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.

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 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 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.

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.