69 results about "Plasmid transfection" patented technology

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

The invention discloses a reverse genetic operating system of fowl adenovirus type 4 and application of the reverse genetic operating system. Belongs to the technical field of virus genetic manipulation. The FAdV-4 full-length genome is amplified in a segmented mode through a PCR method, segmented amplification products are cloned to pOK-CMV vectors through the homologous recombination technology, four library plasmids covering the whole gene are successfully obtained, and LMH cells are transfected to successfully rescue the virus. Meanwhile, the reporter gene NanoLuc is inserted into the FAdV-4 genome, so that the recombinant FAdV-4 for expressing the NanoLuc is successfully rescued. The invention provides an efficient technical platform for deep development of foundation and application research of FAdV-4, and has important scientific application value.

The invention belongs to the technical field of biology, and discloses a construction method of a swine cholera attenuated salmonella recombinant strain capable of simultaneously expressing a haemophilus parasuis outer membrane protein GAPDH gene and an Omp26 gene, a plasmid connected with the haemophilus parasuis GAPDH gene and the Omp26 gene is adopted to transfect a swine cholera salmonella asd gene deleted strain, screening is carried out, and screening is carried out to obtain the swine cholera attenuated salmonella recombinant strain capable of simultaneously expressing the haemophilus parasuis outer membrane protein GAPDH gene and the Omp26 gene. The recombinant attenuated salmonella choleraesuis expressing the haemophilus parasuis GAPDH gene and the Omp26 gene is obtained. The strain can simultaneously express a haemophilus parasuis outer membrane protein GAPDH gene and an Omp26 gene, and compared with a strain which singly expresses Omp26, the strain has the advantage that the immune protection effect is obviously improved. Meanwhile, the invention also provides a construction method and application of the strain.

To provide methods of producing and purifying AAV viral vectors having low empty capsids, low host cell proteins, and / or low contaminating DNA, while retaining high potency; and to provide a pharmaceutical composition containing the vector.SOLUTION: A method of manufacturing an AAV viral vector comprises: a. culturing adherent cells; b. transfecting the adherent cells with one or more plasmids to enable production of the AAV viral vector; c. lysing the adherent cells to isolate the AAV viral vector; d. acidifying and clarifying the cell lysate of c; e. purifying the product of d using cation exchange chromatography; f. filtering the product of e using tangential flow filtration; g. ultracentrifuging the product of f in a cesium chloride buffer; h. collecting the AAV viral vector from the product of g; and i. formulating and filling the AAV viral vector collected in h. to produce a final pharmaceutical product.SELECTED DRAWING: None

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 relates to a cell line capable of stably expressing an orthogonal aminoacyl tRNA synthetase / tRNA pair, constructs an HEK293T cell line capable of stably expressing an EcTyr orthogonal aminoacyl tRNA synthetase / BsTyr tRNA pair and discloses a construction method of the cell line, and the construction method comprises the following steps: stably integrating the orthogonal EcTyr aminoacyl tRNA synthetase and BsTyr tRNA gene into an HEK293T cell genome by virtue of a PiggyBac transposon system and stable plasmid transfection; according to the constructed cell line genome, the EcTyr orthogonal aminoacyl tRNA synthetase and the BsTyr tRNA gene are integrated, so that a stable platform is provided for efficiently introducing non-natural amino acid at any site of protein. The method has the effect of further laying an experimental foundation for exploration of novel protein structures and functions and research and development of protein drugs with certain specific functions.

The invention is applicable to the technical field of biotechnology and cell biology, and provides a method for quickly constructing an IgG antibody high-expression stably-transfected CHO cell strain, which comprises the following steps: S1, constructing antibody plasmids; s2, carrying out cell resuscitation, passage and plasmid transfection; s3, screening positive cells; s4, preparing, namely screening high-expression stably transfected single cell clones; s5, cell feeding batch culture; s6, verifying the stability of the cell strain expressed by the antibody, according to the method provided by the invention, after the plasmid vector enters the cell through electrotransfection and standing culture is carried out for 48 hours, shaking culture is carried out, the cell recovery time can be shortened, the recovery speed is higher than that of traditional standing culture, and the cell state is better than that of a standing state; according to the method, high-density cells are inoculated during cell fed-batch culture, and under the high-density inoculation, the logarithmic phase of the cells is shortened, and the cells enter a protein production phase earlier, so that higher expression quantity can be achieved, the overall production period is greatly shortened, and a large amount of cost is saved.

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 provides an FLNB gene knockout MDCK cell strain as well as a construction method and application thereof. The FLNB gene knockout cell strain is named as MDCK-FLNB-KO, and is preserved in the China Center for Type Culture Collection on November 20, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2024391. Wherein the FLNB gene knockout utilizes a CRISPR / Cas9 technology, sgRNA is designed for a first exon of an FLNB gene of an MDCK cell, and a FLNB gene knockout vector plasmid 1 and a plasmid 2 are constructed; and transfecting the plasmid 1 and the plasmid 2 into the MDCK cell to obtain the FLNB gene knockout MDCK cell strain. Compared with an original MDCK cell strain, the cell strain has the advantages that the adhesion capacity is reduced, and the yield is higher when the cell strain is applied to influenza virus culture.

The invention relates to the technical field of gene engineering, and relates to a construction method of sgRNA with a pBD2 gene knocked out and a construction method of a pig intestine epithelial cell line with the pBD2 gene knocked out. According to a pBD2 gene sequence, sgRNA capable of targeting the gene is designed and synthesized, plasmids of a CRISPR / Cas9 system containing the sgRNA are constructed by using a molecular cloning and recombination technology, the plasmids are transfected into a pig small intestine epithelial cell line, gene editing is performed on a specific position of the pBD2 gene, and a stable monoclonal cell line with the pBD2 gene knocked out is obtained through puromycin drug screening. The homozygous pBD2 gene knockout cell strain obtained by the method is used as a cell model and can be used for exploring the effects of the pBD2 gene in the aspects of immunoregulation, sphingolipid metabolism, cell cycle and the like of small intestine cells.

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 provides a virus-like vector lysis solution and a virus-like vector lysis method. The virus-like vector lysis solution comprises Tris, EDTA (Ethylene Diamine Tetraacetic Acid), NaCl, TritonX-100, sodium deoxycholate, SDS (Sodium Dodecyl Sulfate), Tween 20 and a protease inhibitor mixture. Cas9 protein is wrapped in the viroid vector; the acquisition method of the viroid vector comprises the step of transfecting plasmids containing a structural gene sequence for expressing the viroid vector into virus producing cells to obtain the viroid vector. The cracking method of the viroid vector comprises the following steps: S1, using: carrying out cracking treatment on a viroid vector solution by using the cracking solution; and S2, performing Cas9 protein content detection on substances released after the carrier is cracked. The invention establishes a formula of the special lysate for VLP and a corresponding cracking method, and ensures high cracking efficiency.

The invention discloses a saccharomyces cerevisiae human gene expression plasmid and a construction method and application thereof, the saccharomyces cerevisiae human gene expression plasmid is pRS315-GAP-CYC1, by constructing wild and mutant plasmids and transfecting a saccharomyces cerevisiae strain of a KanMX6 resistance box sequence to replace a homologous gene, pathogenicity prediction of newly found mutations can be realized, the operation is simple and convenient, and the method is suitable for large-scale production. The method is efficient and rapid and has wide application value.

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 present invention discloses a preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The present invention belongs to the field of biomedical technology. The preparation method includes the following steps: cloning the coding sequences of the extracellular region of BAFF-R3 and the adiponectin globular domain; connecting the extracellular region of BAFF-R3 and the adiponectin globular domain through a peptide chain, and inserting the expression sequence of the eBR3-gAD fusion protein into a plasmid using restriction enzymes and DNA ligase; transfecting the above plasmid into cells and screening out a cell line with stable expression; culturing the above cell line, collecting the supernatant, and purifying by gel filtration and chromatography to obtain the soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The preparation method of this novel fusion protein has the advantages of high specificity, high expression level, and simple purification.

The invention relates to the technical field of biology, and discloses a construction method of a cell line for expressing porcine epidemic diarrhea virus S1 protein.The method comprises the steps that a nucleotide sequence of the PEDV S1 protein is inserted into a plasmid, and the plasmid carrying nucleotide of the PEDV S1 protein is obtained; then transfecting the plasmid carrying the nucleotide of the PEDV S1 protein into CHO-K1, and culturing to obtain a cell line for expressing the PEDV S1 protein; the nucleotide sequence of the PEDV S1 protein is shown as SEQ ID NO: 1, the cell line capable of stably expressing the PEDV S1 protein is obtained through the design, and in addition, the invention further discloses the cell line capable of expressing the PEDV S1 protein, application of the cell line and an ELISA detection method.

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 provides a method for efficiently preparing high-purity NudCD3 protein in an in-vitro eukaryotic cell. The method comprises the following steps: synthesizing a target gene; carrying out enzyme digestion on the target gene and the pLEXmMBP vector, and connecting to obtain a recombinant vector; transforming the recombinant vector into competent cells, culturing, and extracting plasmids; transfecting an HEK 293F cell, culturing, and collecting the cell; resuspending the cells in a lysis buffer solution, carrying out ultrasonication and centrifugation, and taking a supernatant; and carrying out affinity chromatography column purification, Prescission protease digestion and molecular sieve purification to finally obtain the NudCD3 protein with the purity of more than 90%. The problems that in the prior art, NudCD3 protein is difficult to express in quantity and insufficient in purity are solved, high-purity protein is provided for structural biology research, drug screening and antibody preparation, and the method has the advantages of being easy to operate, high in yield, high in protein stability and the like.

The invention relates to the technical field of biology. The invention provides a construction method of an E7 cell model for expressing human papilloma viruses 16 and 18, which comprises the following steps: transfecting exogenous plasmids into TC-1 cells in a culture medium inoculated with the TC-1 cells; the culture medium contains G418 genetics mycin; the exogenous plasmid can be used for stably transforming the HPV18E7. The TC-1-HPV18E7 cell model and the mouse model are successfully constructed, and it is proved that the recombinant strain vaccine LM-HPV18E7 shows a good treatment effect in a mouse model application test. According to the TC-1-HPV18E7 cell model, the application of the cell model based on TC-1 can be expanded, and a foundation is laid for subsequent research and development of bivalent and even multivalent therapeutic vaccines.

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.

According to the DKK-1 antibody biological activity detection method provided by the invention, a luciferase expression plasmid containing a TCF-LEF reaction element is transfected into an HEK 293 cell with an active Wnt / beta-catenin signal channel, and a reporter gene detection method based on DKK-1 antibody MOA is successfully established by screening effector cells and optimizing detection conditions. And then carrying out practicability confirmation of the RGA method through condition optimization. The establishment and prediction of the new method can further promote the screening evaluation of DKK-1 antibody drugs and the quality control of the DKK-1 antibody drugs in the subsequent development process, so that an efficient and rapid new method is provided for the biological activity detection of the DKK-1 antibody, and a new thought and direction are provided for the biological activity evaluation of other MOA-based antibodies.

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.

The present invention discloses a specific monoclonal antibody against chlorpyrifos: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:2; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:4. The present invention also discloses an antibody expression plasmid. The variable region sequence genes obtained by the present invention are respectively connected to expression vectors containing heavy chain constant region genes and light chain constant region genes, and two recombinant full-length antibodies (rAb‑13C7 and rAb‑14G4) are obtained based on mammalian cell expression and purification by double plasmid transfection. Indirect competitive ELISA analysis shows that both have detection activities similar to those of the parent ascites antibody. The above variable region sequence can be applied to the stable production of multiple batches of anti-chlorpyrifos recombinant antibodies, providing a reliable and stable core reagent for the construction of immunoassay methods for chlorpyrifos residues in the environment and food and the development of precise and rapid test products.

The present invention belongs to the field of target drug technology, and in particular relates to an application of a SETD1A protein. The amino acid sequence of the SETD1A protein can be found in NCBI under the accession number NP_055527. The SETD1A protein is used as a drug target in the preparation of a drug for treating diseases caused by tobacco-induced DNA damage. By increasing SETD1A expression, for example, SETD1A can be overexpressed by plasmid transfection to repair DNA damage.

Disclosed herein are improved methods of producing recombinant adeno-associated viruses (rAAVs) for gene therapy. The improved method comprises adding low doses of DMSO and valproic acid to a cell transfected with a plasmid encoding the rAAV and a plasmid containing a cofactor for expressing and / or assembling the rAAV capsid.

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.