477 results about "EGFP 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 the technical field of gene transfer materials, particularly a preparation method of nano sulfonated graphene and application of nano sulfonated graphene as a gene transfer material. The preparation method of the nano sulfonated graphene comprises the following steps: 1. preparing graphene oxide; 2. preparing reduced graphene oxide; 3. preparing diazonium salt of sulfanilic acid; and 4. preparing the nano sulfonated graphene. The nano sulfonated graphene can be used as a gene transfer material since positive charges of ions on particle surfaces can be combined with green fluorescent proteins in a non-covalent mode to perform transfection; and when being used as a gene transfer material, the nano sulfonated graphene has the advantages of high transfection effect, favorable cell compatibility and high cell survival rate. The transfection efficiency of the nano sulfonated graphene in human breast cancer cells is up to 40% or so.

The invention relates to the field of biomedical research, in particular to a gastric cancer cell line for expressing green fluorescent protein and luciferase, which is established by introducing the green fluorescent protein and the luciferase into a cell, and an application thereof in a cancer model. The gastric cancer cell line is characterized in that the gastric cancer cell line for stably expressing green fluorescent protein and luciferase is obtained by using a human gastric cancer cell line as mother cell and bringing the green fluorescent protein (GFP) and firefly luciferase (Fireflyluciferase, LUC) genes into the cell by using a slow virus infection method; and different gastric cancer models are established for the cell line by using hypodermic injection, tail vein injection and in situ injection methods. The invention provides establishment and application of the human gastric cancer cell line; and the gastric cancer cell line for expressing green fluorescent protein and luciferase is established by using the slow virus infection method, applied to the cancer model and used for gastric cancer preclinical studies.

We have developed a high through-put screen capable of isolating inhibitors of polypeptide aggregation, such as Alzheimer's Disease polypeptide Aβ aggregation, or other disease state aggregating proteins, from amidst large libraries of candidate inhibitors. The screen uses a fusion of a polypeptide domain that self-aggregates, such as an Aβ42 domain characteristic of Alzheimer's disease plaques, to a reporter construct, such as Green Fluorescent Protein (GFP) or similar fluorescent protein. In the absence of inhibition, the rapid misfolding and aggregation of Aβ42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Aβ42 aggregation enable GFP to fold into its native structure, and can be identified by the resulting fluorescent signal.

The invention relates to a method for screening non-essential regions for replication of a goat pox virus and universal transfer vectors for same. The method comprises the steps of amplifying two-end gene segments of any two regions of a goat pox virus gene by using a PCR (Polymerase Chain Reaction) method; then, inserting an enhanced green fluorescent protein (EGFP) gene and a xanthine-guanine phosphoribosyl transferase (gpt) gene expression cassette into the segments; establishing two universal transfer vectors of the goat pox virus; and acquiring a recombinant virus expressing an exogenous gene stably from the transfer vectors, thereby determining the selected regions to be non-essential regions for replication of the goat pox virus, wherein each universal transfer vector contains one unique restriction enzyme cutting site Sal I and allows gene expression cassettes of other items to insert in. The recombinant virus obtained by means of the two universal transfer vectors provided by the invention not only has a growth performance similar to a parent virus, but also has better safety because a plurality of toxicity related genes in a genome are knocked out in an orientation way, and has the potential to be developed into an attenuated vaccine strain for gene 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.

Disclosed are a polyacrylamide-based method of fabricating surface-bound peptide and protein arrays, the arrays themselves, and a method of using the arrays to detect biomolecules and to measure their concentration, binding affinity, and kinetics. Peptides, proteins, fusion proteins, protein complexes, nucleic acids, and the like, are labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through a copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than 7-fold greater than the nonspecific attachment of non-acrylic labeled GST-GFP. Surface-attached GST-GFP (0.32 ng / mm2) was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng / mm2 using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. The surface attachment strategy is applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.

The invention relates to an alfalfa stress response gene MsNAC3 and an application thereof. A novel NAC (N-Acetyl Cysteine) family related gene is cloned and authenticated from an alfalfa genome by using an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology and is named as MsNAC3 with a nucleotide sequence as shown in SEQ ID No.11, and a fusion expression vector containing an enhanced green fluorescent protein (EGFP) gene is constructed. The expression pattern of the gene MsNAC3 in alfalfa and the relation between the gene and adversity stress (cold damage, salt damage and drought) are analyzed by using a fluorescent quantitative PCR technology, in such a way, people find that the cold resistance, drought resistance and salt resistance of a transgenic tobacco plant can be improved through the overexpression of the gene in tobacco; the gene can be used for genetically transforming other monocotyledons and dicotyledons and improving the stress resistance of the monocotyledons and the dicotyledons.

The invention discloses a genetic transformation method for PEG-medicated fusarium oxysporum sesame special type protoplast, and belongs to the technical field of biology. The PEG-medicated protoplast transformation method is utilized, and through the preparation and transformation of fusarium oxysporum sesame special type protoplast and the screening of transformant, foreign DNA sections containing screening labels are conversed into the fusarium oxysporum sesame special type genome. The transformation of PEG-medicated fusarium oxysporum sesame special type protoplast is achieved, a whole genetic transformation and screening method is established, a great amount of transformated bacterium strains is obtained, wherein the bacterium strains can intensely express hygromycin resistant protein and green fluorescent protein, and the character can be stably inherited; and the transformation efficiency can reach 7667 transformants per mg of DNA. Moreover, the carrier used in the transformation comprises enhanced green fluorescent protein genes and strong promoter gpd in fungus, and thus the strength of fluorescent given off by the transformated bacterium strains is effectively strengthened.

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 belongs to the field of micro-organism animal cell line and relates to a human hepatoma cell line which can emit high-intensity red or green fluorescence and has high transferring ability of lung and lymph node metastasis, and a method for establishing the same. The method comprises the following steps: using the human hepatoma cell line HCCLM3 and HCCLM6 which have high transferring ability of the lung and lymph node metastasis as mother cells, performing cotransfection on plasmid DNA of 239 cells through slow virus packaging plasmids to obtain false slow virus particles by expressing red or green fluorescent protein genes through eucaryon, and infecting liver cancer cell strains of the mother cells to obtain the chromosome integrated hepatoma cell line which has high transferring ability of the lung and lymph node metastasis and can stably expressing the red or the green fluorescence. The human hepatoma cell line which has high transferring ability of the lung and lymph node metastasis in vitro can be applied to the tracer studies on tumor cells, the molecular mechanism studies on the recurrence and transferring of liver cancer, as well as the pre-clinical drug efficacy studies on new anti-tumor drugs, thus the human hepatoma cell line has wide application prospect.

The invention discloses a DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of the DNA and a pichia pastoris expression vector. The DNA has a base sequence as shown in SEQ No.1 (Sequence Number); and the application of the DNA relates to the application of the DNA in construction of the pichia pastoris (Pinchia Pastoris) expression vector. The DNA disclosed by the invention has the constitutive promoter activity, and can activate the transcription of a downstream structural gene without an inductor; the transcriptional activity shows little change in four different culture mediums, namely, ethanol, methanol, glucose and glycerol; the promoter activity is efficient, and the efficiency of the initiation transcription is four times more than the pichia pastoris GAPDH (Reduced Glyceraldehyde-phosphate Dehydrogenase) promoter. The pichia pastoris expression vector, constructed by the DNA disclosed by the invention, can efficiently express the extrinsic protein without methanol induction, and the efficiency of expressing the extrinsic protein (Enhanced Green Fluorescent Protein) is about 6 to 8 times that of the expression system of the GAPDH promoter and 1.5 to 2 times that of the expression system of a TEF1 (Transcription Enhancer Factor 1) promoter.

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 relates to a double-signal transgenic cell sensor for screening a chemopreventive agent and an establishment method thereof. The double-signal transgenic cell sensor is provide with an antioxidant response element (ARE), a TK promoter, enhanced green fluorescent protein (EGFP) and red fluorescent protein (DsRed), wherein expression of the EGFP is regulated and controlled by the ARE. The establishment method comprises the following steps: firstly establishing a eukaryotic reporter vector which is started by the TK promoter and regulated and controlled by four ARE repetitive sequences at upstream; inserting the DsRed and promoter fragments thereof into the vector to establish a double-signal reporter vector; and transfecting the reporter carrier with an HepG2 cell to obtain a transgenic cell, namely, the double-signal transgenic cell sensor of the invention. The established double-signal transgenic cell sensor of the invention which takes the EGFP as a reporter gene and the DsRed as internal reference requires no any substrate and auxiliary reagent, is safe and has convenient operation, short time, low expense and no environmental pollution.

The invention discloses a protein and amorphous metal organic framework compound and a preparation method thereof. The organic framework compound has a 2 nm-50 nm mesoporous structure. The preparationmethod comprises the following step of enabling a protein, zinc ions and an organic ligand to react in a solvent, wherein the organic ligand is a compound containing an imidazole group; the protein is one or a combination of several kinds of cytochrome C, cytochrome P450, horse radish peroxidase, alcohol dehydrogenase, lipase, acetylcholin esterase, laccase, a green fluorescent protein, glucose dehydrogenase, glucose oxidase, trypsin, bacillus subtilis protease, carbonic anhydrase, aldehyde ketone reductase, amylase, saccharase, superoxide dismutase, urease and catalase. The preparation method of the protein and amorphous metal organic framework compound provided by the invention is simple to operate and mild in condition, the obtained product is high in protein embedding rate and good inprotein stability, and the biologic al activity of the protein is reserved to a greater extent.

The present invention provides an isolated, purified and characterized human tyrosine hydroxylase (hTH) promoter nucleic acid sequence. The invention further provides a method of selecting TH positive (TH+) cells by preparing a construct comprising a hTH promoter operably linked to a heterologous nucleic acid sequence, for example, green fluorescent protein encoding sequence, and transfecting cells, particularly stem cells, with the construct. The invention also provides a hTH promoter, useful in gene therapeutic applications in driving therapeutic genes or other nucleic acid sequences operably linked to the hTH promoter. Additionally, the invention provides cell lines and transgenic animals expressing a transgene comprising the hTH promoter operably linked to a heterologous sequence, which cell lines and transgenic animals are useful for isolating TH+ cells for transplantation or for screening of therapeutic agents that affect TH+ function. Methods of producing cell lines and transgenic animals also provided.

The invention discloses an N-acetylneuraminic acid specific biosensor and application thereof and belongs to the field of genetic engineering. The N-acetylneuraminic acid specific biosensor is constructed with transcriptional regulation factor NanR and GFP (green fluorescent protein) gene, from Escherichia coli K12, acting as reporter genes; an effective relationship between N-acetylneuraminic acid and fluorescence intensity is established. The N-acetylneuraminic acid specific biosensor has an important application prospect in screening microorganisms which produce target metabolites.

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 preparation method and an application for a dual-fluorescence reporting system for detecting a micro-RNA (ribonucleic acid) function, wherein the preparation method comprises the following preparation steps of: A, obtaining a mCherry gene sequence via a PCR (polymerase chain reaction) amplification, inserting a rho EGFP-C1 carrier to replace the EGFP (enhanced green fluorescent protein) sequence of the mCherry gene sequence, and constructing a carrier rho mCherry-C1; B, using a RNAi-Ready rho SIREN-RetroQ plasmid as a template, obtaining a human U6 promoter gene via a PCR amplification, and inserting the carrier rho mCherry-C1 to obtain a plasmid rho hU6-mCherry-C1; and C, obtaining a gene sequence carried with a CMV (cytomegalovirus) promoter and an EGFP expression dialog sequence in an interval of 358 to 1944 on a plasmid rho Adtrack-CMV, and inserting the sequence in the plasmid rho hU6-mCherry-C1 to obtain a plasmid rho MGhU6. The plasmid rho MGhU6 is a dual-fluorescence reporting system simultaneously containing a mCherry reporting gene, an internal reference EGFP, a micro-RNA and the target sequence insertion site thereof. The reporting system can be used for detecting the inhibition function of the micro-RNA to the target sequence expression. The detection method is easy, as well as simple and convenient in operation; and a function detection for the micro-RNA can be realized by transfecting one plasmid only. The system also can be used for visually detecting the inhibition function of a micro-RNA in a single living cell to the target thereof by the aid of fluorescence microscopic imaging.

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 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.

The invention discloses a preparation method of a biosensor for detecting L-cysteine and an application of the biosensor in detecting yield of escherichia coli L-cysteine. Main components of the biosensor comprise a transcriptional control gene CcdR coding gene from pantoea ananatis, a promoter region and a terminator region of the transcriptional control gene CcdR coding gene, a promoter region of a gene ccdA from pantoea ananatis, a green fluorescent protein coding gene eGFP and a plasmid framework pTrc-Mob. The biosensor can have a good linear relation under the L-cysteine concentration of 0-32 mmol L<-1> and has the highest fluorescence intensity value and cysteine concentration relation under the L-cysteine concentration of 0.01-8 mmol L<-1>. Through combination with a high-throughput screening system, the biosensor performs real-time detection on the yield of escherichia coli L-cysteine, can screen out high-yield mutants of L-cysteine and mutants for key enzymes in biological synthesis of L-cysteine. Specific, quantitative and real-time detection of L-cysteine can be realized, and the sensitivity is high.

This disclosure provides a method of isolating peptides having cell-penetrating function, wherein the peptides are detected as biotinylated molecules only following their translocation through the cell membrane. The disclosure also provides methods for validating the cell-penetrating function of the peptides, or that may be employed in their own right to isolate such peptides, wherein the peptides are detectable by virtue of their ability to transport a detectable cargo into the cytoplasm, such as a cargo toxin or a fragment of a green fluorescent protein (GFP) that is required for complementation of a functional GFP. The disclosure also provides non-canonical peptides having cell-penetrating function that differ structurally from known CPPs such as TAT, VP22, transportan and penetratin, and that are capable of translocating cell membranes and escaping the endosome. The disclosed peptides have utility in transporting cargo therapeutics and diagnostics into cells.

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 invention relates to a rabies virus Flury-LEP vaccine strain reverse genetic operating system comprising a recombination vector for coding LEP full length genome cDNA and an auxiliary plasmid system thereof, wherein, the auxiliary plasmid system respectively codes nucleoprotein of LEP, phosphoric acid protein P and polyase protein L. the invention also relates to LEP green fluorescent protein recombination viral vector built by the reverse genetic operating system, concretely, the rabies virus Flury-LEP vaccine strain reverse genetic operating system is pCI-LEP and auxiliary plasmid systems pCAGG-N, pCAGG-P and pCAGG-L, the LEP green fluorescent protein recombination viral vector is pCI_LEP_EGEP. The invention also relates to recombination viruses rescued by the rabies virus Flury-LEP vaccine strain reverse genetic operating system and the LEP green fluorescent protein recombination viral vector, and applications thereof.

The invention belongs to bioengineering technical realm. This invention provides a merged protein which is made up of immunoglobulin combined structure field and fluorescent protein as well as the expression of genetic engineering and the method of separation and purification. Using the merged protein which is made up of immunoglobulin combined structure field and fluorescent protein of this invention expressed and purified in biology, medicine. ect realm can do various immunoassay and analysis, such as enzyme joined immunoadsorption analysis, Western Blotting analysis, spot hybridization detecting, immune combinatorial analysis, flowed cytoscopy and so on. This invention also provides two merged fluorescence protein's example of immunoglobulin combined structure field-green fluorescence protein (ZZ-EGF), immunoglobulin combined structure field-red fluorescence protein (ZZ-DsRed).

The invention relates to an expression vector containing a green fluorescent protein gene. The green fluorescent protein gene is a gene sfGFP, wherein the nucleotide sequence of the gene sfGFP is shown in SEQ ID NO: 1. The invention further relates to a construction method of the expression vector containing the green fluorescent protein gene and application of the expression vector containing the green fluorescent protein gene in positive clone screening. According to the expression vector containing the green fluorescent protein gene and the construction method and application thereof, the characteristic that the sfGFP can complete super-folding and illuminate under various conditions is utilized, the advantages of luminous power and luminous intensity of the sfGFP are obviously stronger than those of ordinary proteins, such as GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein) and the like, and are 1.6 times the EGFP, and obvious green fluorescent light can be observed by unaided eyes, so that the screening of positive clones during protein expressed cloning can be facilitated, and the fast and accurate screening for the positive clones is facilitated.

The invention discloses a prodenia litura gene engineering virus No.3 and a building method thereof, in particular relates to a prodenia litura gene engineering virus used for carrying out deficiency and recombination on a wild type virus (SpltMNPV II) by utilizing a gene engineering method. The genome of the virus is deficient in an egt gene, namely an ecdysteroid UDP (uridine diphosphate) glucosyltransferase gene; and at the site of the deficient egt gene, a BmKITa1 gene controlled by a wild type virus, namely an early gene-1(ie-1) starter, and a marker gene, namely an enhanced green fluorescent protein (egfp) gene, which is controlled by the wild type virus, namely a polyhedron gene (ph) starterare inserted. The prodenia litura gene engineering virus No.3 disclosed by the invention has the advantages of faster insecticidal speed, less dosage effect and better field application effect compared with the wild type virus. Compared with the prior art, the prodenia litura gene engineering virus No.3 provided by the invention has less dosage effect and lower LT50 at low dose (105 polyhydral bodies / larva).