35 results about "MCherry fluorescent protein" patented technology

The invention discloses a method for quickly preparing a vaccine by editing pseudorabies virus genomes based on a CRISPR / Cas9 gene editing system and a Cre / lox recombination system and an application of the method. According to the method, the CRISPR / Cas9 gene editing system is used for synchronously and efficiently recombining a GFP gene and a mCherry gene to a pseudorabies virus gE gene site and a TK gene site respectively to obtain conditional deletion strains of a gE gene and a TK gene; after purification, the Cre / lox system is used for cutting off extraneous GFP and mCherry genes in the pseudorabies virus recombinant virus genome so as to perform purification quickly to obtain a live pseudorabies virus vaccine lack of gE / TK genes; multiple genes are operated at the same time, so that multiple rounds of flows for knocking out multiple genes in the conventional method are reduced to one round; and meanwhile, the efficient edition of virus genes by using the CRISPR / Cas9 and Cre / lox systems simplifies about thirty generations of plaque purification processes into 3-4 generations, so that the preparation efficiency of the virus vaccine is greatly improved, and the method provides a strong guarantee for effectively preventing and controlling the larger-range popularization of variant pseudorabies viruses and reducing heavy economic losses.

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 a method for evaluating CRISPR / Cas9 gene editing efficiency or miss frequency, and belongs to the field of bioengineering. The method mainly comprises the following three parts: firstly, selecting a marker gene mCherry to be inserted into an expression vector in order to quickly screen a transgenic plant; secondly, in order to quickly identify the editing efficiency and the miss frequency of the CRISPR / Cas9 gene, inserting fatty acid dehydrogenase FAD2 and FAD3 as target genes into a vector; and finally, utilizing the change of fatty acid components after target gene mutation, and by detecting the composition of C18 unsaturated fatty acid in a transgenic positive plant, quickly and conveniently screening out an edited plant and an unedited plant, so that the editing efficiency and the miss effect of a specific CRISPR / Cas9 gene editing system are quickly, accurately and efficiently determined.

The present invention is a novel biosensor composed of mOrange2 and mCherry fluorescent proteins operably linked via a linker, which provides a distinct color change upon separation of the fluorescent proteins.

Compositions for characterization of Botulinum toxin (BoNT) are described that include a genetically modified cell that is transfected with an artificial construct comprising a nucleic acid sequence that encodes for a hybrid protein having (a) a reporter-containing portion chemically coupled to (b) a cleavage site and (c) a control fluorophore. The cleavage site interacts with a BoNT in a manner that cleaves the reporter-containing portion from remainder of the construct. The cleaved portion is destroyed or otherwise degraded by the local environment, and presence of BoNT is evidenced by reduction in signal from the reporter. The cleavage sequence is all or part of a SNARE protein, the cleavable reporter-containing portion is preferably Yellow Fluorescent Protein (YFP), Citrine, Venus, or a YPet protein and the control fluorophore is preferably CFP, mStrawberry, or a mCherry protein.

The invention discloses a manufacturing method and application of an insect in-vitro protein interaction detecting system. At the positions of amino acid residues at the 159th bit and the 160th bit, mCherry is divided into a segment NmC and a segment CmC through a PCR method, and then the segment NmC and the segment CmC are each fused with a connecting zone sequence, a c-Myc label and a poly(A) sequence to form four fused segments; the four fused segments are each connected with a pIE-MCS plasmid with an autographa californica multiple nuclear polyhedrosis virus ie1 gene promoter and a gp64 gene poly(A) sequence to establish expression plasmids; the expression plasmids are combined to form an insect cell dimolecular fluorescent complementary detecting system. The method is simple and easy to implement, and the established dimolecular fluorescent complementary detecting system expands the application of an insect cell expression system and provides a convenient and effective technological means for insect proteomics and research on protein interaction between insects and pathogenic microorganisms of the insects.

The invention provides a method for establishing embryonic stem cells containing exogenous chromosome. Specifically, the method injects a single chromosome inserted into a mCherry reporter gene into mouse fertilized egg by a microinjection mode, so that the mice embryonic stem cells (ESCs) containing exogenous chromosomes is produced. The method of directly transferring modified chromosomes by single-chromosome microinjection to mediate chromosome engineering is a very effective technique, and can promote the acquisition of cells containing the exogenous chromosomes and chromosome operation.

The invention discloses a novel SynNotch synthetic acceptor and a coding gene thereof. The novel SynNotch synthetic acceptor contains anti-CD19scFV, a zebra fish Notch core segment and a GAL-VP64 segment, the amino acid sequence of the novel SynNotch synthetic acceptor is represented by SEQ ID NO.5, and the corresponding nucleotide sequence of the coding gene is represented by SEQ ID NO.4. According to a SynNotch system modified by virtue of the novel SynNotch synthetic acceptor, a downstream gene can be relatively efficiently expressed. Compared with an original SynNotch acceptor system, theaverage fluorescence intensity for regulating and controlling the expression of a Mcherry fluorescent protein of the modified SynNotch system can be increased by 4.9 times. According to the modified SynNotch acceptor, the application range of SynNotch can be expanded, a novel application prospect is provided for the cell modification, and the novel SynNotch synthetic acceptor has potential important economic values and significances.

The invention discloses a system for rapidly analyzing an RNA (ribonucleic acid) functional element in vivo. The system comprises a bifluorescence reporter vector p35mG, and the vector comprises a coding sequence shown as SEQ ID No.1. Through establishment of the bifluorescence reporter vector p35mG with the coding sequence shown as the SEQ ID No.1, one fluorescent protein mCherry is taken as internal reference, the other fluorescent protein GFP is taken as a reporter gene, a to-be-detected gene segment is inserted into GFP coding sequence to form a recombinant reporter gene GFP-3'UTR, and the functional cis acting element in the RNA is rapidly identified by analyzing influence of different segments on fluorescence intensity ratio of GFP to mCherry; besides, the regulation process of the cis acting element can be controlled through application of different treatment or expression of different effector proteins, and accordingly, the regulation effect of different signals or effector proteins in plants on the RNA is studied.

The invention discloses a constructing method of a stable-expression FGF-1 protein cell line. FGF-1 genes are inserted in Not I and Avr II polyclone sites of a pLenti-CMV-Gag-Mcherry-Puro carrier, co-transfection is conducted on 293T cells through the constructed pLenti-CMV-FGF-1-Emcherry-Puro recombinant plasmids and virus packaging plasmids, and culturing, virus extraction, virus supernate collecting and filtering are conducted to obtain recombinant slow virus liquid. The 293T cells are infected with packaged recombinant slow viruses, and through screening and identifying of puromycin, the stable-expression FGF-2 protein cell line is obtained. The cell line which is mixed with tag proteins and can stably express the FGF-1 proteins is prepared through a recombinant slow virus system, andthe cell line provides a quite good tool for the research of the biological function of the FGF-1 proteins.

The invention discloses a method for marking a protein polymer by using quantum dots and belongs to the field of a nano-biotechnology. The method is characterized by the following steps: selecting a fluorescent protein dimer (TIP1-mCherry dimer) containing a histidine tag sequence (Histag); and mixing protein and the quantum dots according to a certain ratio and coupling to form a protein-quantum dot composite, wherein the structure of the composite is proved to be very stable through imidazole substitution. According to the method, systematic research is performed on the influence of the assembling characteristic of the protein with a three-dimensional structure and the quantum dots, so the combining stability of the protein and the quantum dots is improved and the marked protein polymer can serve as a nanometer fluorescent probe widely applied to a biomarker.

The invention discloses a method for constructing a cell strain for stably expressing a cytochrome C protein. The method comprises the following steps: inserting cytochrome C genes into Not I and BamHI multiple cloning sites of a pLenti-CMV-Gag-Mcherry-Puro carrier; using constructed pLenti-CMV-cytochrome C-Emcherry-Puro recombinant plasmid and virus packaging plasmid for co-transfecting 293T cells; culturing, detoxifying, collecting virus supernatant and filtering, thereby acquiring a recombinant chronic virus liquor; screening and identifying the packaged recombinant chronic virus infected293T cells by using puromycin, thereby acquiring the cell strain for stably expressing the cytochrome C protein. The invention utilizes a recombinant chronic virus system to prepare the cell strain fused with tag protein and capable of stably expressing the cytochrome C protein. The cell strain is capable of supplying an excellent tool for researching the biological function of the cytochrome C protein.

The invention relates to recombinant salmonella capable of expressing red fluorescence protein and a building method of the recombinant salmonella. The clinically isolated salmonella is utilized as a transformation receptor, plasmids pFPV-mCherry with red fluorescence protein gene segments inserted enter a bacterium through electroporation, and the salmonella capable of expressing the red fluorescence protein is built accordingly. The built recombinant salmonella can be used for marking and tracing in-vitro and in-vivo test processes of the salmonella, and the problem that the real-time monitoring cannot be performed in the salmonella test is solved; the recombinant salmonella can express the fluorescence protein and can replace an expensive salmonella specific antibody, not only is the research grant saved, but also the false positive caused by utilization of the antibody is greatly reduced; co-localization can be performed through combination with immunofluorescent staining with related protein, and an effective means is provided for in-depth study of the clinically isolated salmonella.

The invention discloses a method for constructing and applying a cytochrome C gene-carried slow virus expression plasmid. The method comprises the following steps: inserting cytochrome C genes into Not I and BamH I multiple cloning sites of a pLenti-Mcherry-Puro carrier; using a constructed pLenti-cytochrome C-Emcherry-Puro recombinant plasmid for transfecting MDA-MB-453 cells; screening and identifying by using puromycin, thereby acquiring a cell strain for stably transfecting cytochrome C gene. The recombinant plasmid contains red fluorescence protein mcherry, so that positive cells can be conveniently observed under a fluorescence microscope; labels of puromycin resistance gene and ampicillin resistance gene are carried by the recombinant plasmid; ampicillin or puromycin can be added for screening during the process of prokaryotic expression or eukaryotic expression; the plasmid establishes a firm basis for further researching biological characteristics and function of cytochrome Cgene and researching and developing anti-tumor drugs.

The invention discloses a DNA fragment and an application thereof in construction of a recombinant influenza virus expressing a red fluorescent protein gene. An mCherry fluorescent gene is inserted behind an NS1 gene of an H9N2 influenza virus strain, wherein the complete NS gene is formed by sequentially linking an NS1 gene, the mCHerry fluorescent gene and an NEP gene; the gene and the NEP geneare connected by a PTV-1 2A sequence which can be recognized and cut by protease, so a fluorescent protein-labeled NS fusion gene is constructed and has the nucleotide shown in SEQ ID NO:1. The constructed recombinant influenza virus can produce live virus expressing red fluorescent proteins, is relatively stable in the process of virus passage, does not affect the function of the virus itself, has the growth curve and the titer similar to those of a wild virus strain, can be used for subsequent research, and is of great significance on development, screening and production of anti-influenza virus drugs.

The invention discloses a method for detectingprotein interaction by co-immunoprecipitation on the basis of two-color fluorescent tag proteins GFP and mCherry. The method comprises steps as follows: target genes are constructed to N ends of expression vectors respectively, agrobacteria are transformed after plasmid is extracted, agrobacterium infection liquid co-injection model plant is prepared,a co-injection tobacco leaf protein is extracted, target proteins with GFP tags are enriched with GFP-Trap A beads, meanwhile, other target proteins with mCherry tagsare immunized, and finally, expression and interaction conditions of the target proteins before immunization and after immunization are detected by the GFP and mCherry tag antibodies respectively. According to the method, while real-time fluorescent visual monitoring for expression and co-localization of the target proteins in living cells is realized,the protein interaction can be directly and effectively verified through the co-immunoprecipitationby selecting the stage of the highest expression quantity in real time, the success rate of protein interaction verification is greatly increased, and time and economic cost is substantially reduced.

The present invention is a novel biosensor composed of mOrange2 and mCherry fluorescent proteins operably linked via a linker, which provides a distinct color change upon separation of the fluorescent proteins.

The invention discloses an application of a simian virus 40 capsid protein VP1 serving as a cell transmembrane protein. Firstly, the VP1 is revealed to be a cell-penetrating protein, and then the VP1 protein is subjected to fusion expression with other proteins and can carry an exogenous protein to penetrate through a cell membrane so as to enter cells and be localized in cytoplasm. An experiment of the invention proves that the VP1 protein can carry a mCherry fluorescent protein and a cancer suppressor protein to penetrate through a cell membrane so as to enter cells and be localized in cytoplasm. The invention provides a simple and efficient cell transduction system, and realizes subcellular localization in cytoplasm, and a constructed VP1-P53 protein is expected to have potential applications in the aspects of tumor treatment and the like.

The invention discloses a system for rapidly analyzing an RNA (ribonucleic acid) functional element in vivo. The system comprises a bifluorescence reporter vector p35mG, and the vector comprises a coding sequence shown as SEQ ID No.1. Through establishment of the bifluorescence reporter vector p35mG with the coding sequence shown as the SEQ ID No.1, one fluorescent protein mCherry is taken as internal reference, the other fluorescent protein GFP is taken as a reporter gene, a to-be-detected gene segment is inserted into GFP coding sequence to form a recombinant reporter gene GFP-3'UTR, and the functional cis acting element in the RNA is rapidly identified by analyzing influence of different segments on fluorescence intensity ratio of GFP to mCherry; besides, the regulation process of the cis acting element can be controlled through application of different treatment or expression of different effector proteins, and accordingly, the regulation effect of different signals or effector proteins in plants on the RNA is studied.

The invention discloses a manufacturing method and application of an insect in-vitro protein interaction detecting system. At the positions of amino acid residues at the 159th bit and the 160th bit, mCherry is divided into a segment NmC and a segment CmC through a PCR method, and then the segment NmC and the segment CmC are each fused with a connecting zone sequence, a c-Myc label and a poly(A) sequence to form four fused segments; the four fused segments are each connected with a pIE-MCS plasmid with an autographa californica multiple nuclear polyhedrosis virus ie1 gene promoter and a gp64 gene poly(A) sequence to establish expression plasmids; the expression plasmids are combined to form an insect cell dimolecular fluorescent complementary detecting system. The method is simple and easy to implement, and the established dimolecular fluorescent complementary detecting system expands the application of an insect cell expression system and provides a convenient and effective technological means for insect proteomics and research on protein interaction between insects and pathogenic microorganisms of the insects.

The invention belongs to the technical field of biological monitoring, and particularly relates to a streptococcus agalactiae plasmid for expressing fluorescent protein and a construction method and application thereof. The construction method comprises the steps of integrating a promoter of the fish-source streptococcus agalactiae recA gene into the upstream of the mcherry gene through the overlap PCR method, then connecting the fusion fragment to a PBCH carrier, and obtaining the pBCH-RM plasmid. By application of the pBCH-RM plasmid to strains of the fish-source streptococcus agalactiae, red fluorescent protein can be effectively and stably expressed, and the strong toxicity biological characteristics of parent strains are retained; the streptococcus agalactiae plasmid for expressing fluorescent protein can be applied to the basic researches of the route of transmission, fluorescence tracing, interaction between pathogen and host and the like in the streptococcus agalactiae.

The invention discloses a method for marking a protein polymer by using quantum dots and belongs to the field of a nano-biotechnology. The method is characterized by the following steps: selecting a fluorescent protein dimer (TIP1-mCherry dimer) containing a histidine tag sequence (Histag); and mixing protein and the quantum dots according to a certain ratio and coupling to form a protein-quantum dot composite, wherein the structure of the composite is proved to be very stable through imidazole substitution. According to the method, systematic research is performed on the influence of the assembling characteristic of the protein with a three-dimensional structure and the quantum dots, so the combining stability of the protein and the quantum dots is improved and the marked protein polymer can serve as a nanometer fluorescent probe widely applied to a biomarker.

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 a preparation method of vector for specific expression of miR-505 in a central nervous system. The method comprises the following steps: obtaining a target gene by PCR; digesting a GFAP promoter from a pAAV-GFAP-hchR2-mcherry-WPRE vector by appropriate restriction endonuclease; cloning the GFAP promoter and the target gene on a pUC19 vector; digesting a GFAP-EGFP-mir-505 fragment from the pUC vector by appropriate enzyme digestion sites; and cloning the fragment ont a PB vector, thereby obtaining the vector. The vector together with an auxiliary vector expressing transposase can be co-injected into male pronucleus of mouse zygotes, so that a transgenic mouse can be obtained and the transgenic mouse can be used for researching the biological functions (including regulatory sexual development) of miR-505 in a nervous system and especially in neuroglia cells; besides, EGFP proteins can conveniently show the locations of exogenous genes in the nervous system.

The invention discloses a combinant vaccinia virus surface display system vector plasmid and an application thereof. The expression cassette core components of the vaccinia vector plasmid display system include a modified vaccinia virus H5 promoter, an E. coli EM7 promoter, a tandem histidine tag, a streptavidin-binding polypeptide fragment Nano-tag15, red Fluorescent protein mCherry, Zeocin resistance protein, and a vaccinia virus D8 protein transmembrane region. The recombinant vaccinia virus constructed based on the above plasmid can display Nano-tag15 on the surface of the outer membrane of the virus, the recombinant virus can be captured by streptavidin magnetic beads, and the red fluorescent protein mCherry and Zeocin resistance markers can be displayed on the surface, and by singleor combined usage of magnetic bead sorting, fluorescent labeling tracing, flow cytometry sorting and drug resistance, a powerful new means is provided for rapid screening of the recombinant vaccinia virus.

The invention discloses an application of a simian virus 40 capsid protein VP1 serving as a cell transmembrane protein. Firstly, the VP1 is revealed to be a cell-penetrating protein, and then the VP1 protein is subjected to fusion expression with other proteins and can carry an exogenous protein to penetrate through a cell membrane so as to enter cells and be localized in cytoplasm. An experiment of the invention proves that the VP1 protein can carry a mCherry fluorescent protein and a cancer suppressor protein to penetrate through a cell membrane so as to enter cells and be localized in cytoplasm. The invention provides a simple and efficient cell transduction system, and realizes subcellular localization in cytoplasm, and a constructed VP1-P53 protein is expected to have potential applications in the aspects of tumor treatment and the like.

The invention discloses a cultivation method of a transgenic rice sterile line based on an MIL1 gene. The cultivation method includes the following steps: A, obtainment of a rice MIL1 gene expressioncassette; B, obtainment of an mCherry gene expression cassette; C, amplification of a wheat pollen specific promoter Pg47; D, amplification of a wheat pollen lethal gene Ki; and E, linkage of all geneexpression elements. The step E which is the linage of all the gene expression elements further includes the following steps: firstly, linking the complete rice MIL1 gene to a pCAMBIA1390 vector; andsecondly, introducing the mCherry gene expression element to the pCAMBIA1390 vector which is introduced with the MIL1 gene in the first step. An F1 generation heterozygote follows the Mendelian separation law in a self-fertilization process, and produced progenies contain both the heterozygosis capable of maintaining three linkage genes and the sterile line without fertility.

The invention provides a dual-illumination reporting system and application thereof. The dual-illumination reporting system comprises an ECAD promoter fragment and a VIM promoter fragment, and furthercomprises a luciferase gene fragment and a Renilla gene fragment which are connected with the ECAD promoter fragment and the VIM promoter fragment respectively, or further comprises a mCherry gene fragment and an eGFP gene fragment which are connected with the ECAD promoter fragment and the VIM promoter fragment respectively. Molecular targeted medicine screening based on the EMT or the MET can be achieved.