148 results about "Co transfection" patented technology

The invention discloses a D / Yama2019 genetic evolution pedigree influenza delta virus reverse genetic operating system. According to the invention, DNA fragments corresponding to segments of PB2, PB1, P3, HEF, NP, M and NS of a full-length genome of D / JY3002 are amplified by using seven pairs of designed primers, the DNA fragments are seamlessly cloned to a pHW2000 vector and a pCC1-DualPro vector to construct plasmids, the plasmids are co-transfected to a cell line, and the artificially rescued D / Yama2019 genetic evolution pedigree influenza delta virus rD / JY3002 is obtained; the genomic sequence of the rD / JY3002 is completely consistent with the genomic sequence of a naturally separated strain D / JY3002, the infection ability is equivalent, and the rD / JY3002 can be stably subcultured for at least five generations. According to the present invention, the constructed unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 is adopted to obtain the artificially rescued recombinant influenza delta virus rD / JY3002-GFP carrying the green fluorescence reporter gene, and after the host cell is infected, the GFP expression quantity is high and stable; the invention establishes an efficient and stable D / Yama2019 genetic evolution pedigree influenza delta virus reverse genetic operating system which can be used for developing an influenza delta virus vector presenting exogenous gene.

The invention discloses an engineered exosome for promoting hair regeneration and a preparation method and application thereof, Wnt10b gene is inserted into a plasmid vector to construct a target plasmid, the target plasmid and a lentivirus packaging plasmid are co-transfected to packaging cells to generate lentivirus particles, and then target stem cells are infected. Screening to obtain a cell strain capable of stably expressing a target gene, culturing the stably transfected strain, and collecting the engineered exosome generated by the stably transfected strain. The exosomes are rich in Wnt10b protein and can promote proliferation and differentiation of hair follicle stem cells on a molecular level, so that hair follicle tissues in a resting period are activated and enter a growing period again, and hair regeneration is realized. Meanwhile, the exosome is high in safety, and adverse reactions caused by traditional chemical synthesis drugs are avoided. The exosome can be applied to the preparation of hair regeneration products, and the accurate symptomatic treatment capability of the exosome provides a hair growth method aiming at pathogenesis for alopecia patients, and provides a new hope for patients who seek safe and effective hair growth solutions.

The invention discloses a chicken sex identification method based on fluorescence labeling, and belongs to the technical field of gene engineering. The chicken sex identification method comprises the following steps: co-transfecting a CRISPR / Cas9 system and a WPG-EGFP donor plasmid into chicken primordial germ cells PGCs, screening to obtain EGFP positive PGCs, micro-injecting the EGFP positive PGCs into a receptor chicken embryo, hatching to obtain an EGFP reproductive chimera chicken, and identifying the sex through fluorescence labeling, the CRISPR / Cas9 system comprises sgRNA of a targeted chicken W chromosome WPG gene; the WPG-EGFP donor plasmid is constructed by inserting an EGFP gene into a WPG gene locus through a homologous recombination strategy. According to the method, the sex of the chicken can be visually and rapidly identified. The method solves the problems that a traditional sex identification method is complex in operation and depends on sample ages, has the advantages of high efficiency and accuracy, and is suitable for sex control and screening of poultry such as laying hens and broilers.

The invention discloses a recombinant PB transposase mRNA (messenger ribonucleic acid) and PiggyBac vector system and application of the recombinant PB transposase mRNA and PiggyBac vector system. The invention provides a recombinant PB transposase mRNA (messenger ribonucleic acid), the nucleotide sequence of the recombinant PB transposase mRNA is shown as SEQ ID NO: 11, and the 5'end of the recombinant PB transposase mRNA contains a cap structure. The invention provides an mRNA type recombinant PB transposase: PBase-Cap-mRNA, so that the risk that a PB transposase plasmid is inserted into a host cell genome is avoided, and the efficiency of inserting a donor plasmid into the host cell genome is improved. The recombinant PB transposase mRNA is capped, so that the stability of the mRNA is improved, the translation of the mRNA is promoted, and the transposon efficiency is improved. The CHO cells are co-transfected by recombining PB transposase mRNA and donor plasmids, so that the efficiency of transfecting host cells by the donor plasmids is improved, and the construction period of stable cell strains is remarkably shortened.

The invention discloses a construction and verification method of an HEK293T cell line capable of stably overexpressing human ANT2, which comprises the following steps: cloning a human ANT2 gene, constructing the human ANT2 gene into a lentivirus expression plasmid pLVX-TRE3G, co-transfecting the lentivirus expression plasmid pLVX-TRE3G and a helper plasmid pLVX-Tet3G into an HEK293T-ACE2 cell, and carrying out puromycin and G418 pressurized screening and Western blot identification to obtain the HEK293T cell line capable of stably overexpressing human ANT2. A result shows that the expression quantity of the ANT2 is the highest when the HEK293T-ACE2-ANT2 cell is induced for 12 hours at the Doxycycline of 6mg / L; when the ANT2 is induced to express, the virus nucleic acid load and the N protein expression level after SARS-CoV-2 infection are obviously reduced, which indicates that the ANT2 has an inhibition effect on SARS-CoV-2 infection replication, and a cell model and an experimental basis are provided for exploring the effect of the ANT2 in virus infection resistance.

The invention provides a toxoplasma gondii gene deletion strain with an immune protection effect and application of the toxoplasma gondii gene deletion strain, and belongs to the technical field of veterinary parasitology, veterinary immunology and parasite molecular biology. According to the toxoplasma gondii gene deletion strain, a gamma-glutamyl hydrolase gene is deleted. The invention also provides a method for constructing the insect strain. The method comprises the following steps: constructing a circular plasmid pSAG1-Cas9-U6-sgGGH; constructing a repair template containing upstream and downstream homologous arms of the gamma-glutamyl hydrolase gene and a DHFR resistance gene; co-transfecting the annular plasmid and the repair template to a toxoplasma gondii ME49 strain; performing drug screening on pyrimethamine to obtain positive monoclone; and confirming that the GGH gene is completely knocked out through PCR (Polymerase Chain Reaction) detection, sequencing analysis and immunofluorescence detection. The toxoplasma gondii gene deletion strain can provide a remarkable protection effect on toxoplasma gondii infection and is high in safety.

The application discloses a construction method of a fat-targeted gene therapy vector. The method comprises the following steps: obtaining a fat tissue-specific promoter sequence; obtaining a shRNA coding sequence targeting a Tks4 gene; cloning the promoter sequence and the shRNA coding sequence into an adeno-associated virus vector skeleton to construct a recombinant expression vector; and co-transfecting the recombinant expression vector and an auxiliary plasmid into a packaging cell to package and purify a recombinant adeno-associated virus particle. The vector constructed by the application adopts an adiponectin promoter to drive the expression of miR30-shRNA targeting the Tks4, and can be specifically expressed in fat tissues and livers after being injected through a tail vein, significantly inhibits fat cell hypertrophy and systemic fat accumulation induced by a high-fat diet, reduces the serum cholesterol level, does not cause an immune inflammatory reaction, does not affect sugar metabolism, and is not expressed in non-target tissues. The application provides a safe and efficient new strategy for the gene therapy of obesity, hyperlipidemia, fatty liver and related metabolic diseases.

The present invention relates to a nucleic acid nanostructure comprising at least one scaffold chain and a plurality of staple chains wherein the nanostructure, preferably the at least one scaffold chain, comprises at least one nucleic acid sequence encoding a gene. The invention further relates to a composition comprising the nucleic acid nanostructure, and to a set of nucleic acid sequences or a set of plasmids encoding a nucleic acid nanostructure. Furthermore, the present invention relates to a nucleic acid nanostructure or a composition comprising a nucleic acid nanostructure for use in a medicament, preferably in a method for preventing, treating and / or diagnosing a disease or condition. The invention also relates to a method for expressing a gene from a nucleic acid nanostructure, and to the use of a nanostructure or composition for gene expression.

The invention belongs to the field of veterinary virology, and discloses a bovine ephemeral fever virus strain and reverse genetic application thereof, and the reverse genetic steps are as follows: taking pCI plasmid as a skeleton to prepare plasmid for expressing a complete genome of bovine ephemeral fever virus; the method comprises the following steps: respectively preparing auxiliary plasmids for expressing an N protein gene, a P protein gene, an L protein gene and a G protein gene of the bovine ephemeral fever virus by taking pCAGGS plasmids as a skeleton, co-transfecting BHK-21 cells by using the five plasmids, and culturing the cells to obtain the rescued bovine ephemeral fever virus. The rescued bovine ephemeral fever virus has no obvious difference from the reproductive capacity of the parent virus, and the genome is still stable after 10 generations of continuous passage in BHK-21 cells. The invention provides methodological support for genetic modification of the bovine ephemeral fever virus, and has an application prospect.

The invention discloses an AAV2 recombinant gene system for double expression of sFLT-1 and shVEGF as well as a preparation method and application of the AAV2 recombinant gene system, and co-expression of the sFLT-1 and the shVEGF is realized. A target gene (sFLT-1: NM001159920.2, shVEGF: NM001025366.3) is cloned to a pAAV-CMV vector through BamHI, HindIII, EcoRI and MluI sites, a pAAV-CMV-sFLT-1-shVEGF plasmid is constructed, and the plasmid is subjected to PCR (Polymerase Chain Reaction), enzyme digestion and sequencing verification, so that the recombinant plasmid is obtained. And then, co-transfecting HEK293 cells with pAAV-RC2 and pHelper plasmids to produce rAAV particles, carrying out gradient ultracentrifugation purification on the rAAV particles by virtue of iodo-pentose, and storing the rAAV particles at the temperature of 80 DEG C below zero. The AAV2 recombinant gene system has the following advantages that the sFLT-1 and the shVEGF are stably expressed, the immunoreactivity is low, and the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF plays a role for a long time, so that the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF can be applied to various neovascularization diseases.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

The invention provides a Hemsleya chinensis cytochrome oxidase HcCYP87D19 gene, and the sequence of the gene is shown in SEQ NO: 1. This invention constructs a transgenic Saccharomyces cerevisiae engineering bacteria co-transfected with Hemsleya cathayensis cytochrome oxidase HcCYP87D19 and HcCYP81Q58, the bacteria can produce a variety of cucurbitacin intermediates including 11-Carbonyl-cucurbita-5,23-diene-3β,16a,20,25-tetrol (6), 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,23-tetrol (6b), 11-Carbonyl-cucurbita-5,23-diene-16α,20,25-triol-3-one (6a), 11-Carbonyl-cucurbita-5,24-diene-16α,20,23-triol-3-one (6c), it provides a source for cucurbitacin production.

The present application relates to the technical field of genetic engineering, in particular to a T7-293T cell and application of the T7-293T cell in bovine parainfluenza virus type 3 infectious clone rescued virus, and the T7-293T cell provided by the present application has the preservation number of CGMCC No.46335.The T7-293T cell provided by the present application is a 293T cell stably expressing T7 RNA polymerase, and has the advantages of high transfection efficiency and cheap transfection reagent.Further, the present application establishes a method for bovine parainfluenza virus type 3 infectious clone rescued virus based on the T7-293T cell, constructs three genes of NP, P and L of bovine parainfluenza virus type 3 into the same helper plasmid, and co-transfects the T7-293T cell with the full-length gene plasmid of the virus, so as to avoid the low efficiency of co-transfecting four plasmids, and further improve the success rate of bovine parainfluenza virus type 3 infectious clone rescued virus.

The invention relates to a gene delivery system for cytoplasm specific expression of PPM1K as well as a preparation method and application of the gene delivery system, and belongs to the technical field of preparation of gene delivery systems. The gene delivery system is preferably an adeno-associated virus (AAV), and the preparation method of the gene delivery system specifically comprises the following steps: inserting a PPM1K gene or a functional variant thereof into a vector, and adding a detectable label at a C terminal to form a recombinant plasmid; transforming the recombinant plasmids into competent escherichia coli, amplifying, and screening and extracting plasmids to obtain recombinant AAV plasmids; co-transfecting the recombinant AAV plasmid, the helper plasmid and the capsid plasmid into packaging cells by using a transfection reagent, collecting the cells and culturing supernate after transfection, and adding a freeze thawing buffer solution for repeated freeze thawing to obtain a cell lysis solution; centrifuging the cell lysis solution, taking supernate, purifying and filtering. According to the invention, the specific expression of the cell compartment of the therapeutic gene is realized through the vector design, and the obvious therapeutic effect and the safety advantage are shown.

The application discloses a gene editing system for constructing a muscle weakness disease model pig nuclear transfer donor cell with a DOK7 gene mutation and application thereof. The application provides application of DOK7-gRNA2 shown in SEQ ID NO: 16, DOK7-gRNA4 shown in SEQ ID NO: 17 and NCN protein in preparation of a kit. The application further provides a method for preparing a recombinant cell, comprising the following steps: co-transfecting a pig cell with DOK7-gRNA2, DOK7-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with a mutated DOK7 gene. The use of any of the above kits is to prepare a recombinant cell, to prepare a muscle weakness model pig, to prepare a muscle weakness cell model, a muscle weakness tissue model or a muscle weakness organ model. The application has great application value for research and development of muscle weakness disease drugs and revealing the pathogenesis of the disease.

The present application relates to a porcine reproductive and respiratory syndrome virus infectious clone construction and rescue method, and belongs to the technical field of bioengineering. The porcine reproductive and respiratory syndrome virus infectious clone contains a full genome sequence of the porcine reproductive and respiratory syndrome virus, and the nucleotide sequence of the full genome sequence is the 26th to 15384th nucleotide of SEQ ID NO: 1 or a fragment having more than 80% identity with SEQ ID NO: 1 and having the same function. The present application also provides a plasmid pCAGGS-HA-CD163 expressing a porcine CD163 gene. By co-transfecting the aforementioned two plasmids into HEK-293T cells, the virus is rescued in vitro, a first porcine reproductive and respiratory syndrome virus infectious clone rescue platform based on HEK-293T cells is successfully built, and the porcine reproductive and respiratory syndrome virus is successfully rescued.

This invention involves co-transfecting cells with an AAV vector plasmid carrying the target gene, a packaging plasmid providing Rep / Cap proteins, and a helper plasmid providing adenovirus assistance. The transfected cells are cultured under suitable conditions to produce viral particles containing recombinant AAV. Cells and culture supernatant are collected, cells are lysed to release the virus, and the viral particles are purified by centrifugation. A membrane-penetrating peptides or their derivatives are then modified onto the AAV capsid protein. Results show that the gene editing system combined with membrane-penetrating peptides or their derivatives enhances editing efficiency, effectively penetrates ocular surface tissues, and inhibits ocular neovascularization.

The invention discloses a chimeric antigen receptor exosome targeting a CD8T cell, a CAR-T cell and a construction method and application of the chimeric antigen receptor exosome, and relates to the technical field of biomedicine, and the construction method comprises the following steps: co-transfecting three plasmids, namely BRD-PTK-CD8 < + >-Lamp2b, pcDNA3.1-CD63-L7Ae and pcDNA3.1-N6 (SP)-Nb60-T20-C / D box, into a 293F cell, incubating, and purifying to obtain the exosome capable of targeting the CD8T cell. The exosome can deliver CAR-mRNA to T cells and convert CD8 + cells into CAR-T cells, and blood of an infected person does not need to be drawn; cAR molecules aiming at in-vivo malignant cells can be prepared in vivo; chemotherapy and radiotherapy are not needed, and side effects are avoided; and universality is achieved.

The invention discloses a homologous recombination system, a transposition system, a fruit fly animal gene high-throughput screening method and application. The screening method comprises the following steps: firstly, separating and establishing a stable cell line from a fruit fly animal; then co-transfecting a CRISPR / Cas9 mediated homologous recombination system, and obtaining a cell line for stably expressing Cas9 protein through puromycin screening and a limited dilution method; the sgRNAs plasmid library constructed by combining a PiggyBac transposon system can realize efficient screening and functional analysis of a target gene in the Cas9 cell line. The high-throughput screening method provided by the invention is widely applicable to gene function research and target gene screening, is simple in preparation process and convenient to operate, has good universality, and provides effective technical support and solution for related fields.

The invention discloses establishment and application of a flavivirus replication-dependent RNA (Ribonucleic Acid) recombination experiment system, and belongs to the technical field of biology. The RNA recombination system comprises two components: a tembusu virus E gene deleted subgenome replicon CQW1-delta E, and a defective virus genome CQW1-delta NS1 or MM1775-delta NS1 with a part of NS1 gene sequence deleted. The two components cannot generate progeny viruses independently, but after the two components are co-transfected, a large number of recombinant progeny viruses can be generated within several days after transfection due to RNA recombination, the recombination efficiency is extremely high, continuous passage is not needed, the construction period is greatly shortened, and the production cost is saved; meanwhile, the generated progeny virus can form plaques on cells, and by means of plaque counting, the method can be applied to quantitative measurement of the RNA recombination efficiency and analysis of key factors influencing the tembusu virus RNA recombination.

The invention discloses a pearl gentian grouper IgM monoclonal antibody, which is characterized in that screened pearl gentian grouper hybridoma No. 41 is sequenced to obtain nucleotide and amino acid sequences of variable regions of a heavy chain and a light chain of the targeted pearl gentian grouper IgM monoclonal antibody respectively shown as SEQ ID NO.1-12, the heavy chain subtype of the monoclonal antibody is IgG type, the light chain is kappa type, and the light chain subtype of the monoclonal antibody is kappa type. Through codon optimization, recombinant pATX1 vectors of heavy-chain and light-chain antibodies are respectively constructed with a constant region sequence of a murine antibody. The Epinephelus lanceolatus IgM monoclonal antibody is expressed through co-transfection XtenCHO cell in-vitro culture, the chimeric immune globulin is good in reaction characteristic and high in titer (512000 or above) and is easier to store compared with a hybridoma cell mode, and the risk of losing the specific antibody is avoided.

The invention discloses an application of Vietnam ginseng saponin R18. According to the invention, a Ca < 2 + > biosensor is utilized to co-transfect Hela cells with MRGPRX4, the activation effect of Vietnam ginsenoside R18 on MRGPRX4 is detected, and the Vietnam ginsenoside R18 is confirmed to be an agonist of MRGRPX4. The Vietnam ginsenoside R18 acts on a mouse through subcutaneous injection to cause itching of the mouse, so that the Vietnam ginsenoside R18 can be used as a tool medicine for studying itching and is used for screening a G protein coupled receptor antagonist with an itching relieving effect or preparing an itching causing model; the traditional Chinese medicine quality is controlled, and the itching side effect of the traditional Chinese medicine is prevented.

The invention discloses a cartilage cell targeting recombinant adeno-associated virus and a preparation method thereof, and belongs to the technical field of gene engineering. The recombinant adeno-associated virus is obtained by co-transfecting an HEK293 cell by using a recombinant plasmid pSSCMV-Cl2a1-SOX9 and a helper plasmid; and the recombinant plasmid pSSCMV-Cl2a1-SOX9 is obtained by inserting a fusion gene of Col2a1 and SOX9 into a pSSHG-CMV carrier. The preparation method comprises the following steps: step 1, constructing a recombinant plasmid pSSCMV-Col2a1-SOX9, and constructing the recombinant plasmid pSSCMV-Col2a1-SOX9; step 2, culturing the HEK293 cells, and waiting for transfection; and 3, co-transfecting the recombinant plasmid pSSCMV-Cl2a1-SOX9 obtained in the step 1, a pHelper plasmid and a pAAV-RC plasmid into the HEK293 cell cultured in the step 2, culturing, collecting a cell lysis solution, and carrying out iodixanol gradient centrifugation combined with Heparin chromatographic purification to obtain the recombinant adeno-associated virus. The recombinant adeno-associated virus obtained by the invention has targeting property and higher expression level.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for cataract disease models with MIP gene mutations and its applications. This invention provides the application of MIP-gRNA2 (SEQ ID NO: 18), MIP-gRNA3 (SEQ ID NO: 19), and NCN protein in a preparation kit. This invention also provides a method for preparing recombinant cells, comprising the following steps: co-transfecting porcine cells with MIP-gRNA2, MIP-gRNA3, and NCN protein to obtain recombinant cells. The recombinant cells are recombinant cells with a mutated MIP gene. The kits described above are used for: preparing recombinant cells; preparing porcine cataract models; preparing cataract cell models, cataract tissue models, or cataract organ models. This invention has significant application value for the development of cataract drugs and for elucidating the pathogenesis of this disease.

The application discloses a kind of establishment and application of flavivirus replication-dependent RNA recombination experimental system, belong to the field of biotechnology.The RNA recombination system includes two components: tanbusu virus E gene deletion subgenomic replicon CQW1-ΔE, and the defective virus genome CQW1-ΔNS1 or MM1775-ΔNS1 missing partial NS1 gene sequence.The two components cannot produce progeny virus alone, but after co-transfection, due to RNA recombination, a large number of recombinant progeny viruses can be produced within a few days after transfection, the recombination efficiency is extremely high, and continuous passage is not required, greatly shortening the construction cycle and saving production costs;At the same time, the progeny virus can form plaques on cells, and the efficiency of RNA recombination can be quantitatively measured by plaque counting, and the key factors affecting tanbusu virus RNA recombination can be analyzed.

The invention discloses an oryzias latipe spermatogonial stem cell strain capable of stably expressing Cas9 protein as well as a construction method and application of the oryzias latipe spermatogonial stem cell strain. The oryzias latipe spermatogonial stem cell strain is designed and constructed based on a Tol2 transposon system, wherein the transposon vector comprises a coding sequence of a Cas9 gene, a hygromycin B resistance gene and left and right arm sequences of a Tol2 transposon; constructing a Tol2 transposase expression vector; co-transfecting the transposon vector and the expression vector to the spermatogonial stem cells SG3 of the oryzias latipe; screening in an ESM4 culture medium containing hygromycin B to obtain a drug-resistant cell population capable of stably expressing Cas9; selecting monoclonal colonies under a microscope, and carrying out continuous subculture to obtain an oryzias latipe spermatogonial stem cell strain which stably expresses Cas9 protein and is named as oryzias latipe gonad cells SG3-Cas9. The method has the advantages of short construction period, high efficiency, good expression stability and the like, and provides a reliable cell platform for fish cell level gene editing research.

The application provides sgRNA which specifically targets the Rosa26 safe site of a cashmere goat and application of using CRISPR / Cas9 to complete site-directed knockout and site-directed integration of an EGFP gene. The application firstly uses a bioinformatics method to predict the complete sequence of the Rosa26 site, then designs two sgRNA aiming at the site, constructs a targeting vector based on the CRISPR / Cas9 system, verifies the guiding efficiency of the sgRNA, simultaneously constructs a homologous integration EGFP gene vector, co-transfects the sgRNA and the homologous integration vector into cashmere goat fetal fibroblasts, and obtains a cell strain of the Rosa26 site site-directed integration of the EGFP gene. The efficiency of the sgRNA of the application in specifically guiding Cas9 to cut the Rosa26 site reaches about 40%, effectively reduces the off-target phenomenon existing in the CRISPR / Cas9 system, and further reduces the mutation of non-target gene sequences caused by non-specific cutting. The Cas9 / gRNA expression vector can realize the specific knockout or knock-in of the Rosa26 site at the cell, embryo or even individual level, so as to study the expression of specific genes and provide technical support for the cultivation of new goat breeds.

The invention discloses an AQP4-IgG (immunoglobulin G) detection method based on the combination of AQP4-M1 and M23 subtypes. According to the invention, two protein subtypes of AQP4-M1 and M23 are mixed in proportion by simulating physiological conditions and co-transfected to 293T cells, so that heterotetramers are formed and further assembled into a complete orthogonal array (OAP), and more antigen epitopes are exposed, thereby remarkably improving the recognition specificity and detection sensitivity of the AQP4-IgG antibody. Compared with traditional single subtype detection, the system can more truly simulate abundance and distribution of the AQP4 subtype in vivo, reduces false positive and false negative risks, and has a great value of rapidly and accurately screening AQP4-IgG related diseases clinically.

The present invention discloses a RAW 264.7 cell line stably overexpressing SRSF3, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. The present invention constructs a Lenti-Flag-hyg-SRSF3 plasmid overexpressing SRSF3, which forms a three-plasmid expression system with the psPAX2 and pMD2.G packaging vectors, co-transfects 293T cells, collects lentivirus, and then transfects RAW 264.7 macrophages with the lentivirus to obtain a stable cell line overexpressing the SRSF3 gene. The stable cell line overexpressing the SRSF3 gene is detected with a fluorescent antibody against Flag, and an LPS-induced inflammation model is used. Through Western Blot and qPCR detection, the stable cell line overexpressing SRSF3 can inhibit autophagy and reduce the expression level of inflammatory factors, and has the development and application prospects of inhibiting inflammation.

This invention discloses a method for constructing an immune animal model for preparing biofusion enzyme antibodies and its application. The method includes the following steps: designing and screening sgRNAs with high cleavage efficiency based on signal protein genes, and constructing an sgRNA-Cas9 expression vector; linearizing the plasmid by double enzyme digestion, and then ligating it with a signal protein gene fragment containing left and right homologous arms to obtain the Donor plasmid; co-transfecting the sgRNA-Cas9 expression vector and the Donor plasmid into target animal somatic cells, and screening to obtain positive somatic cells that stably integrate the target gene; using the positive somatic cells as nuclear donors for nuclear transfer to construct recombinant embryos, and transferring the recombinant embryos into recipient female animals; after delivery, identifying transgenic animal individuals carrying biofusion enzyme antibodies by genomic PCR. Based on this transgenic animal model, different target antibodies with clinical value can be developed.