, if possible, a GPCR-Rluc / (β-arrestin-GFP complex, and iii) measuring a BRET signal to obtain a BRET ratio, wherein the improvement leads to an increased BRET ratio compared with the ratios obtained by use of the same process employing a β-arrestin-GFP fusion protein wherein the β-arrestin is the wild type β-arrestin, or employing a 13-arrestin-GFP fusion protein, wherein the (β-arrestin is a β-arrestin specifically mutated so that it acts on the receptor independent of the receptors phosphorylation state. The invention further relates to a stable substrate solution for use in an improved BRET assay.

Green fluorescent protein (GFP) is widely used as a reporter in determining gene expression and protein localization. The present invention provides fusion proteins with a half life of ten hours or less with several embodiments having half lives of 4 hours or less. Such proteins may be constructed by fusing C-terminal amino acids of the degradation domain of mouse ornithine decarboxylase (MODC), which contains a PEST sequence, to the C-terminal end of an enhanced variant of GFP (EGFP). Fluorescence intensity of the fusion protein in transfected cells is similar to that of EGFP, but the fusion protein, unlike EGFP, is unstable in the presence of cycloheximide. Specific mutations in the MODC region have resulted in mutants with varying half lives, useful for a variety of purposes.

The invention discloses a method for in vitro detection of the transport of Magnaporthe grisea effector proteins in root tissues of rice. The transport of GFP fused proteins in the root tissues of rice is directly observed through a confocal microscope after disease-resistant rice cultivar Lijiangxintuanheigu root tips of 0.5cm are processed with 5.0mg / ml of prokaryotic expression products of Magnaporthe grisea effector proteins fusing GFP for 12-72h. The method is simple, accurate and rapid.

The invention relates to a nano-antibody to green fluorescent proteins (GFPs). The antibody has the amino acid sequence shown as SEQ ID NO.1. The nano-antibody is a single-domain antibody heavy-chainantibody (namely the nano-antibody) capable of specifically bound with the GFPs, can be used for detection and purification of the GFPs and GFP fusion proteins, for example used for preparing reagents, tools and the like for detection and purification of the GFPs.

The invention discloses a detection method and a detection device for mercury ions, and belongs to the technical field of environmental biology. The detection method for the mercury ions comprises the following steps of adding a mercury ion-containing solution in a Pmerr1-Omerr1-biotin double-stranded DNA system in combination with merR1-GFP fusion protein; standing; taking the solution out; measuring fluorescence intensity; and calculating mercury ion content in the solution by using a standard curve. The detection method for the mercury ions has the following advantages that (1) sample detection can be carried out at a sampling point without transporting the sample for a long distance to a specific detection mechanism or using large expensive equipment; time needed for detection is short, a process from sampling the sample to obtaining data at the end can be obtained in 30 minutes, the detection method is particularly suitable for detecting the mercury content in large-scale wild field oil samples, and manpower and material resources required by the detection can be greatly reduced; and (2) a limit value of the mercury ions detected by the method is 0.3 [mu]g / L, which is very close to that detected by a cold atomic absorption spectrometry, and accuracy is high.

The invention discloses chloroplast expressed fibroblast growth factor 21 protein and a preparation method thereof. The amino acid sequence is shown as SEQ ID NO. 2. The preparation method of the protein comprises the following steps: constructing a tobacco leaf chloroplast expression vector pWYP23406; converting tobacco leaf chloroplast of a fibroblast growth factor 21 gene and carrying out homogenization screening to obtain a homogenized plant of the tobacco leaf chloroplast converted fibroblast growth factor 21 gene; analyzing an expression amount of the fibroblast growth factor 21 in chloroplast transgenic tobacco leaf plants; purifying the fibroblast growth factor 21 to obtain the chloroplast expressed fibroblast growth factor 21. According to the chloroplast expressed fibroblast growth factor 21 protein and the preparation method thereof, GFP fusion protein of the fibroblast growth factor 21 is efficiently expressed in the tobacco leaf chloroplast and commercial production conditions are provided.

The present invention provides fusion proteins including a green fluorescent protein inserted into the internal amino acid sequence of a Gαs protein and further provides method of using the fusion protein construct to follow activation of a G-protein receptor by a candidate drug.

The invention discloses a chloroplast-expressed human epidermal growth factor (hEGF) protein and a preparation method thereof. The amino acid sequence of the protein is shown as SEQ ID NO.2. The invention further discloses a tobacco chloroplast expression vector pWYP23404 for producing the epidermal growth factor hEGF protein. The preparation method of the protein comprises the following steps: building the tobacco chloroplast expression vector pWYP23404, performing tobacco chloroplast transformation and homogenization screening on an EGF (Epidermal Growth Factor) gene to obtain a homogenization plant of a tobacco chloroplast-transformed hEGF gene, and analyzing the hEGF expression amount in the chloroplast-transformed gene tobacco plant; purifying the hEGF protein to obtain the chloroplast-expressed epidermal growth factor hEGF protein. By adopting the chloroplast-expressed hEGF protein and the preparation method thereof, a GFP (Green Fluorescent Protein)-fused protein of the hEGF isexpressed efficiently in a tobacco chloroplast, and commercial production conditions are realized.

The invention belongs to the technical field of biology, and particularly relates to a PK-15 cell line capable of stably expressing CD63-GFP and construction and application thereof, wherein the preservation number of the cell line is CCTCC NO: C2018253. The construction method comprises the following steps: target gene synthesis, target gene and lentivirus carrier double enzyme digestion, recombinant plasmid construction and identification, transfection of 293T cells, infection of PK-15 cells, and cell line screening verification. The PK-15 cell strain capable of stably expressing CD63-GFP can be used for evaluating a content of exudates inside and outside cells. The PK-15 cell line capable of stably expressing CD63-GFP fusion protein can be used for observing green fluorescent protein byfluorescence or conveniently evaluating the content of exudates by a flow cytometry. The establishment of the cell line provides a reliable material for researching the application of the exudates invirus infection of PK-15 cells.

The invention discloses a method for one-step transformation and marking of blue-green algae chromosomes and application. The method comprises the following steps: combining specificity of an inhibiting protein TetR in a tetracycline operon with a manipulated DNA sequence TetO; transferring a TetR-GFP fusion protein and a TetOxn sequence array onto blue-green algae genome chromosomes through one-step genetic transformation; then inducing expression of the TetR-GFP fusion protein by utilizing a promoter; combining the expressed TetR-GFP fusion protein with a TetOxn sequence on the chromosomes; and finally, obtaining a blue-green algae positive strain with the chromosomes emitting green fluorescence. The number and dynamic information of the chromosomes can be observed under a fluorescence microscope, so that the chromosome fluorescence-marked algae strain can be used as an ideal material for researching a copying and separating mechanism of the blue-green algae chromosomes.

The invention discloses a method for regulating seed size by using the ERECTA gene. By cloning the 2037bp promoter of the ATG upstream of the ERECTA gene and the 5526bp ERECTA gene, the Gateway method is used to construct a recombinant vector, and gERECTA can be efficiently and quickly cloned into a binary On the vector pGWB4; use Anti-GFP to detect the expression of gERECTA-GFP fusion protein by Western Blot to ensure the rapid and accurate detection of the expression product. Positive seedlings are screened to homozygous T3 generation to ensure the accuracy of the results and avoid the interference of false positives . The control effect of the invention is remarkable, the result is accurate and reliable, the control method is simple and convenient to operate, can be formulated, and is easy to popularize and apply.

The present invention belongs to biotechnology, and the intracellular analysis and detection process of peptidyl-propyl-cis / trans isomerase activity includes constructing fusion gene through fusion of protein or polypeptide, which contains X-Pro bond and realizes correct folding via cis / trans isomerization under PPIases catalysis, with green fluorescent protein (GFP); expressing fusion protein; fluorescent observation of the green fluorescence of GFP; and indicating and analyzing the activity of PPIases inside microbe, plant and animal cell. The said process may be used in PPIases activity detection inside live cell, and the GFP fluorescence may be observed directly with fluorescent microscope to determine intracellular PPIases activity simply, flexibly and intuitively. The cell introduced into fusion gene may be used directly as the type culture for relevant PPIases research and the type culture may be proliferated and propagated via microbe and cell culturing process for long termuse.

The invention discloses a Puralpha gene segment P4 and application thereof. A nucleotide sequence of the Puralpha gene segment P4 is shown as SEQIDNO:1, the Puralpha gene segment on a pCDNA3.0 vector undergoes eukaryotic expression, the plasmid is transfected to a required study cell line, and the effects on target genes of different structural domains of Puralpha proteins are explored through a protein immunoblot experiment. The Puralpha gene segment on a pEGFP-C1 vector and a green fluorescent protein (GFP) undergo fusion expression, the distribution situation of the Puralpha proteins in cells is observed under a fluorescence microscope, and the expression levels of the different structural domains of the Puralpha proteins in the cells are quantified through the expression level of the GFP protein. In-vitro study can be conducted on the effects on the target genes of the different structural domains of the Puralpha proteins through the eukaryotic expression conducted on the Puralpha protein gene segment on a pGEX vector.

The invention discloses chloroplast-expressed brain-derived neurotrophic factor hBDNFb protein and a preparation method thereof. The invention further discloses the chloroplast-expressed brain-derivedneurotrophic factor hBDNFb protein of which the amino acid sequence is shown as a SEQ ID NO.2. The preparation method of the protein comprises the following steps: constructing a tobacco chloroplastexpression vector pWYP23402; performing tobacco chloroplast transformation and homogenization screening on an hBDNFb gene to obtain a homologous plant of the tobacco chloroplast transformed hBDNFb gene; analyzing the hBDNFb expression quantity in a chloroplast transgenic tobacco plant; purifying the brain-derived neurotrophic factor hBDNFb protein to obtain the chloroplast-expressed brain-derivedneurotrophic factor hBDNFb protein. According to the invention, GFP fusion protein of the hBDNFb is highly expressed in a tobacco chloroplast, and a commercial production condition is available.

The invention discloses a method for observing the transferring of a fusion protein to a rice leafbud at long distance. The method comprises the following steps of: treating a root tip of rice by using a GFP (Green Fluorescent Protein) fusion protein; then, freezing a leafbud of a rice plant with a treated root tip, and slicing; next, dyeing by using a PI (Propidium Iodide) fluorescent dye; and directly observing the transferring condition of the GFP fusion protein from the root tip to the rice leafbud by using a confocal microscopy. By using the method, the position, 0.5cm away from the root tip, of the rice is treated by using the GFP fusion protein, the leafbud is frozen and sliced and then dyed, and the transferring condition of the GFP fusion protein from the root tip to the rice leafbud is observed by using the confocal microscopy, so that the long-distance transferring condition of the GFP fusion protein in the rice plant can be directly and qualitatively known about. The position, 0.5cm away from the root tip of the rice, is directly treated by using the GFP fusion protein, the transferring of the GFP fusion protein to the leafbud is observed by using the confocal microscopy, and thus, a very powerful evidence can be further provided for the fact that an effect protein can induce the defensive reaction of a rice system. The method is simple, convenient, accurate and rapid.

The invention discloses a Pura gene segment P2 and an application thereof. The nucleotide sequence of the gene segment P2 is shown as SEQ ID NO:1. According to the Pura gene segment P2 disclosed by the invention, the Pura gene segment on a pCDNA3.0 vector is subjected to eukaryotic expression, a plasmid is transfected into a required research cell line and Western immunoblotting is conducted so as to discuss the effects of different structural domains of Pura protein on a target gene. The Pura gene segment on the pEGFP-C1 vector can achieve fusion expression with green fluorescent protein GFP, the distribution of the Pura protein in cells is observed by virtue of a fluorescence microscope, and the expression levels of the Pura proteins in the various structural domains are quantified on the basis of the expression level of the GFP protein. By virtue of the Pura protein gene segment on a prokaryotic expression vector pGEX, the effects of the different structural domains of the Pura protein on the target gene can be researched in vitro.

The invention discloses a nano antibody aiming at green fluorescent protein (GFP). The amino acid sequence of the nano antibody is SEQ ID NO. 1. The invention discloses a nano-antibody gene sequence and a host cell, the nano-antibody is a single-domain antibody heavy-chain antibody capable of being specifically bound with GFP, can also be efficiently expressed in escherichia coli, and can be applied to purification and detection of GFP and GFP fusion protein.

The invention discloses a tobacco (Nicotianatabacumcv.SR-1) chloroplast iron transporter gene NtPIC1 and application of encoding protein of the tobacco (Nicotianatabacumcv.SR-1) chloroplast iron transporter gene NtPIC1 in chloroplast iron transport. A nucleotide sequence of the gene is shown in a sequence table SEQIDNO: 1; a corresponding amino acid sequence is shown in a sequence table SEQIDNO: 2; the gene comprises 849bp of open reading frames; 282 amino acids are encoded; the predicted molecular weight is 29.7kDa. The accession number of the nucleotide sequence of the open reading frames on GeneBank is KF640606. By virtue of positioning analysis of GFP fusion protein cells, NtPIC1 protein is positioned on a chloroplast membrane. Proven by a yeast function complementary experiment, expression of the gene in a yeast can be used for effectively transporting iron ions. According to the tobacco (Nicotianatabacumcv.SR-1) chloroplast iron transporter gene NtPIC1 disclosed by the invention, the gene NtPIC1 is introduced into tobaccos so as to obtain transgenic tobaccos with high iron content in the chloroplast; a research result shows that the NtPIC1 gene is involved in the chloroplast iron transport and the chloroplast development process.

The invention relates to a rice glumous fower growing new gene Dh1 and its cloning method. It belongs to rice function gene analytic technique field. Endonuclease Hind III is used to cut Japanese fine corresponding BAC. After electrophoresis object DNA segment is recycled. It is connected to the carrier cutting with the same enzyme to gain one gene Dh1, and used complementary assay to verify its function. The GFP fusion expression carrier is used to study its space-time pattern of manifestation. It lays a foundation for study rice new species in future.

The invention discloses a screening method for a filamentous fungus gene knockout mutant. The method comprises the following steps: (1) constructing an HPH-GFP fusion expression vector, and cloning anHPH-GFP fragment; (2) using a primer with a linker sequence to respectively clone the upstream fragment and the downstream fragment of a gene to be knocked out; (3) forming a chimeric fragment by using the cloned upstream and downstream fragments of the gene to be knocked out and the cloned HPH-GFP; (4) transforming a fungal protoplast by the chimeric fragment to obtain a transformant for later use; and (5) culturing the transformant in a medium containing hygromycin, observing normally grown hyphae by a fluorescence microscope, and selecting the transformant having GFP fluorescence to obtainthe knockout mutant. The method is used to solve the technical problems of low successful rate and high false positive rate of gene knockout in the prior art.

The invention discloses a detection method and a detection device for mercury ions, and belongs to the technical field of environmental biology. The detection method for the mercury ions comprises the following steps of adding a mercury ion-containing solution in a Pmerr1-Omerr1-biotin double-stranded DNA system in combination with merR1-GFP fusion protein; standing; taking the solution out; measuring fluorescence intensity; and calculating mercury ion content in the solution by using a standard curve. The detection method for the mercury ions has the following advantages that (1) sample detection can be carried out at a sampling point without transporting the sample for a long distance to a specific detection mechanism or using large expensive equipment; time needed for detection is short, a process from sampling the sample to obtaining data at the end can be obtained in 30 minutes, the detection method is particularly suitable for detecting the mercury content in large-scale wild field oil samples, and manpower and material resources required by the detection can be greatly reduced; and (2) a limit value of the mercury ions detected by the method is 0.3 [mu]g / L, which is very close to that detected by a cold atomic absorption spectrometry, and accuracy is high.

Green fluorescent protein (GFP) is widely used as a reporter in determining gene expression and protein localization. The present invention provides fusion proteins with a half life of ten hours or less with several embodiments having half lives of 4 hours or less. Such proteins may be constructed by fusing C-terminal amino acids of the degradation domain of mouse ornithine decarboxylase (MODC), which contains a PEST sequence, to the C-terminal end of an enhanced variant of GFP (EGFP). Fluorescence intensity of the fusion protein in transfected cells is similar to that of EGFP, but the fusion protein, unlike EGFP, is unstable in the presence of cycloheximide. Specific mutations in the MODC region have resulted in mutants with varying half lives, useful for a variety of purposes.

The invention relates to the evaluation method of the safety of fruits and vegetables, and belongs to the technical field of fruit and vegetable evaluation. The evaluation method provided by the invention comprises the following steps: (1) constructing a toxin gene and a GFP gene-containing fusion protein expression vector, and transferring the fusion protein expression vector into escherichia coli, thus obtaining escherichia coli for expressing toxin-GFP fusion protein; (2) culturing the escherichia coli, obtained in step (1), for expressing the toxin-GFP fusion protein, to obtain a bacteriasolution, gradually diluting the bacteria solution, inoculating the bacteria solution into the fruits and vegetables, and storing the fruits and vegetables at 4 to 37 DEG C for 24 to 72 h for safety evaluation. The evaluation method provided by the invention can realize evaluation of the safety of different fruits and vegetables to effectively avoid food poisoning.

A recombinant vector capable of expressing MDR1 shRNA and thymidine kinase, and a use thereof. More specifically, provided is a recombinant vector capable of efficiently expressing MDR1 shRNA and thymidine kinase in a host cell, a transfectant cell comprising the same recombinant vector, a composition for treating neoplastic diseases, comprising the same recombinant vector, and a method for imaging of neoplastic lesions using the same recombinant vector. The recombinant vector of the present invention is capable of achieving efficient intracellular expression of MDR1 shRNA and a thymidine kinase-GFP fusion protein within the host cell and is therefore highly effective for combined therapy of anticancer drugs. Further, the recombinant vector of the present invention enables imaging of neoplastic lesions. Therefore, the recombinant vector of the present invention can be used in combination with other anticancer drugs for treatment of neoplastic diseases.

This invention is intended to evaluate the oil-and-fat amount in a seed and genetic changes thereof. A fusion protein of an oil body-specific protein, such as the oleosin-GFP fusion protein, and a fluorescent protein is expressed, and the oil-and-fat amount in a plant seed and changes thereof are evaluated based on the fluorescent intensity in a cotyledon of a fluorescent protein, such as GFP.

Disclosed is a method for observing long-distance transport of fusion protein to the leaf bud of oryza sativa, the method comprising: using GFP fusion protein to treat the root tip of the oryza sativa, freezing and sectioning the leaf bud of an oryza sativa plant having the root tip treated, dying a leaf bud section by using PI fluorescent dye, and using a confocal microscope to directly observe the transport of the GFP fusion protein from the root tip to the leaf bud of the oryza sativa. The method uses the GFP fusion protein to treat the root tip of the oryza sativa for a length of 0.5 cm, freezes and sections the leaf bud, dyes a leaf bud section, and uses a confocal microscope to observe the transport of the GFP fusion protein from the root tip to the leaf bud of the oryza sativa, thus allowing learning directly and qualitatively about the long-distance transport condition of the GFP fusion protein within the oryza sativa plant, and providing convincing evidence for the ability of effector protein to induce the system defense response of the oryza sativa. In addition, the method is easy, accurate, quick, and of important usage value.

The present invention relates to genome editing by the mean of Cas nucleases. The inventors found that the expression of Cas nucleases may be finely controlled by the use of regulatory elements comprising a minimal promoter and at least one amino acid response element (AARE) nucleic acid, which are responsive to a diet deficient in at least one essential amino acid, or tunicamycin. For example, a FLAG-Cas9-GFP fusion and a Cas9-FLAG-RFP fusion could be expressed in 293 T cells. In addition, in the presence of a donor plasmid bearing a puromycin resistant gene, integration of the said puromycin resistant gene may be performed at the site of the safe harbour locus AASV1 on the genome of 293 T cells. Therefore, the invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease in an individual, comprising (i) a regulatory polynucleotide comprising a minimal promoter and from one to twenty AARE nucleic acids, and (ii) a nucleic acid encoding a Cas nuclease, which is placed under the control of the said regulatory polynucleotide.

The invention discloses a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein, including the cloned glyphosate main target EPSP synthase as the receptor protein of glyphosate; the receptor protein and the green Fluorescent protein was fused to construct EPSPS‑GFP, a new protein containing both glyphosate-binding sites and fluorescent labels. It was prokaryotically expressed and purified in Escherichia coli, and used for the detection of glyphosate in water. The invention realizes the qualitative and quantitative detection of the glyphosate herbicide by constructing the fusion protein and using a fluorescence photometer to detect the change of the fluorescence response value and establishing a standard calibration curve.

This invention is intended to evaluate the oil-and-fat amount in a seed and genetic changes thereof. A fusion protein of an oil body-specific protein, such as the oleosin-GFP fusion protein, and a fluorescent protein is expressed, and the oil-and-fat amount in a plant seed and changes thereof are evaluated based on the fluorescent intensity in a cotyledon of a fluorescent protein, such as GFP.