215 results about "Co transfection" patented technology

The invention discloses construction and application of a targeted exosome chimeric antigen receptor molecule for treating HIV (human immunodeficiency virus) infection, and belongs to the technical field of biological medicines. According to the invention, the mRNA for coding the anti-HIV CAR is directionally packaged into the targeting exosome. Nanometer antibody sequences of CD3, CD4 and CD8 < + > T receptors on targeted T cells are fused to the N terminal of LAMP-2B to construct a specific targeting plasmid, so that targeting peptides are expressed on the surface of an exosome membrane, expression plasmids containing RNA binding protein L7A and exosome tag protein CD63 are constructed, and HEK293F cells are co-transfected by the expression plasmids and CAR plasmids containing C / D box to obtain the exosome loaded with CAR mRNA and targeted to the T cells. When the exosome is incubated with PBMC (peripheral blood mononuclear cells) in vitro or is transfused back into a humanized mouse body, human CAR-T cells can be constructed, and the exosome has remarkable activity of killing virus infected cells.

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 belongs to the field of tumor drug research, and particularly relates to a cell vesicle co-delivery system, an anti-tumor drug and application. The cell vesicle-like co-delivery system is constructed according to the following steps: co-transfecting a recombinant expression vector of tumor targeting protein or targeting peptide and a recombinant plasmid of circRNA of effector molecules related to pyroptosis to a tool cell, and collecting to obtain the cell vesicle-like co-delivery system, a recombinant expression vector of the tumor targeting protein or the targeting peptide contains a DNA sequence for coding MCP protein, and a recombinant plasmid of circRNA of an effector molecule related to pyroptosis contains a DNA sequence for coding MS2 protein. According to the cell vesicle co-delivery system provided by the invention, tumor cell death is directly induced by delivering the cell death related effector molecule RNA through the cell vesicles, the killing efficiency is high, the side effect is relatively small, and the problems of mass production and drug loading can be solved.

The invention provides a virus RNAi inhibitor screening vector as well as a construction method and application thereof, and belongs to the technical field of viruses. The virus RNAi suppressor screening vector comprises a green fluorescent protein coding gene expression cassette and a shRNA expression cassette targeting a green fluorescent protein coding gene, the nucleotide sequence of the shRNA is as shown in SEQ ID NO: 2 or SEQ ID NO: 3. According to the invention, the green fluorescent protein coding gene and the shRNA are cloned into the same expression vector, so that the consistency of transfection efficiency is ensured. The invention further provides a screening method of the virus RNAi suppressor, the virus RNAi suppressor screening vector and the recombinant vector for expressing the virus RNAi suppressor to be screened are co-transfected into cells, and the screening result is judged according to the green fluorescence intensity. According to the technical scheme, operation is easy and convenient, the screening result is accurate and reliable, and wide application and popularization value is achieved.

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 method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase report system, and relates to the technical field of biological medicine. The method comprises the following steps: constructing an insect dsRNA degrading enzyme into a pAc5.1 / V5-His B plasmid to form a recombinant plasmid, co-transfecting the recombinant plasmid and a psiCHECKTM-2 plasmid into an insect cell for culturing, then respectively adding dsLuc and dsGFP into the cell, continuing culturing, finally collecting the cell, and detecting the enzyme activity of the cell. The system is constructed based on a dual-luciferase report system, and qualitative and quantitative enzyme activity is realized through three steps of cell culture, transfection and detection. Compared with in-vitro heterologous protein expression, agarose detection after in-vitro degradation reaction or RNA extraction and then fluorescent quantitative PCR (RT-qPCR) detection, the method provided by the invention is faster and more accurate, and is not limited to the first step in which the protein is difficult to obtain.

The invention relates to an oncolytic virus VSV-aPDL1 / aCD137 for expressing a bispecific antibody as well as a construction method and application of the oncolytic virus VSV-aPDL1 / aCD137. The preparation method comprises the following steps: firstly designing aPDL1 / aCD137, then synthesizing an aPDL1 / aCD137 gene in vitro, amplifying the aPDL1 / aCD137 gene, connecting the aPDL1 / aCD137 gene to a pT7-VSV whole genome plasmid to obtain a virus whole genome plasmid pT7-VSV-aPDL1 / aCD137, and then co-transfecting the plasmid and a helper plasmid to a 293T cell to obtain the VSV-aPDL1 / aCD137 oncolytic virus. According to the present invention, the local anti-tumor immune response of the tumor is activated through the virus, the infiltration of the lymphocyte to the tumor tissue is recruited, the anti-PD-L1 / anti-CD137 bispecific antibody is expressed, the inhibition of the PD-1 / PD-L1 pathway on the T cell is blocked, and the inhibition signal of the PD-1 / PD-L1 pathway on the tumor cell surface is reversed into the CD137 signal of the activated T cell, such that the functional failure of the T cell caused by the PD-1 / PD-L1 pathway can be blocked, and the anti-tumor immune response of the tumor can be inhibited; the function and viability of T cells can be further improved, long-term immune surveillance on tumors is realized, and long-acting anti-tumor immune response is generated. Experiments show that the oncolytic virus can significantly prolong the lifetime of tumor-bearing mice, and a new strategy is provided for development of solid tumor drugs.

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 belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a mouse recombinant monoclonal antibody for specifically recognizing African horse pestivirus VP7 protein. According to the invention, a hybridoma cell strain 2C12 for African horse pestivirus VP7 protein is obtained through screening by using a hybridoma cell technology, total RNA is extracted and is subjected to reverse transcription to form cDNA, the cDNA is used as a template and is amplified by using a nested PCR method to obtain light chain and heavy chain variable region sequences of mouse IgG antibody molecules, the light chain and heavy chain variable region sequences are constructed to a plasmid vector containing a mouse source constant region, and the mouse IgG antibody is obtained. And co-transfecting 293F suspension cells for expression and purification to obtain the murine recombinant monoclonal antibody for specifically recognizing the African horse pestivirus VP7 protein. The recombinant antibody can specifically recognize the recombinant VP7 protein of the African horse plague virus, but does not recognize the recombinant VP7 protein of bluetongue virus and epidemic hemorrhagic fever virus, so that support is provided for further establishing a specific African horse plague diagnosis technology.

The invention relates to a construction method and application of a recombinant oncolytic virus VSV-sPD1IL15RaIL15. The recombinant oncolytic virus VSV-sPD1IL15RaIL15 is constructed by using homologous recombination, and the recombinant oncolytic virus VSV-sPD1IL15RaIL15 can be used for preparing the recombinant oncolytic virus. The preparation method comprises the following steps: firstly designing sPD1 and IL15RaIL15, then fusing the sPD1 and the IL15RaIL15, synthesizing a sPD1IL15RaIL15 gene, amplifying the sPD1IL15RaIL15 gene, inserting the sPD1IL15RaIL15 gene into an ATU region of a VSV whole genome cDNA cloning plasmid pT7-VSV by using a homologous recombination method to obtain a virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15, and co-transfecting the virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15 and an auxiliary plasmid to a 293T cell to prepare the VSV-sPD1IL15RaIL15 oncolytic virus. According to the oncolytic virus, specific anti-tumor immunity is activated, and sPD1IL15RaIL15 is locally expressed in a tumor, so that a long-acting anti-tumor immune response is generated. When the oncolytic virus is used for treating solid tumors, the functions of tumor specific immune cells in a tumor microenvironment can be improved, infiltration of CD8 + T cells and NK cells can be promoted, and the lifetime of tumor-bearing mice can be remarkably prolonged.

The invention discloses a preparation system of RAB5A-induced targeted recombinant protein exosomes and application of the preparation system in delivery of recombinant biological drugs. The preparation method comprises the following steps: constructing pIRES-RAB5A-Insulin and pKEX2-alpha-Rt-hst3 plasmids, carrying out co-transfection on cells, and carrying out extraction, so as to obtain the target type exosome. The vesicle gene RAB5A can enable insulin to be expressed on an exosome membrane when early exosome vesicles are formed, the insulin can target insulin receptor cells, KEX2-alpha can be combined with the RAB5A to enable Rt-hst3 to be wrapped into the early exosome vesicles, and the insulin receptor targeted exosome wrapping hst3 protein is formed. Further research finds that the exosome can obviously improve the mitochondrial function. Therefore, the hst3-insulin exosome constructed by the invention realizes targeted delivery of recombinant drugs, and also provides a new way for treatment of diseases related to mitochondrial dysfunction.

The present invention relates to the field of drug delivery, and in particular to an extracellular vesicle (EV) delivery platform loaded with a target protein or polypeptide and a use thereof. The present invention provides a plasmid combination for preparing an EV loaded with a target protein or polypeptide, wherein the plasmid combination comprises a pCAG-GOI plasmid and a pMD2.G plasmid, and the GOI refers to a gene sequence corresponding to the target protein or polypeptide. According to the EV delivery platform loaded with the protein or polypeptide provided by the present invention, target proteins or polypeptides can be efficiently loaded into EVs by applying double-plasmid co-transfection cells, the use of EVs as carriers to deliver target substances such as proteins or the polypeptides to exert therapeutic effects into clinical practice is further promoted, and by means of the good structural stability, the innate advantages of transporting biologically active substances and excellent biocompatibility of the EVs, the systemic toxic and side effects caused by conventional delivery carriers are reduced.

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 relates to sgRNAs for demethylation editing of an fgf2 gene of oryzias melastigma, an editing system and application, and belongs to the technical field of gene editing, and the sgRNAs are sgRNA1 + sgRNA2 + sgRNA3 or sgRNA2 + sgRNA3 + sgRNA4 + sgRNA5; the system comprises a Fuw-UbC-dCas9-tet1 plasmid and a pGL3-U6-sgRNAs plasmid, and the sequence of a conserved structural domain of a tet1 gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). Through co-transfection of Mix1 or Mix2, the DNA methylation level of the fgf2 gene promoter can be effectively reduced by about 20.41% at most, the off-target efficiency is extremely low, the cell growth speed is remarkably increased, and stable inheritance is achieved.

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.

To provide recombinant herpes simplex virus-2 (HSV-2) vaccine vectors and compositions and vaccines containing them, and methods of use thereof.SOLUTION: Disclosed is a process for producing a vaccine vector targeting a heterologous antigen, the process comprising the steps of: providing a HSV-2 genome nucleic acid comprising: (i) a gene encoding an intact or partially deleted HSV-2 glycoprotein D and (ii) a promoter-FP construct; b) co-transfecting a host cell with: (i) the HSV-2 genome and (ii) a linear DNA encoding the heterologous antigen under conditions where allelic recombination will occur between the HSV-2 genome of a) and the DNA fragment; c) screening plaques resulting from b) and identifying plaques not showing fluorescence under excitation light which elicits fluorescent protein fluorescence; and d) recovering a recombinant HSV-2 virus or virion from plaques not showing fluorescence in c).SELECTED DRAWING: Figure 2

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.

The invention discloses a mini-chromosome state characterization cell model for drug screening. The mini-chromosome state characterization cell model is a transfected cell obtained by co-transfecting a hepatocyte line with prcccDNA-HiBiT-HBx and a plasmid Cre; the invention also discloses an application of the compound C20H14F3N3S or the pharmaceutically acceptable salt thereof in preparation of an HBV (hepatitis B virus) inhibitor or a medicine for treating hepatitis B virus infectious diseases or symptoms. According to the invention, a novel anti-hepatitis B virus small molecule compound is screened by utilizing a constructed cell model, and experiments prove that the novel anti-hepatitis B virus small molecule compound can obviously reduce the expression levels of HBV total RNAs, HBeAg, HBV pgRNA and HBV nucleoprotein.

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.