24 results about "Plasmid transfection" patented technology

The application belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus recombinant strain, a construction method and application. The S gene sequence of the existing epidemic porcine epidemic diarrhea virus PEDV CH / SX / 2016 strain is replaced by the S2015 gene sequence shown in SEQ ID NO. 1 to construct a PEDV CH / SX / 2016 strain infectious clone. A porcine epidemic diarrhea virus recombinant strain rCH / SX / 2016-S 2015 is successfully rescued by transfecting cells with a plasmid containing the infectious cDNA shown in SEQ ID NO. 4. The problem that the original strain cannot be successfully rescued by reverse genetics engineering is solved, and the recombinant virus strain rCH / SX / 2016-S 2015 has an activity comparable to that of the original strain, and can be used for preparing a PEDV diagnostic reagent and / or vaccine.

The invention relates to the related technical field of molecular biology and cell engineering, in particular to an FLP-IN-PIEC cell line and a construction method and application thereof, and the construction method comprises the following steps: step 1, constructing CRISPR / Cas9 site-specific cleavage plasmid containing a pig HIPP11 safety site sequence; step 2, constructing a donor plasmid Donor-FRT containing a homologous arm of a safety site of the HIPP11; 3, jointly transfecting plasmids in the step 1 and the step 2 into a porcine hip artery endothelial cell line (PIEC) to obtain an FLP-IN-PIEC cell line for expressing red fluorescence; and 4, under the action of Flp recombinase, transfecting an exogenous gene donor plasmid containing an FRT site to the FLP-IN-PIEC cell line obtained in the step 3, and driving the expression of a target gene through an optimized strong promoter to realize fixed-point transfer and efficient expression of the exogenous gene in the pig (PIEC) cell line. Different from an existing Flp-In system depending on a random integration strategy, the construction method disclosed by the invention emphasizes fixed-point integration and promoter optimization, and improves the homogeneity, stability and screening efficiency of exogenous gene expression.

The invention discloses a blocking agent for cell immunofluorescence detection by a slide method and a use method of the blocking agent. The blocking agent is a homologous no-load cell lysis solution without transfection plasmids, and the protein concentration is greater than or equal to 30mg / mL. The blocking agent is completely the same as cell cytoplasm and cell membrane components of cells to be detected except for plasmids-expressed antigens, when in use, the blocking agent is pre-mixed with a blood cleaning sample in advance, interfering antibodies except target antibodies in serum can be purified, and meanwhile, due to the fact that no antigen protein expressed after plasmid transfection exists, the blocking agent can be used for detecting the cells to be detected. And the protein concentration is equivalent to the globulin concentration in serum, so that the target antibody cannot be lost in the serum, and the slide method CBA detection sensitivity can be greatly improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to an agrobacterium rhizogenes-mediated coastal chrysanthemum indicum tissue culture-free genetic transformation method and application thereof in enhancing the salt tolerance of a root system. The agrobacterium rhizogenes-mediated coastal chrysanthemum indicum tissue culture-free genetic transformation method comprises the following steps: transfecting agrobacterium rhizogenes with plasmids containing to-be-transformed gene expression cassettes to obtain recombinant agrobacterium rhizogenes, transfecting stems with terminal buds of coastal chrysanthemum indicum with the recombinant agrobacterium rhizogenes, and then cutting. The method provided by the invention is short in required period, and the transgenic plant can be obtained only in 30-60 days. The problems of long chrysanthemum conversion rate period, complex operation and tedious process are solved. A tissue culture mode is not needed, so that the tedious process of tissue culture is reduced; local materials can be used according to the characteristic of cutting rooting of chrysanthemum, chrysanthemum seedlings do not need to be cultivated from seeds, and time cost is reduced.

The invention discloses an FRET (Fluorescence Resonance Energy Transfer) biosensor as well as a preparation method and application thereof. The FRET biosensor is obtained by transfecting cells with plasmids for expressing a molecular sensor; the molecular sensor contains NEK7 protein, a fluorescence donor and / or a fluorescence receptor, and the fluorescence donor or the fluorescence receptor is connected to the N end or the C end of the NEK7 protein through a flexible chain. According to the invention, the FRET biosensor is designed, and multiple conditions are optimized, so that the signal response efficiency of the FRET biosensor is improved, and meanwhile, real-time monitoring of NLRP3 oligomerized living cells is realized, and screening of an NEK7-NLRP3 protein interaction interference inhibitor is realized.

The invention relates to the technical field of medicine and biology, and particularly discloses a stress-inducible virus and application thereof.The stress-inducible virus comprises a stress-inducible adenovirus or a stress-inducible lentivirus, the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 1, and the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 2. The preparation method comprises the following steps: constructing a virus vector, wherein the virus vector has a nucleotide sequence as shown in SEQ ID NO: 1; and virus packaging: transfecting HEK293T cells by adopting shuttle plasmids containing SEQ ID NO: 1 and skeleton plasmids containing viral genome sequences, and collecting a virus stock solution to obtain the stress induced virus. The invention also discloses application of the stress-induced virus in preparation of medicines for preventing and treating oral tumors. Controllable release of the foreign protein under a specific signal is realized. The therapeutic protein produced by the invention can maximize the curative effect of drugs such as CD47 targeted protein and the like, and meanwhile, the toxic and side effects of the drugs on other tissues of the whole body are reduced.

The invention discloses a high-affinity anti-carbendazim monoclonal antibody which comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, and the amino acid sequence of a coding gene of the heavy chain variable region is shown as SEQ ID NO: 2; the amino acid sequence of the light chain variable region coding gene is as shown in SEQ ID NO: 4. The invention also discloses a heavy chain expression plasmid of the anti-carbendazim full-length IgG recombinant antibody. The heavy-chain and light-chain variable region gene sequences of the antibody obtained by the invention are respectively connected to expression vectors containing heavy-chain constant region genes and light-chain constant region genes, and the anti-carbendazim full-length IgG recombinant antibody is obtained by adopting mammalian cell expression mediated by a double-plasmid transfection system. Indirect competitive ELISA proves that the expressed recombinant antibody has recognition activity similar to that of a parent monoclonal antibody.

The application belongs to the technical field of biology, and discloses a construction method and application of a subtilin expression strain, wherein the recombinant strain is obtained by transfecting a plasmid carrying a spaBTCSIFEGRK whole gene sequence of bacillus subtilis ATCC6633 and containing or not containing an original promoter into BS168 competent cells, so as to obtain a recombinant strain with high expression of subtilin, wherein the recombinant strain has very high safety, a clear genetic background, is easy to be high-density fermented, and has strong protein secretion capacity compared with the original strain.

The present invention relates to a method for rescue of Vesicular Stomatitis Virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth comprising (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises (i) an expression cassette comprising a VSV genomic cDNA; (ii) at least one expression cassette encoding VSV nucleoprotein (N) protein, VSV phosphoprotein (P) protein, and VSV large (L) protein; and (iii) an expression cassette encoding SV40 Large T antigen; (c) culturing the transfected cells; and (d) harvesting the cell culture supernatant comprising the rescued VSV. Also provided is the use of a HEK293 cell line or a HEK293 cell line adapted to suspension growth for rescue of Vesicular Stomatitis Virus (VSV) or the use of a plasmid encoding SV40 Large T antigen for rescue of Vesicular Stomatitis Virus (VSV) in a HEK293 cell line or a HEK293 cell line adapted to suspension growth HEK293-F cells by means of transient transfection.

Provided are an FLNB gene-knockout MDCK cell line, a construction method therefor and the use thereof. The FLNB gene-knockout cell line is named MDCK-FLNB-KO, and has been deposited in China Center for Type Culture Collection (CCTCC) on November 20, 2024, and the accession number is CCTCC NO: C2024391. The FLNB gene knockout uses CRISPR / Cas9 technology, wherein a sgRNA is designed for exon 1 of the FLNB gene of MDCK cells, an FLNB gene knockout vector plasmid 1 and plasmid 2 are constructed, and the plasmid 1 and the plasmid 2 are transfected into the MDCK cells to obtain the FLNB gene-knockout MDCK cell line. Compared with the original MDCK cell line, the obtained cell line has a lower adhesion ability and a higher yield when applied to influenza virus culture.

The invention relates to the related technical field of molecular biology and cell engineering, in particular to an FLP-IN-DF-1 cell line and a construction method and application thereof, and the construction method comprises the following steps: step 1, constructing CRISPR / Cas9 site-specific cleavage plasmid containing a chicken HIPP11 safety site sequence; step 2, constructing a donor plasmid Donor-FRT containing a homologous arm of a safety site of the HIPP11; 3, jointly transfecting plasmids in the step 1 and the step 2 into a chicken DF-1 cell line to obtain an FLP-IN-DF-1 cell line for expressing red fluorescence; and step 4, under the action of Flp recombinase, transfecting an exogenous gene donor plasmid containing an FRT site to the FLP-IN-DF-1 cell line obtained in the step 3, and driving the expression of a target gene through an optimized strong promoter to realize site-specific transfer and efficient expression of the exogenous gene in the chicken DF-1 cell line. Different from an existing Flp-In system depending on a random integration strategy, the construction method disclosed by the invention emphasizes fixed-point integration and promoter optimization, and improves the homogeneity, stability and screening efficiency of exogenous gene expression.

The invention discloses an in-vitro stable subculture construction method of a yak ovarian granular cell line. Relates to the technical field of cell culture. Comprising the following steps: carrying out subculture and amplification on primary yak ovarian granular cells to obtain yGCs; and carrying out pCI-neo-hTERT plasmid transfection and establishment of a monoclonal cell. The immortalized yGCs constructed by using an hTERT plasmid method can provide infinitely proliferated homogenized cells, reduce damage to yaks and avoid individual differences, can retain the core function of regulating follicular development of yGCs, provides a high-quality model for analyzing molecular mechanisms such as seasonal oestrus and low reproduction efficiency of yaks, and has theoretical and application values.

The invention relates to the technical field of non-coding RNA (Ribonucleic Acid) of parasites, and discloses a method for researching regulation and control of variant antigen genes of plasmodium non-coding RNA, which comprises the following steps: S1, constructing a modified plasmid pLN-ENR-GFP by overexpression plasmid: replacing a promoter region with a plasmodium falciparum U6 promoter; after PCR amplification, carrying out HindIII / EcoRI double enzyme digestion on a product, and inserting the product into a pLN-ENR-GFP carrier; adding a 9 * T terminator at the 3'end, introducing EcoRI / ApaI enzyme cutting sites at the two ends, and carrying out enzyme cutting connection: inserting an RUF6-15 fragment into the modified pLN-ENR-GFP vector; s2, synchronization of plasmodium and plasmid transfection and synchronization: taking a plasmodium falciparum in-vitro culture with a plasmodium falciparum body proportion of more than 60% in a cyclic body stage, and adding 5% of D-sorbitol (1 ml of cell volume: 14 ml of sorbitol); according to the method for researching the regulation and control of the variant antigen gene of the plasmodium non-coding RNA, a plasmodium falciparum RUF6-15 transgenic overexpression strain (138nt) is constructed, efficient and stable expression of target ncRNA is realized through optimization of a U6 promoter (SEQ ID NO: 1) and a PolyT terminator, and the technical bottleneck of functional verification of medium-length ncRNA of the plasmodium is broken through.

The application discloses a preparation method of cat induced pluripotent stem cells, which comprises the following steps: constructing piggyBac transposon plasmids carrying cat source OCT4, SOX2, KLF4 and c-MYC four factors in series, piggyBac transposon plasmids carrying cat source or pig source NANOG and LIN28 two factors in series and piggyBac transposon plasmids carrying monkey kidney virus SV40LT plasmids, and transfecting the plasmids into cat fetal fibroblasts; picking up cloned cells with embryonic stem cell morphologies, and carrying out subculture of cell clones meeting the characteristics of embryonic stem cells through identification and screening, so that the cat induced pluripotent stem cells are obtained. The preparation method of the cat induced pluripotent stem cells is safe, efficient and has good differentiation potential, lays a scientific foundation for researching in-vitro culture conditions of feline ESCs and establishment of feline iPSCs, and lays a scientific foundation for further cultivating cloned cats and gene modified cats and researching in related disease mechanisms, screening drugs and regeneration and repair.

The application discloses a construction method of a human adenovirus type 4 protein V knockout mutant strain and application thereof, and belongs to the technical field of biotechnology. The application precisely knocks out a protein V coding sequence in a human adenovirus type 4 full-genome infectious clone by means of a Red / ET homologous recombination system combined with a ccdB reverse screening technology, and obtains a recombination plasmid pBR322-Ad4-△V-EGFP which is clear in genetic background and highly homozygous in sequence. After the plasmid is transfected into HEK-293 cells, the human adenovirus type 4 protein V knockout mutant strain obtained exhibits a typical “replication-deficient” phenotype: although a particle containing a viral genome can be packaged, the physical titer (4.76×10 6 copies / μL) of the particle is about one order of magnitude lower than that of a wild type, and the progeny virus completely loses secondary infection ability. Transcriptome analysis reveals that the deletion mutation can specifically down-regulate a host proteasome pathway and activate a natural immune response. The mutant strain is high in safety and strong in immunogenicity, and can be used as an ideal attenuated vaccine carrier or a gene therapy tool.

The invention discloses a cell line for stably expressing porcine delta coronavirus S protein, a porcine delta coronavirus IgA antibody detection antigen and a kit. The method comprises the following steps: based on an S protein sequence of a CH / XJYN / 2016 strain, selecting an extracellular domain part D20-N1077, adding a human source septoglobin family 1D meber 1 signal peptide sequence MRLSVCLLLTLALCCYRANA to an amino terminal of the S protein, adding a 6 * His tag sequence to a carboxyl terminal, carrying out codon optimization according to a CHO expression system, and constructing into a pCDNA3.1 (+) eukaryotic expression vector to obtain a recombinant eukaryotic expression plasmid pcDNA3.1 (+)-PDCoV-S; then transfecting CHO cells with the plasmid, and carrying out two rounds of heredity mycin G418 pressurized screening, limited dilution cloning and continuous subculture to obtain an expiCHO-PDCoV-S stably transfected cell line through screening; and finally, constructing the PDCoV IgA antibody detection kit based on the cell line. The kit has no cross reaction with other common porcine disease virus positive serum, has intra-batch and inter-batch variation coefficients of less than 10%, has good specificity, repeatability and sensitivity, and can be used for early prevention and control of PDCoV.

The invention belongs to the technical field of biology, and particularly relates to a method for preparing human lysozyme exosome fusion protein based on immortalized bovine mammary epithelial cells and application of the human lysozyme exosome fusion protein. The method comprises the following steps: acquiring and purifying primary bovine mammary epithelial cells; transfecting an hTERT gene to construct an immortalized cell line; constructing an expression plasmid containing the fusion gene of the CD47 ankyrin and the human lysozyme; transfecting plasmids into immortalized cells, and screening positive clones; and finally, separating the human lysozyme exosome fusion protein from the cell culture supernatant by using a Ni-NTA chromatographic column to obtain the human lysozyme exosome fusion protein. The fusion protein has lysozyme activity, can effectively dissolve bacteria, has antibacterial and anti-inflammatory functions, is high in stability and low in antigenicity and is suitable for preparing antibacterial or anti-inflammatory drugs, immortalized cells are used as a bioreactor, efficient and continuous secretion of human lysozyme is achieved, and the human lysozyme can be used for preparing antibacterial or anti-inflammatory drugs. The problems of low yield, poor activity and insufficient stability in the traditional method are solved.

Disclosed in the present invention is an optimized cell transplant. The optimized cell transplant is formed by performing gene induction and modification on a mesenchymal stem cell in the form of a small molecule and protein composition. The expression levels of CD200 gene, Galectin-9 gene and VISTA gene can be increased synchronously after cell culture. Vector virus infection and plasmid transfection are not required in the cell preparation process, so that high biological safety and great clinical application value of cells are achieved. The optimized cell transplant is suitable for the technical field of mesenchymal stem cells applied to cell transplantation therapy, and the therapeutic effect of the optimized cell transplant is more excellent than that of the non-modified mesenchymal stem cell.

The application aims to provide a preparation method and application of an oral engineering probiotic system for producing active substances in situ metabolism of tumors. First, the probiotic is cultured, the CES gene is inserted into a plasmid as a target gene, the plasmid containing the CES gene is transfected into a probiotic competent cell by a heat shock method, the engineering probiotic capable of continuously and stably expressing carboxylesterase is obtained, and the oral engineering probiotic system is prepared by coating the engineering probiotic with a natural high molecular material, mercapto hyaluronic acid. The preparation method is scientific and reasonable, raw materials are abundant, and the preparation is easy to produce. The preparation method enhances the chemotherapy effect of colorectal cancer and reduces the toxic side effects caused by the distribution of chemotherapy drugs in other tissues and organs, provides new technical support for the treatment of colorectal cancer, is an innovation in oral microecological preparation drugs, and has great economic and social benefits.

The invention belongs to the field of gene engineering, and relates to preparation and application of a recombinant human (THP) 2-mFc fusion protein. The recombinant human (THP) 2-mFc fusion protein is a plasmid pCGS3-GS-(THP) 2-mFc, wherein the plasmid pCGS3-GS-(THP) 2-mFc is formed by fusing a tumor targeting peptide sequence THP and an mFc structural domain of human IgG1 by using a genetic engineering technology. The plasmid is transfected into a CHOZN cell, a stably transfected monoclonal cell strain CHOZN-(THP) 2-mFc is obtained through screening, and the THP 2-mFc fusion protein is prepared through expression and purification of the stably transfected monoclonal cell strain CHOZN-(THP) 2-mFc. A series of in-vitro experiments prove that the (THP) 2-mFc fusion protein disclosed by the invention has the effects of targeted recognition and tumor cell killing, has an obvious anti-tumor effect and has a relatively good application prospect.

The invention discloses a yeast heterologous expression recombinant human collagen III and a preparation method thereof. The yeast heterologous expression recombinant human collagen III comprises an amino acid sequence as shown in SEQ ID No. 1. The preparation method comprises the following steps: cloning a nucleotide sequence as shown in SEQ ID No.2 into a vector to construct a recombinant expression vector plasmid I; the method comprises the following steps: cloning a nucleotide sequence shown as SEQ ID No.3 into a vector, and constructing a recombinant expression vector plasmid II; transfecting the recombinant expression vector plasmids I and II into host cells, and screening to obtain double-gene transformed recombinant bacteria; fermenting and culturing the double-gene transformation recombinant bacteria; and purifying the yeast heterologous expression recombinant human III type collagen. On the basis of retaining the hydrophobic structure and activity of the original human-derived III-type collagen, the stability and hydrophilicity of the protein structure are improved; a P4H sequence is introduced, so that the defect that a pichia pastoris expression system cannot be hydroxylated is overcome.

Adeno-associated virus (AAV) is a promising tool for gene therapy, but its high manufacturing costs, driven by the complexity of the traditional triple plasmid transfection process, limit its widespread use. Even though traditional AAV production have relied in the transfection of three plasmids encoding essential components, like the inverted terminal repeats (ITRs), with the Rep and Cap genes, and the helper genes; putting these elements together in a single plasmid can reduce process complexity. Thus, simplifying the AAV production process by using a single plasmid could reduce costs and streamline production, making gene therapies more accessible. The present application discloses a single-plasmid system for the production of a recombinant parvovirus in a host cells. Host cells and kits comprising the plasmid, as well as methods for producing recombinant parvovirus using the single-plasmid system, are also disclosed.

This invention relates to the fields of biotechnology and cell biology, and provides a method for rapidly constructing a stable CHO cell line with high IgG antibody expression. The method includes the following steps: S1. Antibody plasmid construction; S2. Cell resuscitation, passage, and plasmid transfection; S3. Positive cell screening; S4. Preparation and screening of stable single-cell clones with high expression; S5. Feed-and-carry batch culture; S6. Verification of the stability of the antibody-expressing cell line. The method provided by this invention, after the plasmid vector enters the cells via electroporation, performs shaking culture after 48 hours of static culture, which accelerates cell recovery time, resulting in faster recovery and better cell condition compared to traditional static culture. During feed-and-carry batch culture, high-density cell inoculation shortens the logarithmic growth phase, allowing cells to enter the protein production phase earlier, thereby achieving higher expression levels, significantly shortening the overall production cycle, and saving substantial costs.

The invention belongs to the field of gene engineering, and particularly relates to a reverse genetic system of an rSARS2-MHV-S recombinant strain and application of the reverse genetic system. According to the invention, a reverse genetics technology is utilized, a yeast recombination system is adopted, and pYES1L is adopted as a carrier to construct plasmids containing a full-length viral genome. The recombinant virus with infectivity can be obtained by transfecting the plasmid into a natural host cell DBT cell of the MHV-A59 virus, the whole operation process is simple and convenient, complex experimental equipment is not needed, and the experimental period is short.