48 results about "MCherry" patented technology

The invention discloses a recombinant protein for detecting a para-tumor Yo antibody through a CBA method and application, and belongs to the technical field of biomedical engineering.The amino acid sequence of the recombinant protein sequentially comprises a secretory signal peptide, a CDR2 protein partial sequence, a CDR2L protein partial sequence, a transmembrane region and a fluorescent label, and the secretory signal peptide is human albumin signal peptide ALB; the transmembrane region is a CD8a hinge; and the fluorescent label is mCherry. A novel recombinant protein which can be stably over-expressed on a cell membrane of an eukaryotic cell is constructed by intercepting specific partial sequences of CDR2 protein and CDR2L protein and redesigning and fusing a fluorescent label by using a secretory signal peptide, a transmembrane sequence and a connecting peptide, and the recombinant protein retains respective core antigen regions of the CDR2 protein and the CDR2L protein, so that the specific partial sequences of the CDR2 protein and the CDR2L protein can be stably over-expressed on the cell membrane of the eukaryotic cell. The kit can effectively overcome the defects in the aspects of sensitivity and specificity, and when a CBA method is adopted for detection, the detection rate of the para-tumor Yo antibody can be remarkably increased, and the false positive rate is reduced, so that the requirements of clinical detection are better met.

The invention relates to the technical field of gene engineering, in particular to a transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on a Tol2 transposition subsystem and a construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry. The preparation method comprises the following steps: S1, constructing a Tol2 transposon expression vector pTol2-ef1a-tjp1a-P2A-mCherry which contains an ef1a promoter, a tjp1a gene, a P2A peptide sequence and an mCherry reporter gene; s2, carrying out in-vitro transcription to prepare Tol2 transposase mRNA; s3, mixing the expression vector with Tol2 transposase mRNA, and microinjecting the mixture into the single-cell stage embryo of the zebra fish; s4, performing fluorescence screening on the F0 generation embryos surviving after injection to obtain the Founder fish with positive transgenosis; s5, after the F0-generation positive fish is bred to be sexually mature, an F1 generation is obtained through mating, transgenic positive individuals with stable inheritance are screened out after identification, and a transgenic zebrafish strain is established. According to the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system and the construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system, efficient integration of exogenous genes is achieved by utilizing the Tol2 transposon system, and the transgenic zebrafish line capable of being stably inherited to the F1 generation is successfully obtained.

The invention discloses a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector as well as a construction method and application of the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector. The construction method comprises the following steps: mixing an mCherry gene segment and an IRES2 gene segment which are respectively obtained by PCR (Polymerase Chain Reaction) amplification in equal mass, then carrying out fusion amplification to obtain an IRES2-mCherry fusion gene segment, and carrying out homologous recombination on the IRES2-mCherry fusion segment and a pCDNA-3xHA-hTERT linearization vector. On the basis of a pCDNA-3xHA-hTERT stent vector, a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector with red fluorescence is successfully constructed, and the fusion expression vector can be used as a fusion expression vector of the pCDNA-3xHA-hTERT-IRES2. The construction method is simple and efficient, enzyme digestion is not needed, and the constructed pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector can be subsequently used for transfecting bovine endometrial epithelial cells, so that the immortalized bovine endometrial epithelial cells are obtained.

The invention provides a recombinant lentivirus capable of indicating bovine spermatogonial stem cell differentiation, a spermatogonial stem cell line and application, and belongs to the technical field of cell biology. The invention provides a method for constructing a spermatogonial stem cell line for indicating bovine sperm differentiation on the basis of a lentivirus transduction technology, and a visual detection system for differentiating spermatogonial stem cells into spermatoblasts is constructed by regulating expression of mCherry protein by utilizing a PRM1 promoter. According to the method, the expression of PRM1 is indicated by fluorescent protein mCherry, so that the differentiation process of the bovine spermatogonial stem cells is indicated, and a good tool is provided for the induction of differentiation of the bovine spermatogonial stem cells to sperms, the research of a development mechanism and the optimization of a differentiation system of the bovine spermatogonial stem cells. The method is a new way for solving the neck clamping problem that good-variety bovine sperms are difficult to obtain in scientific research and production, can also be used for optimization of a spermatogonial stem cell differentiation system, sorting and purification of sperms, even downstream research of drug screening and the like, and has a good application prospect.

The application provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

The invention belongs to the field of biomedicine, and discloses a method and a kit for monitoring a lysosome acidification process mediated by mitochondrial-lysosome interaction. According to the method, an LYSO-PZ probe with a specific structure is used for marking cell lysosome contents, a pH rodo dye and a lysosome membrane marker sfGFP-LAMP1-mCherry are combined for monitoring the pH value and membrane positioning of the lysosome respectively, then a three-channel fluorescence image is obtained through a structural illumination microscope (SIM), and the fluorescence intensity ratio is quantitatively analyzed. In addition, the method further comprises a step of forming mitochondrial-lysosome contact (MLC) through optogenetics regulation, related protein binding is induced through blue light, fluorescence intensity changes before and after MLC formation are compared, and MLC-mediated proton transport is verified. A complete monitoring system is established for the first time, lysosome acidification can be accurately monitored, information can be obtained in multiple dimensions, and the method has important clinical application value and wide market prospects.

The application belongs to the field of biomedicine, and discloses a method and a kit for monitoring a process of mitochondrion-lysosome interaction-mediated lysosome acidification. The method is characterized in that a cell lysosome content is first labeled with a LYSO-PZ probe with a specific structure, a pHrodo dye and a lysosome membrane marker sfGFP-LAMP1-mCherry are combined to monitor the pH value and membrane localization of the lysosome, respectively, then three-channel fluorescence images are acquired through a structured illumination microscope (SIM), and the fluorescence intensity ratio is quantitatively analyzed. In addition, the method further comprises a step of optogenetic regulation of mitochondrion-lysosome contact (MLC) formation, the fluorescence intensity change before and after the formation of MLC is compared through blue light induction of related protein binding, and the proton transport mediated by MLC is verified. The application first establishes a complete monitoring system, can accurately monitor lysosome acidification, and has important clinical application value and broad market prospect.

The invention discloses a bifluorescence reporter plasmid, construction of the plasmid and application of the bifluorescence reporter plasmid in detection of bacterial extracellular itaconic acid level. The reporter plasmid is composed of a resistance gene, a promoter, a first reporter gene, a terminator, a transcriptional regulation factor BM590RS11055, a bidirectional promoter and a second reporter gene. The promoter Ptac controls the expression of the first reporter gene mCherry, is used for detecting the plasmid background expression level, and can eliminate the influence of the plasmid copy number on the itaconic acid level detection result; the bidirectional promoter PIct controls the expression of the second reporter gene EGFP and is used for detecting the extracellular itaconic acid level, the expression of the first reporter gene and the expression of the second reporter gene are not interfered with each other, and the detection result is more accurate and more reliable. The bifluorescence reporter plasmid constructed by the invention can be used for detecting the extracellular itaconic acid level of bacteria, the operation is simple, and the result is reliable.

The invention discloses a genetically engineered bacterium for producing D-pantothenic acid based on a corynebacterium glutamicum CPSPC system as well as a construction method and application of the genetically engineered bacterium. Wild type C.glutamicum ATCC 13032 is taken as a chassis strain, and a functional combination of a glycine ribosome switch BsGR from bacillus subtilis and a promoter Ptuf / Ph36 is verified through fluorescent protein mCherry; a GCS system of escherichia coli and bacillus subtilis is introduced to the strain DPAg-15, genes related to synthesis of 5, 10-dimethyltetrahydrofolic acid are tested, it is found that serA delta197GTG / ATG significantly increases the yield of D-pantothenic acid, the yield of D-pantothenic acid of the constructed strain DPAj-2-tuf is increased to 23.06 g / L (63 h), the yield is 0.37 g / L / h, and the conversion rate is 0.17 g / g; the engineering strain DPAj-2-tuf is obtained by dynamically regulating and controlling a D-pantothenic acid synthesis path through a CPSPC system and combining plasmid overexpression serA delta197GTG / ATG, the yield of the engineering strain DPAj-2-tuf is increased by 2.1 times compared with that of a traditional method, and an efficient technical scheme is provided for producing D-pantothenic acid through a microbial fermentation method.

The invention relates to the technical field of saccharomyces cerevisiae expression foreign protein, and discloses a saccharomyces cerevisiae strain with ASFV p14.5 displayed on the surface and a construction method and application thereof, and the method comprises the following steps: (1) synthesizing a delta site homologous arm sequence; (2) seamlessly connecting the homologous arm sequence of the delta site to a carrier pET23a, connecting a selective marker tryptophan biosynthetic gene TRP1 to the 5'end of the upstream homologous arm sequence of the delta site, and sequentially connecting an Aga2 gene, an E120R gene, a T2A peptide gene and an mCherry gene to the 5 'end; (3) carrying out enzyme digestion on two sides of the delta site through restriction endonuclease salI, and recovering a linearized fragment through agarose gel; and (4) integrating the recovered fragments into a host saccharomyces cerevisiae genome through a chemical conversion method, and culturing until bacterial colonies grow. The surface display type saccharomyces cerevisiae strain obtained by the invention not only has the stability of integrative saccharomyces cerevisiae for expressing foreign protein, but also can be used for more easily and more intuitively screening a high-expression quantity saccharomyces cerevisiae strain by combining the T2A peptide with mCherry.

This invention belongs to the field of genetic engineering technology, specifically disclosing a method for preparing and applying gene-modified homozygous cells of chicken progenitor cells (PGCs). Using a dual-fluorescent marker gene, two donor plasmids carrying green fluorescent protein (EGFP) and red fluorescent protein (mCherry), respectively, are transfected into PGCs. Cells simultaneously expressing red and green fluorescence are obtained using flow cytometry, thus forming homozygous cell lines. Then, the dual-fluorescent marker gene is deleted using the Cre-LoxP system combined with electroporation, and residual LoxP fragments are deleted using ssODN as a donor template, achieving traceless gene editing in recipient chicken embryos. This invention does not introduce any exogenous genes, increasing the safety of gene-modified chickens and is of great significance for eliminating the biosafety risks associated with gene-edited chickens.

The application belongs to the technical field of biology, and particularly relates to a mitochondrion function evaluation method and application thereof. The application provides an innovative mitochondrion function evaluation method, which reports the ATP concentration change in the mitochondrion matrix and the mitochondrion morphology in real time, in situ and quantitatively through ATP response fluorescent protein Mito-AT1.03 with a mitochondrion positioning signal, directly reflects the mitochondrion energy metabolism and the mitochondrion fusion / fission state; meanwhile, a fusion protein Parkin-mCherry is constructed by using Parkin protein, a key starting factor of mitochondrion autophagy, as an early specific mark of autophagy initiation. The two are co-transfected into cells, and the signal level and positioning condition of the two fluorescent proteins are observed, so that the mitochondrion function can be accurately and timely evaluated, and a new idea is provided for the mechanism research of mitochondrion dysfunction and the methodological development of the screening of mitochondrion function regulators.

The invention relates to the technical field of cell engineering, in particular to a cell strain causing pathogenic splicing mutation of a female HA patient and a construction method of the cell strain. A construction method of the T point mutation cell strain comprises the following steps: designing an sgRNA sequence based on a mutation site of a target gene F8; the method comprises the following steps: based on an sgRNA sequence and a CRISPR / Cas9 gene editing technology, constructing a plurality of pX458-sgRNA plasmids by taking a pX458 plasmid as a vector; the method comprises the following steps: constructing an LA-pMD-18T-pGK-puro-mcherry-RA donor plasmid by taking a pMD-18T-pGK-puro-mcherry plasmid as a vector on the basis of an sgRNA (small guide ribonucleic acid) sequence and a pX458-sgRNA plasmid; the preparation method comprises the following steps: simultaneously transfecting a pX458-sgRNA plasmid and an LA-pMD-18T-pGK-puro-mcherry-RA donor plasmid into a cell, and carrying out drug screening, so as to obtain a stable cell strain. In addition, the invention also provides application of the mutant cell strain. The discovery of the novel pathogenic splicing mutation not only enriches an F8 gene mutation database, but also provides a new strategy for precise treatment of female HA patients in the future.

The invention discloses a construction method of a crisprcas9 visual model based on fluorescent Escherichia coli, a red fluorescent protein gene is introduced into an Escherichia coli BL21 genome to construct a mcherry Escherichia coli model, and an easy-to-use biological model capable of verifying a Crisper / cas9 knockout effect and fluorescent Escherichia coli which can be quantified, is easy to observe, is high in sensitivity and is low in background are obtained.

The invention belongs to the technical field of biology, and particularly relates to a mitochondrial function evaluation method and application thereof. The invention provides an innovative mitochondrial function evaluation method which comprises the following steps: reporting ATP concentration change and mitochondrial morphology in a mitochondrial matrix in real time, in situ and quantitatively through ATP response fluorescent protein Mito-AT1.03 with a mitochondrial positioning signal, and directly reflecting mitochondrial energy metabolism and mitochondrial fusion / division states; meanwhile, a mitochondrial autophagy key initiation factor Parkin protein is utilized to construct a fusion protein Parkin-mCherry, and the fusion protein Parkin-mCherry is used as an early-stage specific marker for autophagy initiation. The two fluorescent proteins are co-transfected into cells, and the signal levels and positioning conditions of the two fluorescent proteins are observed, so that the mitochondrial function can be accurately evaluated in real time, and a new thought is provided for methodology development of mitochondrial dysfunction mechanism research and mitochondrial function regulator screening.

A method for constructing a mouse model for rapid degradation of Myostatin (MSTN) protein is provided. The method includes: inserting sequentially a 5′ homologous arm, 3 X (Glu-Ala-Ala-Ala-Lys) (3xEAAAK), an FK506-binding protein (FKBP1A) gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein monomeric Cherry (mCherry) sequence and a 3′ homologous arm into a Donor vector backbone to obtain a Donor recombinant vector; designing a Guide Ribonucleic Acid (gRNA) based on a third exon of the Mstn gene; mixing the gRNA, CRISPR-associated protein 9 (Cas9) protein, and the Donor recombinant vector and co-injecting a mixture of the above into mouse fertilized eggs, culturing the fertilized eggs in vitro and transplanting to obtain the F0 generation mice; and hybridizing the F0 generation mice with wild-type mice, or further breeding hybridized offspring to obtain a knock-in mouse model.

The invention relates to an application of 2-methyl-4 (3H)-quinazolinone in preparation of an anti-angiogenesis medicine. The invention discloses the application of the 2-methyl-4 (3H)-quinazolinone compound as an angiogenesis inhibitor in anti-angiogenesis drugs for the first time, and the 2-methyl-4 (3H)-quinazolinone compound has a remarkable inhibition effect on internode blood vessels and dorsal longitudinal anastomosis angiogenesis of a Tg (flk: mCherry) fluorescent transgenic zebrafish strain. Meanwhile, the growth of the zebrafish embryos of the strain is not obviously influenced, and the toxicity is extremely low. The compound not only can be extracted from streptomyces, but also can be obtained through an artificial synthesis process, has a reliable and stable source, and has a good application prospect in the field of anti-angiogenesis drugs.

The application discloses a method for constructing a fluorescently labeled piwil1 gene knockout sterile zebrafish line, which utilizes a CRISPR / Cas9 system to construct a piwil1 gene knock-in vector carrying P2A-EGFP or P2A-mCherry, respectively, to obtain two hybrid strains through microinjection, and then to screen a homozygote expressing EGFP and mCherry simultaneously by mating the two strains. The homozygote shows male sterility and can be used for reproductive development research and ecological prevention and control of transgenic fish. The application realizes visual labeling of the function of the piwil1 gene and accurate screening of the sterile phenotype, and is simple and efficient to operate.

The invention relates to the technical field of genetic engineering and model animals, in particular to a visual myocardial specific gene knockout zebrafish model and a construction method and application thereof. According to the technical scheme, the visual myocardial specific gene knockout zebrafish model comprises a model body, the model body is transgenic zebrafish, and a genome of the model body comprises a Cas9 protein coding sequence driven by a myocardial specific promoter; a Tg transgenic zebrafish strain is constructed, a cmlc2 promoter with high cardiac muscle cell specificity is utilized, expression of Cas9 protein optimized by zebrafish codons and expression of red fluorescent protein mCherry are driven at the same time, the Cas9 protein and the red fluorescent protein mCherry are connected through a P2A self-cleavage peptide sequence, it is ensured that independent and complete functional protein is generated through translation after transcription, and the expression of the Cas9 protein and the red fluorescent protein mCherry is promoted. The expression of the Cas9 protein is completely coupled with an mCherry fluorescence signal.

The present application relates to a kind of construction method of macrophage specific fluorescent reporter mouse model, belong to genetic engineering and genetic modification technical field.The present application is first by CRISPR / Cas9 genome editing technology to C1qc gene site is edited after obtaining a kind of brand-new C1qc-CreChy gene knock-in mouse model, realizes the expression of Cre and mCherry is directly driven by C1qc promoter, and it is verified by flow cytometry that mCherry only in the microglia of mouse brain and the macrophage in spleen has very high expression, and mCherry expression is not detected in other immune cells, to realize the tracing of the two kinds of macrophages, for studying its function.The present application provides a kind of usable animal model for studying the heterogeneity of macrophage, and provides a kind of new animal model for studying the complex immune function in body.

The invention relates to the field of bioengineering, in particular to an artificial codon optimized nucleic acid molecule for coding red fluorescent protein. The invention provides a nucleic acid molecule for coding red fluorescent protein, the red fluorescent protein is mCherry, compared with the nucleotide sequence of the existing red fluorescent protein, the number of CpG sites of the nucleic acid molecule is increased from 56 to 92, and the content of G / C (GC3) at the third site of a codon is not less than 99%. The nucleotide sequence provided by the invention is an mCherry synonymous codon substitution sequence obtained by combining increasing the number of CpG sites and using an optimal codon strategy, and the fluorescence intensity of the synonymous codon substitution sequence in mammalian (human / mouse) cells is improved by 1.3-2.4 times compared with that of a sequence before modification; nucleic acid molecules which do not exist in the nature and can encode red fluorescent protein with higher fluorescence intensity are obtained by using a codon optimization method, and the development of living cell imaging is promoted.

The invention discloses an establishment method and application of an agrobacterium rhizogenes mediated woody plant non-genotype dependent root tiller regeneration transformation system, and belongs to the technical field of plant transgenosis. In order to solve the technical problem that the molecular breeding process of poplar is restricted due to lack of a poplar efficient genetic transformation system, a set of efficient and universal poplar genetic transformation system is established, specifically, a fluorescent labeled gene (EGFP / mCherry) is innovatively adopted to replace a traditional antibiotic screening marker, the screening period is shortened to be within 2 weeks from conventional 4-6 weeks, and the screening efficiency is greatly improved. The screening efficiency is obviously improved; meanwhile, a traditional multi-step transformation process is simplified into a single rooting culture medium and a bud induction culture medium by systematically optimizing a culture medium formula, so that the operation process is simplified, and the experiment cost is reduced. According to the method, data reference and technical support are provided for establishment of an efficient genetic transformation and gene editing system of all woody plants with sprout tillering capacity.

The invention relates to the field of vaccine preparation, and discloses a saccharomyces cerevisiae strain for displaying African swine fever virus protein on the surface and a construction method and application of the saccharomyces cerevisiae strain. Sequentially connecting TRP1, Aga2, African swine fever virus protein, T2A peptide and gene segments of mCherry to the 3'end of the upstream homologous arm sequence of the delta site to obtain a recombinant plasmid; and (2) carrying out enzyme digestion on upstream and downstream regions of the delta site by using incision enzyme, separating by adopting agarose gel, transforming the obtained target DNA fragment onto a saccharomyces cerevisiae genome, culturing to obtain a transformed bacterial colony, and observing a single bacterial colony with red fluorescence, namely the saccharomyces cerevisiae strain with the surface displaying the African swine fever virus protein. The saccharomyces cerevisiae strain capable of displaying ASFV p54 or ASFV j18L on the surface is successfully constructed, the strain not only can stably express ASFV protein, but also can rapidly screen positive clone through red fluorescence, and the protein expression verification mode is simple.

The invention provides a high-fidelity Cas9 variant, a tissue targeting lipid nanoparticle delivery system, a CRISPR-Cas9-mediated genome precise editing method and application thereof, and belongs to the technical field of genetic engineering, the high-fidelity Cas9 variant HyperFi-Cas9 is adopted, four mutations of R691A, N497A, R661A and Q695A are introduced, and the off-target effect is reduced to 0.025% or below while the wild type targeting activity is kept not lower than 85%; a tissue targeting intelligent lipid nanoparticle delivery system is adopted, and by optimizing lipid composition and ligand modification, 30-60% of cell delivery efficiency and 16-80% of in-vivo editing efficiency are achieved; the integrated double-fluorescence real-time monitoring system comprises an FRET (fluorescence resonance energy transfer) molecular beacon probe and an mCherry-P2A-EGFP (enhanced green fluorescent protein) integrated report module, and is used for realizing dynamic monitoring and quantitative evaluation of an editing process; and a homologous recombination enhancement strategy is adopted.

The invention provides an efficient genetic transformation method of Agrobacterium tumefaciens-mediated bamboo reed callus, and belongs to the technical field of plant genetic engineering. The method comprises the following steps: transforming a plant expression vector containing a red fluorescent protein mCherry gene and a nolserine resistance gene nat into Agrobacterium tumefaciens; drying the bamboo reed callus until the surface is shrunk, cutting, carrying out oscillation, ultrasonic and vacuum infection by using the obtained agrobacterium solution under a dark condition, carrying out dark culture on the infected callus on a co-culture medium with sterile filter paper, washing with water, and recovering the obtained callus, and selecting calluses emitting red fluorescence, and carrying out differentiation culture and rooting culture on the calluses. According to the invention, the efficient agrobacterium genetic transformation method of the bamboo reed based on the nolserine selection marker is successfully established at home and abroad for the first time, and the method is simple and efficient, and is particularly important for breeding and variety improvement of the bamboo reed.

The present application relates to the field of bioengineering, and particularly relates to a kind of artificial codon optimization nucleic acid molecule of the red fluorescent protein coding.The present application provides nucleic acid molecule of red fluorescent protein coding, and the red fluorescent protein is mCherry, compared with the existing red fluorescent protein nucleotide sequence, the number of CpG site is increased from 56 to 92, the content of the third position of codon G / C (GC3) is not less than 99%.The nucleotide sequence provided by the present application is the mCherry synonymous codon substitution sequence obtained by combining the increase of the number of CpG sites and the use of optimal codon strategy, which is 1.3-2.4 times higher than the fluorescence intensity of the sequence before modification in mammalian (human / mouse) cells;The present application uses the method of codon optimization to obtain the nucleic acid molecule of the red fluorescent protein that does not exist in nature, which can encode the red fluorescent protein with stronger fluorescence intensity, and promotes the development of live cell imaging.

The invention relates to a high-performance RNA (Ribonucleic Acid) off-target detection report system as well as a construction method and application thereof. According to the invention, several modules of an MS2-MCP system, deoxyadenosine deaminase, mCherry and BFP are fused, and a fluorescence report system for indirectly detecting the miss of deoxyadenosine deaminase RNA through fluorescence and a sequencing report system for directly detecting the miss of deoxyadenosine deaminase RNA through sequencing are developed. The RNA off-target detection report system comprises an RNA off-target fluorescence report system and an RNA off-target sequencing report system. The RNA off-target detection report system can be effectively applied to RNA off-target risk assessment of the ABE base editing tool. The RNA off-target detection report system obtained through the method is convenient to operate, short in period, low in cost and high in sensitivity, effectively makes up for the shortages of RNA off-target detection in the current base editing field, and has very wide application prospects.

The invention discloses a photoresponsive macrophage as well as a preparation method and application thereof. The method comprises the following steps: (1) constructing an adenovirus vector containing an mCherry-LOV2-CD3 zeta photosensitive element; (2) introducing the mCherry-LOV2-CD3 zeta photosensitive element into the macrophage by using an adenovirus vector, so as to obtain the blue light activated macrophage CD3 zeta-M; and (3) co-incubating the up-conversion nanoparticles UCNP and the CD3 zeta-M to obtain the photolocalization activated macrophage U (at) CD3 zeta-M. The prepared U (at) CD3 zeta-M cell remarkably improves the treatment effect of a macrophage therapy on melanoma, and remarkably inhibits tumor growth compared with normal saline.

The invention relates to the technical field of gene editing, and discloses a gene editing efficiency detection vector based on double-color fluorescent protein as well as a construction method and application thereof. The construction method comprises the following steps: synthesizing a full-length DNA fragment containing a specific targeting sequence, an mCherry reporter gene and a C-terminal fused PEST degrading peptide sequence; performing double enzyme digestion and purification to obtain a target fragment; connecting the target fragment with the vector skeleton subjected to enzyme digestion linearization through T4 DNA ligase; the gene editing efficiency detection vector pLV3-CMV-RGFP-PEST-Neo is prepared by the following steps: carrying out gene editing efficiency detection on the gene editing efficiency vector pLV3-CMV-RGFP-PEST-Neo, transforming the gene editing efficiency vector pLV3-CMV-RGFP- The system containing the gene editing efficiency detection vector co-expresses red and green, during editing, an mCherry coding sequence is cut off, cells are converted from double positive (yellow) to single positive (green), cell lysis is not needed, the editing efficiency is quantified by calculating the change of the green / red fluorescence ratio, and the method is convenient and fast.

The invention discloses a construction method and application of an acetylcholin esterase inhibitor high-throughput screening cell line. The invention belongs to the technical field of biology, and particularly relates to a construction method and application of an acetylcholin esterase inhibitor high-throughput screening cell line. The recombinant cell constructed by the invention contains a fluorescent label expression module and an acetylcholin esterase gene expression cassette, wherein the fluorescent label expression module contains a cpGFP-inserted muscarinic-like receptor (M3R) encoding gene and a red fluorescent protein mcherry encoding gene carrying a CAAX membrane positioning sequence. When the recombinant cell is used for screening the acetylcholin esterase inhibitor, acetylcholin esterase does not need to be additionally added, the reaction step of acetylcholin esterase hydrolysate and auxiliaries is reduced, the use of toxic and unstable DTNB and other reagents is avoided, the system is simpler, and the time cost and the economic cost are lower.