377 results about "Green fluorescent protein" patented technology

A sensor system detects organophosphorus pesticide residue by inducing the aggregation of gold nanoparticles. A method comprises aggregating gold nanoparticles by a reaction between an organophosphorus pesticide and imidazole or a green fluorescent protein (GFP), and detecting the organophosphorus pesticide based on a absorption spectral change resulting from the aggregation. The system for detecting pesticide residue is useful as a biosensor for analyzing pesticide residue in situ, because the optical change of the reagent by the presence of an organophosphorus pesticide is distinct, the detection speed is fast, and the range of detection limits is broad.

The invention relates to methods and compositions utilizing Renilla green fluorescent proteins (rGFP), and Ptilosarcus green fluorescent proteins (pGFP). In particular, the invention relates to the use of Renilla GFP or Ptilosarcus GFP proteins as reporters for cell assays, particularly intracellular assays, including methods of screening libraries using rGFP or pGFP.

The invention relates to a method for preparing a chitosan-silk fibroin composite nano-fiber multifunctional patch for promoting myocardial tissue regeneration and monitoring stem cells. The method comprises the following steps: preparing a cellulose nano-fiber basal plate by an electrostatic spinning technology; alternately assembling positively charged chitosan (CS) and negatively charged silk fibroin (SF) to the surface of nano-fibers layer by layer by adopting a layer-by-layer assembly technique, and assembling 5.5-10.5 layers to form a CS-SF composite nano-fiber membrane; and planting seed cells of adipose tissue-derived stromal cells or cardiac progenitor cells labeled by green fluorescent protein and firefly luciferase on the surface of the CS-SF composite nano-fiber membrane, preparing the patch by three-dimensional co-culture. The patch has excellent biocompatibility, can be used as a cell vector, has an effect of resisting oxidative stress to improve the survival rate and treatment efficiency of stem cells, and is capable of evaluating the number, distribution and function states of transplanted stem cells, effectively preventing occurrence of post-myocardial infarction heart failure and reducing the death rate of ischemic cardiomyopathy.

The invention discloses a preparation method of a recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity, and application, including (1) the preparation of the recombinant pseudorabies virus for expressing green fluorescin with high sensitivity; (2) the application to marking of a neural circuit. The recombinant pseudorabies virus for expressing the green fluorescin with high sensitivity is successfully prepared by using a platform. The recombinant pseudorabies virus for expressing reverse neural circuit tracing of green fluorescin with high sensitivity is successfully obtained. Wide application value is realized in aspects of neural circuit marking, medicine screening platform building, medicine inhibition virus action mechanism, viral vaccine and diagnostic reagent invention and development, animal model building, virus replication, pathogenic mechanism analysis and the like.

The invention discloses a tetravinyl-based Gemini type amphiphilic compound as well as a preparation method and application thereof. The compound disclosed by the invention is mainly prepared through McMurry coupling reaction, nucleophilic substitution reaction and Click reaction; the structure of the compound is also confirmed through infrared, nuclear magnetic and mass-spectrum ways; in an aqueous solution, molecules of a derivative of the compound disclosed by the invention self-assemble to form aggregation-induced fluorescence-enhanced micelles (AIE micelles); the compound acts with a nucleic acid, and then can co-aggregate to form nano particles easy for cellular uptake; green fluorescent protein (GFP) and luciferase (Luciferase) expression assays prove that the compound self and a liposome formed with dioleoyl phosphatidyl ethanolamine (DOPE) can be used as non-viral gene vectors; meanwhile, the derivative is successfully used for tracing the cellular uptake and release processes of pGL-3 and FAM-DNA by utilizing the reversible transformation between the self-assembly of the compound and the co-assembly with DNA (Deoxyribonucleic Acid).

The invention discloses a gateway cloning technology entry plasmid vector. The vector contains a photoinducible promoter (Prbcs) of a small subunit gene of 1,5-bisphosphate carboxylase (Rubisco) and a green fluorescent protein (GFP) reporter gene. The invention also discloses a construction method and application of the vector. Through the vector, the gateway cloning technology can be utilized to quickly construct a photoinducible plant expression vector of a target gene to realize the high level expression of the target gene in plant leaves, the expression of the target gene is adjusted by light intensity, and expressed target protein can be positioned into chloroplast or cytoplasm.

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineering to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

A delivery system employing nanoparticles of thiolated chitosan which complex with nucleic acids or other drugs. The system utilizes an approximately 33 kDa thiol-modified chitosan derivative resulting in highly effective gene delivery. Thiolation of chitosan resulted in reduced density of positive charges and DNA binding capacity. However, thiolated chitosan carrying a plasmid DNA expressing a green fluorescent protein (GFP) showed significantly higher GFP expression in various cell lines and in vivo in mice. Sustained delivery of plasmid DNA from thiolated chitosan was achieved by crosslinking thiolated chitosan / plasmid DNA nanocomplexes through inter- as well as intramolecular disulfide bonds under the physiological conditions. Thiolated chitosan nanoparticles have a great potential for gene therapy and tissue engineering.

The invention discloses a carbon nanometer tube decorated by polyethyleneimine and a compound of plasmid DNA thereof. The plasmid DNA adopted in the invention comprises P53DNA, green fluorescent protein expression (JPC) and unmethylated oligomerization deoxynucleotide plasmid DNA. The carbon nanometer tube decorated by the polyethyleneimine and the compound of the plasmid DNA have the advantages of high transfection efficiency and less toxicity to cells; in particular, when the concentration is 30 g / ml, the transfection efficiency is larger than 90 percent, and the validity of the cells is larger than 80 percent. The carbon nanometer tube decorated by the polyethyleneimine and the compound of the plasmid DNA have wide application prospect during the process of preparing medicaments for remedying genes outside transfecting osteosarcoma cell bodies and in animal bodies; and the carbon nanometer tube decorated by the polyethylene imine / the plasmid DNA / an adriacin nanometer compound also have wide application during the process of preparing medicaments for restraining the growth of tumor.

This invention relates to a method for increasing the conversion efficiency of pollen tube pathway method by using agrobacterium tumefaciens virulence gene. The method comprises: combining or mixing agrobacterium tumefaciens virulence gene and the target gene to be converted (green fluorescent protein gene) in vitro, packaging with liposome, and converting plants, especially cotton by using pollen tube pathway method. The method combines agrobacterium-liposome-pollen tube pathway technique, and has obviously increased protoplast conversion efficiency. Plasmids are not only protected by liposome when entering embryo sacs, but also encapsulated by the toxic protein to be expressed when entering egg cells, which can improve the stability of exogenous DAN in cell conversion process, realize precise cutting in T-DNA region, and increase the conversion efficiency.

The invention discloses a foot and mouth disease virus recombinant virus sample particle as well as a preparation method and application thereof. The recombinant virus sample particle is commonly assembled and expressed by components in a DNA molecule composition. The DNA molecule composition contains an O-type foot and mouth disease VP0 gene, an O-type foot and mouth disease VP1 gene and an O-type foot and mouth disease VP3 gene and further contains a green fluorescent protein gene. By virtue of the property that FMDV VLPs is self-assembled through VP0, VP1 and VP3, a construction method of a baculovirus recombinant vector is improved, a green fluorescent protein label is added into the vector, and FMDV VLPs is successfully prepared by virtue of a pFBDM Bac-to-Bac system, so that a theoretical foundation is laid for the further development of safe and efficient O-type FMDV genetic engineering vaccines.

The invention relates to a method for using ultrasonic wave with agricillin to transfer the cotton germ to cultivate gene-transferred cotton. The inventive method comprises that planting the disinfected and skin-removed cotton on the germ-free cultivate medium; removing two fresh blades to be arranged on the germ-free filter paper; adding 10ml of 0.5 OD600nm agricillin solution into the plastic eccentric tube or glass triangle bottle, then putting the germ into the agricillin solution, transferring the treated germ into the share cultivate medium to be cultivated; washes with germ-free water which contains cefradine, absorbs the surface water, to be transferred to the return cultivate medium, under sunlight to be returned and cultivated to obtain the germ; then transfers it to the selecting cultivate medium to obtain the antibody germ; cuts off the antibody germ to be transferred to the rooting cultivate medium to induce the root or transplanted to the cotton stock, to obtain the regenerated gene transferred plant. The invention can improve the instant expression rate of GFP (green fluorescence protein) gene at blast to 100 times, realizes the gene transferred plant of island cotton, and obtain anti-sect cotton. The test has proved that the external gene is stably transferred into the cotton gene to be expressed and generated.

The invention relates to a gene drug co-carrier system using cation liposome to establish a gold nano-spherical shell and a preparation method. The method comprises: using phosphatides such as DPPC, MPPC and DPPE-PEG and the derivatives thereof to form the cation liposome via membrane extrusion; carrying the hydrophilic drug hydrochloric acid adriacin into the liposome in the forming process; forming the drug-carried cation liposome; adding chlorauric acid solution into cation liposome solution; adsorbing on the surface of the cation liposome; adding ascorbic acid solution for reduction; forming the gold nano-spherical shell structure; reducing sulfhydryl out of the single chain of green fluorescent protein silent gene with a disulfide bond, grafting onto the surface of the gold nano-spherical shell; and forming the gene drug co-carrier system. The gold nano-spherical shell has low toxicity and unique optical properties. The gene drug co-carrier system using cation liposome to establish the gold nano-spherical shell can provide carrier for the co-treatment of drugs and genes, can select light to illuminate the gold nano-spherical shell, and utilize light-heat conversion property to realize the co-release of the drugs and genes.

The invention discloses a CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an amicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treatingacute myelogenous leukemia (AML) clinically is outstanding.

The invention relates to four green fluorescent protein (GFP) nano antibody encoding genes as well as a preparation method and application thereof. The invention establishes a GFP nano antibody library. By using a phage display technique, four nano antibodies which are specifically combined with the GFP are screened from the antibody library, which are respectively named as A12, E6, D5 and B9. Nucleotide sequences of the four nano antibodies are obtained through sequencing, the nucleotide sequences are as shown in SEQ ID NO:1. SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequences have corresponding amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A gene A12 is cloned to a transformed expression vector pADL-10b-His, and introduced intoan SS320 strain; genes E6, D5 and B9 are respectively cloned to transformed expression vectors pBAD 24-Flag-His, and are respectively introduced into a TOP10 strain, and then four prokaryotic expression vectors and strains of four nano antibodies are obtained. Four nano antibodies are expressed and purified, tests show that the four GFP nano antibodies can be specifically combined with GFP, and the genes can be applied to detection on GFP in basic research.

The invention discloses a recombinant vector for gene knock-in. The recombinant vector comprises a DNA (deoxyribonucleic acid) fragment structure IL17A-IRES-luc, wherein IL17A is an encoding gene of interleukin 17A, IRES is internal ribosome entry site, and luc is an encoding gene of secreting luciferase. The invention also provides a method for preparing the gene knock-in recombinant vector and a mouse model prepared by using the recombinant vector, wherein the mouse model can be used for detecting the expression of the IL17A gene by detecting the expression of the secreting luciferase in blood or urine; a fluorescent microscope or other complicated equipment is not needed; the secreting luciferase emits light by virtue of an oxidizing reaction of substrate coelenterazine in the catalysis of luciferase, and laser light is not needed, so that the fluorescent background is far lower than GFP (green fluorescent protein) fluorescence, the detection sensitivity is higher, the background is cleaner, and the experiment results are more accurate.

A mammalian Green Fluorescent Protein (GFP) Reconstruction Across Synaptic Partners (mGRASP) method and system is provided by optimizing synaptic transmelectron microscopicbrane carriers for mammalian synapses. The method and system in one form integrates molecular and cellular approaches with a novel computational strategy to reliably reconstruct neurons in 3D. The method and system allows mGRASP to be applied to both long-range and local microcircuits, analysis of synaptic distribution at the level of single neurons and dendritic compartments within neural cells.

Described herein are devices and methods for extracting cellular material from living cells and then depositing them into to a receptacle in a nanoliter scale. Using a nanopipette integrated into a scanning ion conductance microscope (SICM), extraction of mitochondrial DNA from human BJ fibroblasts and Green Fluorescent Protein (GFP) transcripts from HeLa / GFP cells was achieved with minimal disruption to the cellular milieu and without chemical treatment prior to obtaining the isolated sample. Success of the extraction was confirmed by fluorescence microscopy and PCR analysis of the extracted material. The method and apparatus may be applied to many different cell types and intracellular targets, allowing not only single cell analysis, but single subcellular compartment analysis of materials extracted in their native state.

The invention relates to a bicistronic mRNA coexpression gene transporter and a preparation method thereof. The gene order of the gene transporter is SEQ ID No. 15; the gene transporter from 5' end to 3' end comprises bovine Nuclear matrix binding region MAR, artificially constructed combined promoter CAG, Profilin gene FSTN, internal ribosome entry site (IRES), green fluorescent protein AcGFP gene and Rabbit globin poly A signal region; the preparation method comprises the following steps: constructing a carrier pCAG-IRES2-AcGFP1; obtaining FSTN gene and inserting the pCAG-IRES2-AcGFP1 carrier; obtaining the sequence of MAR and inserting the pCAG-IRES2-AcGFP1 carrier; constructing a plasmid carriers so as to obtain the bicistronic mRNA coexpression gene transporter. The gene transporter is not only pure and safe gene transporter but also realizes dual-gene coexpression in any combination, achieves the purpose of multi-gene coexpression through repeated utilization of IRES, and provides new thinking and route for improving the shape of dual or multiple gene control such as economic character.

The invention relates to the use of scaffold proteins, particularly green fluorescent protein (GFP), in fusion constructs with random and defined peptides and peptide libraries, to increase the cellular expression levels, decrease the cellular catabolism, increase the conformational stability relative to linear peptides, and to increase the steady state concentrations of the library peptides and peptide library members expressed in cells for the purpose of detecting the presence of the peptides and screening peptide libraries. N-terminal, C-terminal, dual N- and C-terminal and one or more internal fusions are all contemplated. Novel fusions utilizing self-binding peptides to create a conformationally stabilized fusion domain are also contemplated.

The invention relates to a method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers. The method comprises the following steps: first, cloning green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT) on a marker gene carrier, placing a target gene in other T-DNA carrier, mixing strains of agrobacterium tumefaciens carrying the marker gene carrier and strains of agrobacterium tumefaciens carrying the target gene carrier and converting callus of the rice, performing PCR (polymerase chain reaction) detection on the disease-resistance genes of the target gene carrier by a specific primer, and screening to obtain co-transformation plants (T0); then, screening the marker gene plants with positive GFP rapidly and massively by means of a desk lamp fluorescence detector in segregative generations (T1 or T2) of the co-transformation plant for removing, and performing PCR detection for disease-resistance genes to the plants with negative GFP so as to obtain the individuals without selective markers of the transgenic disease-resistance genes. The method can be applied to transgenic breeding without selective markers of rice blast-resistant genes or other functional genes of the rice, and enhances the disease resistance of the rice or improves other agronomic traits.

The invention discloses a method for efficiently separating and instantaneously converting a protoplast of artemisia annua L., comprising the following steps: 1) selecting leaves of young and tender artemisia annua and removing lower epidermis, to obtain the leaves of the artemisia annua L. with the lower epidermis removed; 2) placing the leaves of the artemisia annua L. with the lower epidermis removed in an enzymatic hydrolysate for enzymatic hydrolysis to obtain a mixed solution of enzymatic hydrolysis; 3) filtering and centrifuging the mixed solution of enzymatic hydrolysis to obtain a protoplast precipitate, and resuspending to obtain a protoplast; and 4) instantaneously converting the protoplast. The method for separating and converting the protoplast is constructed in the artemisia annua L. for the first time, to obtain the protoplast with yield of 2.749 is multiplied by 10<5> / g FW, viability of 96%, and conversion efficiency of green fluorescent protein of 80%, and the obtained protoplast has large yield and high viability. The protoplast of the artemisia annua L. is utilized as a receptor for conversion, and green fluorescent protein GFP, luciferase LUC and sea cucumberluciferase REN can be successfully expressed.

This invention relates to a universal transfer vector of poxvirus avium, recombinant poxvirus avium and its preparation. Duplicate nonessential region fragment of Poxvirus avium is used to amplificate and clone for pPCI carrier with PCR. Gene cassette of green fluorescent protein(GFP) is constructed with early or late composed promotor of avian molluscum roup virus, and loxP sequence is transferred into its two sides, and then inserting into pPCI together to obtain universal transfer plasmid vector pHBP, which together with Poxvirus avium genome DNA cotransfecting CEF to obtain recombinant virus rFPVGFP.

The biomedicine PTD4-GFP-Apoptin fusion protein for treating tumor has its synthesized PTD4 utilized to introduce Apoptin protein into cell, and its Apoptin further utilized to induce tumor apoptosis, so as to inhibit tumor growth. At the same time, green fluorescent protein (GFP) is utilized for observing the intracellular distribution of the fusion protein and showing the aggregation of the medicine in the target position, realizing the effective monitoring and timely regulation of the treating process. Experiment shows that the fusion protein can penetrate biomembrane to kill tumor cell effectively while generating no damage to health cell. The present invention is applied in treating tumor.

The invention relates to a method for detecting activity of a chloroplast promoter on the basis of a tetracycline regulation system, belonging to the technical field of plant plastogene engineering research. The method comprises the following steps: on the basis of the tetracycline gene and tetracycline specific recognition sequence core control region, biosynthesizing a prrnO1O2 promoter with the tetracycline core control region, verifying the activity of the promoter in prokaryotic cells, and carrying out GFP (green fluorescent protein) gene expression under the driving action of the promoter; establishing a tetracycline-induced expression vector, and screening out the tetracycline suitable for bacterium growth, of which the maximum concentration is 5 mu g / ml; and finally, establishing the GFP expression vector under the tetracycline regulation system, wherein the function of the prrnO1O2 promoter is inhibited when the tetracycline is not added, and the GFP gene expresses green fluorescent proteins after the tetracycline is added. The tetracycline regulation system is utilized to control the activity of the chloroplast promoter, thereby avoiding the regulation interference of the nuclear genome on the plastome, and providing an effective method and way for further plastogene engineering breeding.

The invention relates to a lead ion sensitive whole-cell biosensor with a gene cascade amplification expression system and an establishment method. The establishment method comprises the following steps: connecting an artificial full-synthetic bidirectional promoter Ppbr and a lead ion specific binding protein PbrR such as a nucleotide sequence of SEQ ID No. 1 and a nucleotide sequence of SEQ ID No. 4 of a self-feedback regulation and control protein LuxR of plasmid pGN68 in a digestion mode, and screening positive cloning so as to obtain a target recombinant vector pET28a-pbr-lux; sequentially connecting a self-feedback promoter PluxR1, a green fluorescent protein nucleotide sequence and a self-feedback regulation and control protein LuxR nucleotide sequence of the plasmid pGN68 in the digestion mode; performing co-transformation on the recombinant vector and the plasmid pGN68 by using escherichia coli E.coli DH5alpha as a host, thereby obtaining the lead ion sensitive whole-cell biosensor.

The invention discloses application of a bifluorescence reporting system in tumor stem cell targeted drug screening and a method thereof. Red and green fluorescence proteins are utilized to respectively mark colony tumor cells and tumor stem cells, and the targeting properties of the drug for the tumor cells and tumor stem cells can be respectively evaluated by using a high-content imaging system. No targeted drug screening methods for tumor stem cells exist at present. The tumor stem cell screening method established by the invention is hopeful to be used for screening novel antineoplastic drugs for tumor stem cells, thereby enhancing the screening effect of the antineoplastic drugs.

The invention discloses a method for reducing toxicity of a Vip3 protein to a transgenic plant through gene fusion. The method comprises the following steps: firstly, a C-end of a polypeptide gene is directly connected with an N-end of the gene vip3; and a connecting peptide consisting of four amino acids is added between the C-end and the N-end so as to connect the C-end and the N-end; and secondly, the fusion protein of the fusion gene in plant cell expression is not concentrated on a cell membrane of the plant. The Vip3 protein is any one of Vip3 protein types of a Bt vegetative period insecticide protein, a amino acid sequence in SEQ ID NO:1 and an amino acid sequence which is 70% similar to the SEQ ID NO:1; the polypeptide is a GFP (Green Fluorescent Protein); and the connecting peptide is Gly-Asp-Pro-Thr. The obtained fusion gene is transplanted into plants so as to obtain transgenic zoophobous.

The invention discloses an agrobacterium tumefaciens-mediated barley stem apex transformation method. The transformation method comprises the following steps: A) culturing barley stem apex and constructing a carrier, wherein the specific steps are as follows: 1) selecting barley caryopses, removing hulls, stripping mature embryos and downwards placing scutella on a germination culture medium for germination; and 2) obtaining a maize ubiquitin promoter and the related sequence from an intermediate carrier pMBL-3 and a green fluorescent protein gene (GFP) through enzyme digestion and a PCR (polymerase chain reaction) method; and B) mediating the genetic transformation of the barley stem apex by utilizing agrobacterium tumefaciens, wherein the specific steps are as follows: 1) obtaining the barley stem apex; 2) dip-dyeing the stem apex; 3) using the stem apex and the agrobacterium tumefaciens GV3101 to co-culture the culture medium: taking out the stem apex which is dip-dyed with the agrobacterium tumefaciens and sucking up surface bacterial liquid with sterile filter paper; 4) performing restoring culture on the stem apex by using a restoring culture medium; 5) screening the stem apex on a screening the culture medium; and 6) vernalizing and cultivating a regenerated plant so as to obtain a candidate transformed plant. The method is easy to operate and simple to operate and has the advantages of short cycle, high efficiency and simple procedure, and the result is reliable.

The invention discloses a mercaptyl-terminated (2-dimethylamino ethyl) polymethacrylic ester polymer, a self-assembled and modified nano-gold compound (PDMAEMAGNPs) of the polymer as well as a compound of the polymer and plasmid DNA; and the invention also discloses preparation methods of the polymer and the compounds as well as the application of all in carrier as genes. When the concentration ofthe compounds of PDMAEMAGNPs and GFP DNA (green fluorescent protein expression DNA) is 30Mug / mL, the transfection efficiency to HEK 293T cell is 30 percent, and the survival rate to HEK 293T cell, HeLa cell and 293 cell is more than 80 percent. The PDMAEMAGNPs / CpG ODN (human CpG oligodeoxynucleotide) compound has remarkable effects in gene therapy in vitro and in vivo. The suppression ratio to tumor growth of the PDMAEMAGNPs / p53 DNA (tumor suppressor gene) is 30-37 percent.