149 results about "Antibody expression" patented technology

The present invention provides a dual expression vector, and methods for its use, for the expression and secretion of a full-length polypeptide of interest in eukaryotic cells, and a soluble domain or fragment of the polypeptide in bacteria. When expressed in bacteria, transcription from a bacterial promoter within a first intron and termination at the stop codon in a second intron results in expression of a fragment of the polypeptide, e.g., a Fab fragment, whereas in mammalian cells, splicing removes the bacterial regulatory sequences located in the two introns and generates the mammalian signal sequence, allowing expression of the full-length polypeptide, e.g., IgG heavy or light chain polypeptide. The dual expression vector system of the invention can be used to select and screen for new monoclonal antibodies, as well as to optimize monoclonal antibodies for binding to antigenic molecules of interest.

Nucleic acid molecules modified to enhance recombinant protein, e.g., antibody, expression and / or reduce or eliminate mis-spliced and / or intron read-through (IRT) by-products are disclosed. The invention also provides methods for producing proteins devoid of mis-spliced and / or intron read-through by-products by the use of such vectors in host cells under cell culture conditions suitable for recombinant protein expression.

The present invention provides a dual expression vector, and methods for its use, for the expression and secretion of a full-length polypeptide of interest in eukaryotic cells, and a soluble domain or fragment of the polypeptide in bacteria. When expressed in bacteria, transcription from a bacterial promoter within a first intron and termination at the stop codon in a second intron results in expression of a fragment of the polypeptide, e.g., a Fab fragment, whereas in mammalian cells, splicing removes the bacterial regulatory sequences located in the two introns and generates the mammalian signal sequence, allowing expression of the full-length polypeptide, e.g., IgG heavy or light chain polypeptide. The dual expression vector system of the invention can be used to select and screen for new monoclonal antibodies, as well as to optimize monoclonal antibodies for binding to antigenic molecules of interest.

Cysteine engineered antibodies comprising a free cysteine amino acid in the heavy chain or light chain are prepared by mutagenizing a nucleic acid sequence of a parent antibody and replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody.

The invention relates to a method for manufacturing an anti-RhD recombinant polyclonal antibody composition (anti-RhD rpAb). The method comprises obtaining a collection of cells transfected with a library of anti-RhD antibody expression vectors, wherein each cell in the collection is capable of expressing from a VH and VL comprising nucleic acid segment, one member of the library, which encodes a distinct member of anti-RhD recombinant polyclonal antibody composition and which is located at the same site in the genome of individual cells in said collection. The cells are cultured under suitable conditions for expression of the recombinant polyclonal antibody, which is obtained from the cells or culture supernatant. The nucleic acid segments encoding the anti-RhD rpAb is introduced into the cells by transfection with a library of vectors for site-specific integration. The present method is suitable for manufacturing anti-RhD rpAb, thereby making available a superior replacement of plasma-derived prophylactic and therapeutic immonoglobulin products.

The invention relates to using a mouse able to generate human IgGl heavy chain- k light chain to prepare humanized monoclonal antibody and its application. The characteristic: respectively knocking in huma k-chain and gl-chain constant gene at the sites of gene in mouse k- light chain constant region and heavy chain gl constant region; the humanized monoclonal antibody: the constant region is fully the same as that of human antibody and the variable region is mouse sourece, and the preparing steps: obtaining mouse k-chain and gl-chain gene groups; constructing a knoncking-in carrier; making homologous recombination in ES cell; producing the mouse with light-heave conversion; further mating to obtain a mouse with complete chimeric gene; the application: taking spleen and marrow cells of an immunized chemeric mouse to make fusion and culture; screening hybridomas of humanized monoclonal antibody generating antigenic specificity; preparing the humanized monoclonal antibody with antigenic specificity. The advantages is easy to obtain high-affinity human-mouse chimeric antibody.

Cysteine engineered antibodies comprising a free cysteine amino acid in the heavy chain or light chain are prepared by mutagenizing a nucleic acid sequence of a parent antibody and replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody. Certain highly reactive cysteine engineered antibodies were identified by the PHESELECTOR assay. Isolated cysteine engineered antibodies may be covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

The present invention relates to the production of a transgenic bovine which comprises a genetic modification that results in inactivation and loss of expression of its endogenous antibodies, and the expression of xenogenous antibodies, preferably human antibodies. This is effected by inactivation of the IgM heavy chain expression and, optionally, by inactivation of the Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of non-bovine antibodies, preferably human antibodies.

The invention relates to a combined reagent for detecting acute myelocytic leukemia cells and a system thereof, wherein the combined reagent and the system thereof belong to the field of medical technology. The combined reagent comprises at least one selected from the following antibody combinations: a first antibody combination which comprises CD38, CD13, CD34, CD117, CD33, CD19, HLA-DR and CD45antibodies; a second antibody combination which comprises CD38, CD64, CD34, CD123, CD56, CD14, HLA-DR and CD45 antibodies; and a third antibody combination which comprises CD38, CD7, CD34, CD5, CD11b,CD15 and CD45 antibodies. The antibody combinations of the invention cover the expression marks of three systems of granulocyte, single cell and lymphocyte. A normal antibody expression mode is established. Tumor cells can be identified maximally. Furthermore, through a large number of experiment data, the antibodies in each combination have no problem of mutual expression inhibition. FurthermoreAML-MRD can be comprehensively and quickly detected with high sensitivity through multi-parameter flow type cell analysis.

The invention relates to a killing cell for high-efficiency and stable expression of antibodies and a use thereof. Specifically, the invention provides the transgenic killing cell; a genome of the killing cell is stably integrated with an expression cassette containing an encoding sequence of human Fc segment-containing antibodies, or containing an encoding sequence of chimeric antigen receptors and inhibitory antibodies or activated type antibodies, and both ends of the expression cassette contain inverted terminal repeated sequences of a transposon. The killing cell can stably express the human Fc segment-containing antibodies or the chimeric antigen receptors and antigen-binding fragments derived from interested antibodies with high level while maintaining cell killing toxic effect. In addition, for preventing systemic toxicity and autoimmune diseases caused by over expression of antibodies due to in-vivo continuous proliferation of immune cells for stable expression of the antibodies, a molecular braking system is also introduced. With use of monoclonal antibody agents appearing on the market, the killing cell integrated with the antibody expression cassette can be quickly eliminated, and the safety of treatment is effectively improved.

The invention discloses a cell culture method for reducing acidic peak content of an antibody and improving glycoform of the antibody. In the method, GS-CHO cells are used as an expression system, glutamine is added into a basic medium for antibody-expressed cell strain culture. After glutamine is added to the basic medium, it is possible to effectively reduce acidic peak content of the antibody and improve glycosylation level, thus modifying the activity and efficacy of antibody drugs and improving the quality of the antibody so that the quality is close to that of a standard product; the method is adapted to the development and study and later large-scale production of new antibody drugs and has a promising application prospect and good economic benefit.

The invention relates to the field of molecular medicaments and gene therapy and specifically relates to an AAV (adeno-associated virus) vector expression system which is capable of efficiently expressing full genes of a human antibody and is constructed through an AAV2 / 1 shuttling expression vector pSNAV. The expression system not only has a good antibody expression effect at a cell level, but also realizes long-period and high-efficiency expression in a mammal body. The expression system provides a good platform for the application of the AVV vector for expressing the full genes of the antibody in the aspects of antibody therapy, high-yield cell strains and the like.

The present invention provides a bispecific antibody, a method and applications thereof. The bispecific antibody has a first antigen binding domain capable of specifically binding to human CD3 and a second antigen binding domain capable of specifically binding to GPC3 antigen, can specifically recognize and bind to GPC3 antigen and GPC3 overexpression tumor cells including liver cancer and other GPC3-positive tumor diseases, can further bind to T cell receptors, and can mediate T cells to provide killing effects, such that GPC3 infections can be cleared, and great application values can be provided in diagnosis, treatment, prevention and detection of GPC3-related diseases. The present invention further provides minicircle DNA having a nucleotide sequence encoding the bispecific antibody. According to the present invention, the bispecific antibody and the minicircle DNA can be used for preparing antibody pharmaceutical compositions or GPC3-positive tumor cell diagnosis reagents.

The invention discloses an enzyme linked immunosorbent assay kit for detecting a diethoxy organophosphorus pesticide based on a nano antibody. The enzyme linked immunosorbent assay kit contains the nano antibody as a detection antibody, and an amino acid sequence of the nano antibody is shown as SEQ ID NO.1. The kit is the enzyme linked immunosorbent assay kit, and the enzyme linked immunosorbentassay kit comprises an enzyme label plate containing organophosphorus pesticide antigen, a horseradish peroxidase-labelled secondary antibody solution, a diethoxy organophosphorus pesticide standard solution, substrate liquid, a substrate buffer solution, a stopping solution, and condensation washing liquid. The expression level of the diethoxy organophosphorus pesticide based on the nano antibodyis high, the stability is strong, the kit can be used for detecting a plurality of the diethoxy organophosphorus pesticides, realizes broad spectrum specific identification of the diethoxy organophosphorus pesticide, has the advantages of accurate detection result, good effect, good stability, simple operation, high sensitivity, and low cost, and can accurately detect the residues of the diethoxyorganophosphorus pesticide in an agricultural product.

The invention relates to the technical field of cellular immunity and discloses a construction method and application of a chimeric antigen receptor (CAR) based on a BMCA nano-antibody sequence. The construction method comprises the following steps: step 1: constructing a BMCA single domain antibody library; step 2: screening a BMCA specific nano-antibody sequence; step 3: preparing chimeric antigen receptor T cells. The chimeric antigen receptor is mainly applied to tumor treatment medicines; tumors are tumor diseases related to blood and the tumor diseases related to the blood mainly comprise various leukemia and malignant lymphomas. According to the construction method and application of the chimeric antigen receptor based on the BMCA nano-antibody sequence provided by the invention, the prepared nano-antibody expression sequence is shorter and a limited lentiviral vector space is saved; the chimeric antigen receptor T cells which are constructed based on the antibody have better specificity and a stronger tumor killing capability.

The invention discloses a method for culturing animal cells for producing anti CD20 antibody, and specifically discloses inoculation quantity, medium formula and various culture process control parameters. The medium is a serum-free medium, so that no side effect is caused by serum in the process of cell culture, and antibody extraction and purification are further facilitated. By using the method for producing the anti CD20 antibody, differences between different batches are small, antibody expression quantity is high, production cost is low, and safety is good.

The invention discloses a nano vaccine taking an SARS-CoV-2 virus S protein RBD region as an antigen and a preparation method of the nano vaccine. An expression vector with an SARS-CoV-2 virus S protein RBD region gene sequence is transfected into 293T, 293i and CHO cell lines for antibody expression, and the required vaccine is obtained after protein extraction, ultrafiltration enrichment, antibody affinity chromatography purification, protein collection after purification, PBS dialysis and aluminum hydroxide adjuvant mixed incubation. The vaccine is a protein for expressing an amino acid sequence of the SARS-CoV-2 virus S protein RBD region, 24 monomers in a cell can be self-assembled into a sphere structure, and an SARS-CoV-2 virus S protein RBD peptide fragment subjected to fusion expression is displayed on the surface of a sphere. The vaccine has high neutralizing activity on the SARS-CoV-2 virus, and can effectively inhibit infection of the virus on vero cells. A toxicity attacking experiment shows that the vaccine can obviously and effectively protect the infection and damage of the SARS-CoV-2 to rhesus monkeys, and an excellent candidate vaccine is provided for the prevention and control of the current COVID-19 epidemic situation.

The invention relates to a chimeric antigen receptor and an expression gene thereof, a double-antigen regulated type T cell modified by the chimeric antigen receptor, and application thereof. The expression gene of the chimeric antigen receptor comprises a first fusion protein expression gene and a second fusion protein expression gene, wherein the first fusion protein expression gene comprises a mucoprotein-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcription activating protein expression gene which are sequentially connected; the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and an anti-mesothelin expression gene which are sequentially connected. By adopting the innovative design, the chimeric antigen receptor has the advantages that the chimeric antigen receptor can be successfully imported into the T cell to form the T cell modified by the chimeric antigen receptor; the immune reaction cannot be produced until the mucoprotein-1 and the anti-mesothelin signals simultaneously exist, so as to reach the controllable immune reaction of the chimeric antigen receptor, fewer side effects in treatment, and high specificity.

The invention relates to a NK cell capable of efficiently and stably expressing an antibody and application thereof. Specifically, the invention provides the transgenic NK cell, and the genome of the transgenic NK cell is stably integrated with an expression cassette containing a coding sequence of an antibody of a human Fc segment, wherein two ends of the expression cassette contain inverted terminal repeats of transposons. The transgenic NK cell provided by the invention can maintain cell killing toxicity and stably express the antibody of the human Fc segment at a high level. Moreover, a molecular brake system is introduced to prevent systematic toxicity and autoimmune diseases caused by excess expression of antibodies due to continuous propagation of stably expressed antibodies in immune cells. Commercially available monoclonal antibody drugs can be used for rapidly removal of killer cells integrated with the expression cassettes of antibodies, so treatment security is effectively improved.

The invention discloses a neutralizing monoclonal antibody resisting to tetanus toxin. Hybridoma cells are obtained from tetanus toxin heavy chain C fragment immune mice, light chain and heavy chain variable region genes of the antibody are taken, human-mouse chimeric complete antibody expression vectors are built, CHO cell transfection is carried out, purification is carried out, and then the antibody is obtained. The antibody has the activity of specific binding to tetanus toxin, the monoclonal antibody can partially protect mice against attack of tetanus toxin, and four antibodies can completely protect mice against a twofold lethal dose of tetanus toxin in combination.

The invention provides a specific double-targeted antibody as well as a method and application thereof. The specific double-targeted antibody has a first antigen combination structural domain with the specificity combined with human CD3 and a second antigen combination structural domain with the specificity combined with an HBV (Hepatitis B Virus), can specifically identify and be combined with HBV antigen positive cells, can be also combined with TCR (T Cell Receptor), mediates T cells to develop the killing effect so as to clear away chronic HBV infection, and has extremely large application value in diagnosis, treatment, prevention and detection of HBV related diseases. The invention further provides a minicircle DNA with a nucleotide sequence for coding the double-targeted specific antibody. The specific double-targeted antibody and the minicircle DNA which are provided by the invention can be used for preparing an antibody medicine composition or an HBV diagnosis reagent.

The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.

The invention relates to the field of biotechnology, in particular to a cell culture medium for improving antibody purity and a cultivation method, discloses a self-made feeding culture medium, and the culture medium contains 90mM to 500mM cysteine, and further provides a cell cultivation method. The cell cultivation method comprises the following steps: mammalian cells containing encoded antibody nucleic acid are inoculated into a basal culture medium; after the cells enter into a cellular proliferative stage, the self-made feeding culture medium is added; and cell density is monitored and incubation time is adjusted. By controlling the concentration of the cysteine in the self-made feeding culture medium, the adding moment and the adding quantity, the antibody purity can be improved remarkably, while the antibody purity is improved, the antibody expression quantity and normal glycosylation level can be maintained, and the antibody efficacy is ensured.

The invention provides a specific dual-targeting antibody as well as a preparation method and application thereof. The specific dual-targeting antibody comprises a first antigen binding structural domain specifically bonded with human CD3, and a second antigen binding structural domain specifically bonded with a cMet antigen, and is capable of specifically identifying and bonding tumor cells with overexpression of the cMet antigen, wherein the tumor cells are of liver cancer, other cMet-positive tumor diseases and the like; furthermore, the specific dual-targeting antibody is also capable of bonding a T cell receptor, and mediates T cells to play a killing role, thus clearing cMet infection; therefore, the specific dual-targeting antibody has a great application value in diagnosis, treatment, prevention and detection of cMet-related diseases. The invention also provides microcircular deoxyribonucleic acid (DNA) encoding the nucleotide sequence of the specific dual-targeting antibody. The specific dual-targeting antibody and the microcircular DNA which are provided by the invention can be used for preparing antibody-based pharmaceutical compositions or cMet-positive tumor cell diagnostic reagents.

The present invention relates to a bicistronic expression vector for antibody expression, an animal cell transfected with the expression vector, and a method for producing an antibody including culturing the animal cell, in which the expression vector includes a first expression cassette including ‘promoter-UTR-intron-antibody light chain gene-polyA’ and a second expression cassette including ‘promoter-UTR-intron-antibody heavy chain gene-internal ribosome entry site (IRES)-amplification gene-polyA’. An expression vector capable of expressing a desired antibody with high efficiency can be constructed using the bicistronic expression vector including an intron for antibody expression according to the present invention, and the expression vector can produce the antibody by culturing the transfected animal cell with stability and high efficiency.

The invention discloses a serum-free culture medium which comprises the following components of DMEM / F12 and growth factors of 0.6-300 ng / mL, hormone of 15-120 [mu]g / L, protein of 3-150 mg / mL, amino acid and peptide of 0.1-1.7 g / L, lipid compound of 6.51-13.1mg / L, trace elements of 22-220 [mu]g / L, and vitamin of 0.5-5.9 mg / L, wherein the growth factors of 0.6-300 ng / mL, the hormone of 15-120 [mu]g / L, the protein of 3-150 mg / mL, the amino acid and peptide of 0.1-1.7 g / L, the lipid compound of 6.51-13.1mg / L, the trace elements of 22-220 [mu]g / L, and the vitamin of 0.5-5.9 mg / L are added in the DMEM / F12. The invention relates to a cell culture medium, in particular to a culture medium for culturing suspension cells, the culture medium for the culturing suspension cells is used for the expression of recombinant proteins, the culture medium of the invention does not contain serum components at all, and the purity of the produced antibodies is greatly improved; meanwhile, the culture mediumhas high amplification rate and high antibody expression capacity; and the culture medium can maintain cell viability for a long time, and the cell culture time is greatly prolonged so that the production time can be greatly prolonged, and the antibody production is improved.

The invention discloses a charge-reversed DNA (Deoxyribose Nucleic Acid) nano-carrier and a preparation method and application thereof, and belongs to the fields of gene treatment and DNA vaccine antibody expression. The charge-reversed DNA nano-carrier comprises a compound prepared from methylsuccinic acid-3-dimethylamino-1-propyl ester quaternary ammonium salt and pDNA, has different quantities of electric charges under different pH conditions, and can be subjected to charge reversing under a corresponding PH condition. The prepared charge-reversed compound has a pH response, has different quantities of electric charges under different pH conditions through the methylsuccinic acid-3-dimethylamino-1-propyl ester quaternary ammonium salt, and is subjected to charge reversing under a certain PH condition, and a nano-compound carries positive charges when the compound passes through the surface of a cell membrane carrying negative charges, so that the compatibility with the cell membrane is very high, the compound can be effectively internalized, the electric charges are reversed under different pH conditions in cells, and a carrier carrying the negative charges can circulate for a long time more easily and is more suitable to be applied in gene treatment and antibody production.

The invention discloses a variable region sequence of a specific anti-pyraclostrobin antibody. The amino acid sequence of a heavy chain variable region encoding gene is disclosed by SEQ ID NO: 2. Theinvention also discloses an anti-pyraclostrobin recombinant overall-length antibody, which comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region encoding gene is disclosed by SEQ ID NO: 2. The invention also discloses an antibody expression plasmid.A sequence gene obtained by the invention is independently connected to expression carriers containing a heavy chain constant region gene and a light chain constant region gene, mammal cell expressionof a double-plasmid system is adopted to obtain the recombinant overall-length antibody, the recombinant overall-length antibody has consistent identification activity with a murine parent antibody,the anti-pyraclostrobin antibody can be stably produced on a large scale, and a reliable core reagent is provided for various immunoassay methods for detecting pyraclostrobi residues.

The invention discloses an antibody variable region coding gene. Based on the coding gene, a targeting antibody expression level is substantially improved. An external biological function evaluation result shows that a targeting antibody obtained based on the antibody variable region coding gene can specifically recognize a human vascular endothelial growth factor (VEGF) antigen and can substantially inhibit tumor tissue neovascularization thereby inhibiting tumor growth. The invention also discloses a preparation method of the above targeting antibody.