107 results about "Co transfection" patented technology

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 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 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 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 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 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 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.

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.

The application discloses a kit for constructing an ATM gene mutation ataxia-telangiectasia model pig nuclear transfer donor cell. The application provides a kit comprising ATM-gRNA1 shown in SEQ ID NO: 16, ATM-gRNA4 shown in SEQ ID NO: 17 and NCN protein. The application also provides a method for preparing a recombinant cell: co-transfecting a pig cell with ATM-gRNA1, ATM-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with ATM gene mutation. The kit is used for: preparing a recombinant cell; preparing an ataxia-telangiectasia model pig; preparing an ataxia-telangiectasia cell model or an ataxia-telangiectasia tissue model or an ataxia-telangiectasia organ model. The application has great application value for research and development of ataxia-telangiectasia drugs and revealing the pathogenesis of the disease.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for ALS models with SOD1 gene mutations and its applications. This invention also provides a method for preparing recombinant cells: SOD1- g RNA1 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 18), SOD1- g RNA6 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 19), SOD1-mutant-ss160 (shown in SEQ ID NO: 20), and NCN protein (Cas9 protein or a fusion protein containing Cas9 protein) were co-transfected into porcine cells to obtain recombinant cells. This invention utilizes CRISPR / Cas9 technology combined with ssODN homologous recombination technology to perform point mutation gene editing of the SOD1 gene, mimicking the natural pathogenesis and genetic characteristics of ALS, and obtaining single-cell clones with precise point mutations in the SOD1 gene. This lays the foundation for later breeding of ALS disease model pigs using somatic cell nuclear transfer animal cloning technology.

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 application provides a CD45 recombinant antibody, which belongs to the technical field of recombinant antibodies, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ_1, the amino acid sequence of the light chain variable region is shown in SEQ_2, the nucleotide sequence of the coding gene of the heavy chain variable region is shown in SEQ_3, and the nucleotide sequence of the coding gene of the light chain variable region is shown in SEQ_4; the recombinant antibody is obtained by connecting the light chain and the heavy chain to expression vectors respectively and then co-transfecting host cells; the application can produce more stable antibodies and overcome the problems caused by non-specificity (unreliability) and batch difference of traditional antibodies.

The application discloses a kind of targeted exosome chimeric antigen receptor molecules for treating HIV infection and its application, belong to the field of biological medicine technology.The application is packaged into targeted exosome by coding anti-HIV CAR mRNA directionally.The nano antibody sequence of targeted CD3, CD4, CD8+T receptor on T cell is fused to the N-terminal of LAMP-2B to construct specific targeting plasmid, so that the targeted peptide is expressed on the surface of exosome membrane, the expression plasmid containing RNA binding protein L7A and exosome tag protein CD63 is constructed, and the exosome loaded with CAR mRNA and targeted to T cell is obtained by three-plasmid co-transfection of HEK293F cells containing C / D box CAR plasmid.The exosome can construct human CAR-T cell when incubated with PBMC in vitro or back into humanized mouse in vivo, and has significant killing virus infected cell activity.

The invention relates to a Rosa26 site-targeted Vero cell line as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The invention provides a Vero cell line targeting a Rosa26 site. The Vero cell line comprises an attP sequence integrated into the Rosa26 site of a genome of the Vero cell line. Researches show that by co-transfecting donor plasmids expressing exogenous genes and attB sites and Bxb1 expression plasmids to the Vero cell line, the exogenous genes can be rapidly integrated to a genome of the Vero cell line, and then the Vero stably transfected cell line capable of stably expressing the exogenous genes for a long time is constructed. According to the Vero cell line provided by the invention, the construction time of the Vero stable transfection cell line for expressing an exogenous gene is greatly shortened, and based on insertion of the Rosa26 site, the Vero cell line provided by the invention can maintain the stability and consistency of a genome for a long time, so that long-term stable expression of the exogenous gene is ensured.

The invention provides a method for rescuing a novel measles recombinant coronavirus. The pGEM3Zf (+)-T7RNAP plasmid constructed by the invention can be subjected to in-vitro reverse transcription processing to obtain T7RNA polymerase mRNA (messenger Ribonucleic Acid). The method comprises the following steps: by taking an MRC-5 cell as a cell matrix, co-transfecting the MRC-5 cell by using a pT7-MVEZ-COV2S.B.1.617.2 plasmid constructed by the invention and a helper plasmid in a physical electric shock manner, and simultaneously transfecting T7RNA polymerase mRNA (messenger ribonucleic acid). T7RNA polymerase mRNA translates and expresses T7RNA polymerase in an MRC-5 cell, the T7RNA polymerase further drives a T7 promoter, transcription of a full-length plasmid containing all genetic information of the virus and expression of N, P and L genes are started, virus packaging and proliferation are completed in a host cell, and the paramyxovirus with infection activity is obtained.

The invention belongs to the technical field of antibody detection, and particularly relates to a multi-subtype antigen co-transfected cell immunofluorescence staining method and application. The method is established for the purpose of a CBA detection method, the basic principle is similar to that of an overexpression antigen cell immunofluorescence method for existing anti-GFAP-IgG antibody detection, and the difference is that three GFAP subtypes are adopted for cotransfection design, antigen subtypes are covered, and the problem of leak detection is avoided. According to the invention, the high-quality expression of the antigen is realized by self-constructing the plasmid based on the pCDH lentiviral vector. According to the invention, by optimizing the schemes of fixation, transparent preservation and the like after cell transfection culture, the dyeing performance is enhanced, and the preservation effect is improved.

The invention relates to the field of biomedical research, and provides a lung cancer cell strain (H1299) capable of stably and low-expressing Integrin alpha6 protein and a construction method of the lung cancer cell strain (H1299). The preparation method comprises the following steps: designing and synthesizing an shRNA (short hairpin Ribonucleic Acid) sequence for specifically knocking down gene expression aiming at a human Integrin alpha6 gene, forming a double-chain fragment through annealing, and inserting the shRNA sequence into a pLKO.1-puro lentiviral vector by adopting a connection independent cloning (LIC) method, so as to construct a pLKO.1-ITG alpha6-shRNA recombinant plasmid. Furthermore, the recombinant plasmid and lentivirus packaging plasmids psPAX2 and pVSVG are co-transfected to an HEK293T cell by utilizing a lentivirus packaging system, and the lentivirus is produced by packaging. And infecting a target lung cancer cell strain H1299 by using the obtained lentivirus, and then carrying out puromycin resistance screening and continuous subculture to finally obtain the stable and continuous low-expression Integrin alpha6 protein H1299 cell strain. A fluorescence microscope, a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) and an immunoblotting technology are used for verifying that the gene silencing efficiency of the Integrin alpha6 in the cell strain can reach more than 80%. The stable cell strain constructed by the invention provides a reliable and efficient tool cell model for researching the molecular mechanism of the Integrin alpha6 in the processes of occurrence and development, invasion and metastasis, energy metabolism, immune escape and the like of lung cancer, and also provides a new experimental material for screening antitumor drugs targeting the Integrin alpha6.

The present invention provides heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved. Interface residues were mutated such that each light chain strongly favoured its cognate heavy chain when two different heavy chains and two different light chains were co-transfected and co-expressed in the same cell to assemble a functional, heterodimeric antibody or fragment thereof.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

The present application relates to a kind of cytoplasm-specific expression PPM1K gene delivery system and its preparation method and application, belong to gene delivery system preparation technical field.The gene delivery system is preferably adeno-associated virus (AAV), and its preparation method specifically includes the following steps: PPM1K gene or its functional variant is inserted into vector, detectable label is added at C-terminal, and recombination plasmid is formed;Recombinant plasmid is transformed into competent e. coli and is amplified, after plasmid is screened, recombination AAV plasmid is obtained;Recombinant AAV plasmid, auxiliary plasmid and capsid plasmid are co-transfected into packaging cell using transfection reagent, after transfection, cell and culture supernatant are collected, freeze-thaw buffer is added repeatedly freeze-thaw, and cell lysate is obtained;After centrifugation of cell lysate, supernatant is taken, and purified filtration is carried out.The present application realizes the cell compartment specificity expression of treatment gene by vector design, and shows significant treatment effect and safety advantage.