45 results about "Antibody expression" patented technology

The invention discloses a nano antibody expression vector, which is characterized in that pET28a (+) plasmid is used as a vector skeleton, a nano antibody gene is connected to the downstream of a T7 promoter of the vector skeleton, and a maltose protein gene is connected or not connected between the T7 promoter and the nano antibody gene; the vector expressed by using the nano antibody expression vector can have excellent living activity without purification. The invention further provides a method for displaying the nano-antibody library by using escherichia coli based on the nano-expression vector, and compared with the existing method for displaying the nano-antibody library by using bacteriophage, the method for displaying the nano-antibody library has the advantages that the yield is high; the library diversity is higher, the amino acid loss rate is low, the efficiency is high, the cost is low, the period is short, and the specific nano antibody can be obtained without screening by a flow instrument.

This invention provides an anti-CD16 antibody, the amino acid sequence of which is selected from SEQ ID NO. 1-3 in the heavy chain CDR region and from SEQ ID NO. 4-6 in the light chain CDR region. This invention belongs to the field of antibodies. It provides a protein sequence of an anti-human CD16 mouse monoclonal antibody, and designs a gene expression base sequence based on the sequenced protein sequence. A stable CHO cell line for recombinant expression of this gene is established. The purified antibody can specifically recognize the human CD16 antigen, and the antibody expression yield is much higher than that of antibodies prepared using traditional hybridoma cells. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, a stable CHO cell line for recombinant expression of the rabbit monoclonal antibody is established. The purified antibody maintains its specific recognition of the human CD16 antigen.

The application provides a kind of MFGE8 monoclonal antibody and its preparation method and application, the heavy chain and light chain variable region nucleotide sequence of the MFGE8 monoclonal antibody is obtained by constructing rabbit phage library, panning, monoclonal screening and identification, then the heavy chain and light chain variable region nucleotide sequence obtained is respectively subcloned into the expression vector containing IgG constant region nucleotide sequence, is constructed into complete IgG antibody expression vector and is introduced into eukaryotic cell to express and purify, and five strains of monoclonal antibodies with strong affinity are screened out by ELISA experiment.The application also pairs five strains of monoclonal antibodies two by two to screen the antibody pair with the strongest antigen binding capacity, and uses the antibody pair to prepare MFGE8 ELISA detection kit for quantitatively detecting the content of MFGE8 in human biological samples, to assist early diagnosis and screening of breast cancer.

The invention discloses a high-sensitivity wide-spectrum specific antibody for resisting parathion, parathion-methyl and fenitrothion, the high-sensitivity wide-spectrum specific antibody 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 the heavy chain variable region is as shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region coding gene is shown as SEQ ID NO: 4. The invention also provides an antibody expression plasmid. The obtained variable region sequence gene is respectively connected to an expression vector containing a heavy chain constant region gene and a light chain constant region gene, and a new recombinant full-length antibody is successfully obtained through an HEK293 (F) mammalian cell expression system. Based on the characteristics of stability and easy preservation of recombinant expression plasmids, the recombinant expression plasmids can be used for multi-batch repeated production of prohibited and limited organophosphorus pesticide recombinant antibodies for resisting parathion and the like; a stable and reliable core reaction reagent is provided for construction of an immunoassay method for multiple residues of organophosphorus pesticides such as parathion in a development environment and a food sample and development of a field screening product.

The invention relates to the technical field of recombinant protein expression and cell culture, in particular to a serum-free culture medium additive, a serum-free cell culture medium and application. The myricetin provided by the invention is used as a serum-free culture medium additive, is particularly used for CHO cell recombinant antibody expression, and is particularly used for suspension culture, the final concentration in the CHO serum-free culture medium is 3-6 [mu] M, normal growth and metabolism of cells are not influenced, the myricetin acts on the CHO cells by controlling oxidative stress, and the growth of the CHO cells is inhibited. And the myricetin can change the quality attribute of the adalimumab, so that the production cost of biological products is reduced.

The invention discloses an anti-muscovy duck parvovirus VP3 protein recombinant nano antibody, an expression engineering strain and application thereof, and belongs to the field of biology. The anti-Muscovy duck parvovirus VP3 protein recombinant nano antibody is obtained by screening through technologies of computational biology, molecular docking simulation, deep learning and the like. The method comprises the following steps: firstly, screening a heavy-chain antibody variable region sequence VP3-Nb66, then constructing a recombinant eukaryotic expression plasmid by adopting the heavy-chain antibody variable region sequence VP3-Nb66, and further constructing a recombinant expression engineering strain by adopting the recombinant eukaryotic expression plasmid. And further preparing the Muscovy duck parvovirus VP3 protein recombinant nano antibody by adopting the recombinant expression engineering strain. The yield of the recombinant nano antibody reaches 1.01 mg / L, the ELISA result OD450 is equal to 1.66, and the recombinant nano antibody has remarkable binding activity and can be used for preparing a nano antibody preparation for detecting the muscovy duck parvovirus disease.

The invention provides a separated nucleic acid molecule. The nucleotide sequence of the separated nucleic acid molecule is shown as SEQ ID NO. 1. The invention also provides an antibody coded by the nucleic acid molecule. The invention also provides a carrier which contains the nucleic acid molecule. The invention also provides a method for producing the antibody. The method comprises the following steps: transfecting antibody expression plasmids in competent cells, culturing, collecting cell supernatant, centrifuging to remove precipitate, purifying antibody protein, collecting eluent, and concentrating to obtain the antibody. The invention also provides an application of the antibody, the carrier or the pharmaceutical composition in preparation of drugs for treating autoimmune diseases. The antibody disclosed by the invention can be specifically combined with FcRn and Fc gamma Rs, is a potent inhibitor for FcRn-mediated IgG recirculation and Fc gamma R-dependent immune complex activation, is a therapeutic drug for autoimmune diseases, and has huge potential.

The application belongs to the technical field of molecular biology, and particularly relates to an anti-CD28 nanobody and a preparation method and application thereof. The anti-CD28 nanobody provided by the application is prepared in the following manner: constructing a CD28 antigen to immunize a llama, constructing a phage library to the PBMC of the immunized llama, screening the phage library, selecting correct sequences to construct and express and purify, and finally obtaining the anti-CD28 nanobody through cell function detection screening. The nanobody provided by the application has specific recognition and binding capacity for CD28, has the advantages of high specificity, high sensitivity and the like, effectively reduces the development and production cost of the CD28 antibody, and shortens the expression time of the 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.

The invention discloses an engineered exosome for expressing a complete antibody and a preparation method of the engineered exosome, and belongs to the field of biotechnology and drug delivery. Aiming at the problem that a complete antibody is difficult to efficiently and stably express on the surface of an exosome membrane in the prior art, the engineering exosome with the complete antibody displayed on the surface is extracted from a culture supernatant after a target antibody and a natural antibody membrane-bound transmembrane region (TMD) sequence are fused, a recombinant lentiviral expression vector is constructed and a host cell is transfected. According to the method, TMD is used as an anchoring element, accurate positioning and efficient expression of an antibody on an exosome membrane are achieved, and the targeting recognition capacity of the exosome and the application potential of the exosome in the fields of targeting delivery, disease treatment and the like are remarkably improved. Experimental results show that the obtained exosome is typical in form, uniform in particle size and high in antibody expression efficiency, and a new technical scheme is provided for a targeting vector system based on the exosome.

The application belongs to the technical field of biological medicine, and particularly relates to an anti-FOLR1 nanobody and a preparation method and application thereof. The anti-FOLR1 nanobody comprises a framework region and a complementarity determining region, and the complementarity determining region amino acid comprises CDR1, CDR2 and CDR3. The application adopts automatic panning and a mammalian expression system, significantly improves the screening efficiency and antibody expression quality, greatly shortens the development cycle, and provides an efficient tool for FOLR1 targeted diagnosis and treatment. The obtained anti-FOLR1 nanobody exhibits excellent specific recognition and binding capacity.

The application provides a monoclonal antibody specifically recognizing HCGP-39 and a preparation method and application thereof, and relates to the technical field of biological medicines.The application provides two monoclonal antibodies 6F6 and 6D9 of HCGP-39, and the titers are good.Based on the complementarity determining region sequences of the variable regions of the heavy chains and the light chains of the monoclonal antibodies, the variable region sequences are respectively constructed on corresponding antibody expression vectors, and the recombinant cells expressing the recombinant monoclonal antibodies can be obtained by transfecting cells.The monoclonal antibody vector obtained by the application is easy to preserve and easy to control the quality in the antibody production process; the monoclonal antibody can recognize the HCGP-39 protein, has biological activity, can be used for HCGP-39 antigen detection and the like, and has very good application value and very important scientific research guiding significance.

This invention discloses a porcine sperm-specific antibody, the coding sequence of which is shown in any one of SEQ ID No: 1-3. This invention prepares a nanobody library by immunizing alpacas with whole porcine sperm. The library can then be screened to identify antibodies that specifically bind to X or Y sperm, thus achieving X and Y sperm sorting. Furthermore, the screened antibodies are nanobodies, only 10% the size of traditional antibodies, with a simpler structure, high stability, strong specificity, and low immunogenicity. This allows for large-scale antibody expression, significantly reducing antibody production costs and facilitating industrial application.

The application provides a recombinant oncolytic virus targeting a tumor hypoxic microenvironment and an application thereof. A genome of the recombinant oncolytic virus comprises at least one nucleic acid fragment selected from a first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment; wherein the first nucleic acid fragment comprises genes encoding an immunosuppression relief and T cell local activation unit and genes encoding a T cell function enhancement and chemotactic expansion unit; the second nucleic acid fragment comprises genes encoding a hypoxia sensing unit and genes encoding a T cell activation antibody expression unit; and the third nucleic acid fragment comprises at least one gene encoding an immunomodulatory unit. The recombinant oncolytic virus has three action mechanisms of a high remodeling of a solid tumor microenvironment, a universal in vivo T cell adapter and a virus conditional replication regulation, and can be effectively used for tumor treatment.

The application provides a CD93 monoclonal antibody and a preparation method and application thereof, the heavy chain and light chain variable region nucleotide sequences of the CD93 monoclonal antibody are obtained by screening and enrichment through constructing a rabbit phage library, then the obtained heavy chain and light chain variable region nucleotide sequences are respectively subcloned into an expression vector containing an IgG1 constant region nucleotide sequence, an entire IgG1 antibody expression vector is constructed and introduced into a eukaryotic cell for expression and purification, and the CD93 monoclonal antibody is obtained; three strains of CD93 monoclonal antibodies with the strongest affinity are screened out through ELISA detection. The application further pairs the three strains of screened monoclonal antibodies two by two to screen out an antibody pair with strong antigen binding capacity, and prepares a CD93 ELISA kit from the antibody pair and uses the antibody pair for quantitative detection of the CD93 content in a human biological sample, thereby assisting in the diagnosis and prognosis evaluation of hepatocellular carcinoma.

The application provides a Claudin 6 antibody containing a non-natural amino acid modification and a preparation method and application thereof, the sequence of the antibody is obtained by replacing original amino acids with non-natural amino acids on the basis of SEQ ID NO: 1 and 6; the replacement site of the non-natural amino acid is selected from any one or a combination of at least two of the following: heavy chain: S114, T115, P148, G156, A157, T159, Q191, V149, L158 or G161; or light chain: T109, A112, K169, S202, S203, L154 or S156. The application introduces non-natural amino acids into specific sites of the antibody and expresses the antibody by using a cell expression system, evaluates the influence of non-natural amino acid insertion at different sites on the expression level, structural stability and binding capacity of the antibody, and provides a new idea for rational design and functional optimization of the antibody.

The present application belongs to the technical field of genetic engineering, and provides an autophagy regulation gene for improving recombinant antibody expression of CHO cells and a cell line construction method and application thereof, wherein the vector for improving recombinant protein expression of CHO cells is any one of a Gadd45b expression vector, a Cdk6 expression vector, a Rrn3 expression vector and a Sirt1 expression vector. The above autophagy-related gene overexpression vector constructed by the present application can realize efficient expression of a transgene in CHO cells when being co-transfected into CHO cells alone or in a certain ratio, and can also improve the expression amount of a recombinant protein when being applied to construction of a CHO cell expression system, thereby helping to break through the bottleneck of low recombinant protein expression level of CHO cells and providing a theoretical basis for recombinant protein production.

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.

This invention provides a peptide-human serum albumin (HSA) conjugate drug, its preparation method, and its application. The peptide-human serum albumin conjugate drug comprises a target tumor cell membrane protein peptide, human serum albumin, and a cytotoxin. This invention innovatively introduces an HSA and covalently conjugates the target tumor cell membrane protein peptide and cytotoxin to the HSA, resulting in a conjugate drug with good stability and minimal cytotoxin loss. This peptide-human serum albumin conjugate drug overcomes the technical shortcomings of large molecular weight and cumbersome preparation of ADCs and insufficient drug loading of traditional PDCs, achieving multiple technical advantages such as high drug loading, small molecular weight, and easy preparation, demonstrating significant advantages and promising application prospects. The preparation method provided by this invention is simple and easy to implement, requiring no complex antibody expression and purification processes, resulting in lower production costs.

The present invention relates to an in vitro method for culturing one or more antibody-expressing cells. The method comprises culturing one or more antibody-expressing cells obtained from peripheral blood in the presence of IL-2, IL-21, and non-cell surface-presented CD40 stimulants and in the absence of feeder cells. Furthermore, this specification provides a method for producing antibodies, including the step of culturing one or more antibody-expressing cells according to the method of the present invention, novel CD40 stimulants and their use, and cell culture media.

The present invention relates to the use of tRNA in promoting the protein-coding ability of mRNA. The expression level of a target protein is improved by means of overexpressing tRNA, and a codon corresponding to the tRNA can promote or improve the stability of the mRNA. Further provided is a new tRNA+mRNA immunopotentiating vaccine. By means of introducing one or more tRNA molecules, the antigen protein encoding ability of an mRNA vaccine is enhanced, thereby eliciting stronger humoral and cellular immune responses in vivo. Further provided is a recombinant cell for producing an antibody. The recombinant cell overexpresses tRNA capable of increasing the expression level of the antibody, and the tRNA comprises a tRNA isodecoder family. Further provided is a recombinant cell for producing or packaging recombinant AAV, wherein the recombinant cell overexpresses tRNA capable of improving the AAV packaging efficiency.

The application provides a porcine pseudorabies virus antigen and a preparation method and application thereof, relates to the technical field of biotechnology, and the antigen comprises a porcine pseudorabies virus gD protein; the amino acid sequence of the porcine pseudorabies virus gD protein is shown as SEQ ID NO. 5; experiments prove that the porcine pseudorabies virus antigen has good antigenicity, is easy to express highly, and has good broad-spectrum property; and the antibody produced by the porcine pseudorabies virus antigen has high neutralization capacity for the currently popular strains. The porcine pseudorabies virus antigen provided by the application is easy to express highly, and the expression amount of the antigen in a fermentation liquor is significantly improved after fermentation culture of recombinant cells. The technical problem of low expression amount of antibodies generated by immunization of commercial vaccines in the prior art is solved.

The invention belongs to the technical field of gene engineering, and particularly relates to application of a TP63 gene in improving the expression quantity of exogenous recombinant protein, and the nucleotide sequence of the TP63 gene is shown as SEQ ID NO.1. According to the invention, the TP63 overexpression vector is creatively applied to construction of a recombinant protein expression system, the TP63 overexpression vector is transfected into CHO cells to construct a CHO cell line for overexpressing TP63, and the CHO cell line can significantly improve the expression level of TP63 protein. Meanwhile, the CHO cell line is used as a host cell for recombinant protein expression, compared with a wild type CHO cell line, the expression quantity of recombinant protein can be increased, and the problem that the expression level of antibody expression quantity in an existing CHO cell expression system is low is effectively solved.

The present disclosure is directed to methods for treating a neuropsychiatric disorder associated with expression of autoantibodies to ionotropic glutamate receptor, NMD A 2A (GiuN2A), expression of anti-double-stranded (ds) DNA antibodies that cross react with one or more subunits of the NMD A receptor, or a mutation in the ionotropic glutamate receptor NMD A type subunit 2A (GRIN2A) gene. The methods involve administering a memantine, or a pharmaceutically acceptable salt thereof, to a subject suffering from a neuropsychiatric disorder associated with expression of GluN2A or a mutation in the GRIN2A gene.

The invention provides an SP monoclonal antibody and a preparation method and application thereof. A heavy chain variable region nucleotide sequence and a light chain variable region nucleotide sequence of the SP monoclonal antibody are obtained by constructing a mouse bacteriophage library through screening and enrichment. Respectively subcloning the obtained heavy chain and light chain variable region nucleotide sequences into an expression vector containing an IgG1 constant region nucleotide sequence to construct a complete IgG1 antibody expression vector, and introducing the complete IgG1 antibody expression vector into eukaryotic cells for expression and purification to obtain an SP monoclonal antibody; and a monoclonal antibody with the strongest affinity and high specificity is screened out through ELISA (Enzyme-Linked Immunosorbent Assay) detection. Meanwhile, the screened SP monoclonal antibody and the screened CGRP monoclonal antibody are combined for use, so that the expression of macrophage interferon and the expression of inflammatory factors are improved, and the virus infection resistance of individuals is improved.

This invention belongs to the field of molecular biology technology, specifically relating to an anti-CD28 nanobody, its preparation method, and its application. The preparation process of the anti-CD28 nanobody provided by this invention is as follows: CD28 antigen is constructed to immunize alpacas; a phage library is constructed from the immunized alpaca PBMCs; the phage library is screened, and correct sequences are selected for construction, expression, and purification; the anti-CD28 nanobody is finally obtained after screening by cell function detection. The nanobody provided by this invention has specific recognition and binding ability for CD28, exhibiting advantages such as high specificity and high sensitivity; simultaneously, it effectively reduces the development and production costs of CD28 antibodies and shortens antibody expression time.

The invention provides a signal peptide combination for improving antibody expression quantity, a recombinant expression vector, a recombinant cell and application, and belongs to the technical field of bioengineering. According to the invention, three signal peptides SP-A, SP-B and SP-C with good expression are combined pairwise and fused with an Inflixi or human immune globulin antibody in Chinese hamster ovary cells for expression. Compared with the original carrier signal peptide, the combined signal peptide has the advantages that the antibody expression quantity is obviously improved, and the problem of low antibody expression quantity is solved. The antibody produced by the method is high in yield and convenient for industrial application.

The application relates to the technical field of biological medicine, and particularly discloses a monoclonal antibody combined with human CD4 and a preparation method and application thereof. The antibody is obtained by combining single B cell sorting, gene cloning and mammalian cell expression technology after rabbit immunization of a CD4 polypeptide antigen. The antibody has high affinity and good specificity, and can effectively recognize CD4 positive T cells in human peripheral blood. The antibody is verified by ELISA and flow cytometry, and has high titer, low background and a signal-to-noise ratio superior to that of existing mouse-derived antibodies. Further, the amino acid sequences of the light chain and the heavy chain variable region of the antibody are obtained, so that controllability and repeatability of antibody expression are realized. The antibody can be widely applied to scenes such as immunodetection, HIV auxiliary diagnosis, T cell subgroup research and antibody engineering development, and has important scientific research and industrial values.

The present invention relates to an in vitro method for cultivating one or more antibody expressing cell(s). The method comprises cultivating one or more antibody expressing cell(s) obtained from peripheral blood in the presence of IL-2, IL-21 and a non-cell surface presented CD40-stimulating agent and in the absence of feeder cells. Moreover, herein provided are methods for producing antibodies comprising the step of cultivating one or more antibody expressing cell(s) according to the method of the invention, a novel CD40-stimulating agent and uses therefrom as well as a cell culture medium.

This invention belongs to the field of antibody preparation technology, specifically relating to an anti-NKG2D nanobody, its preparation method, and its application. The method is as follows: NKG2D protein expression is constructed, and alpacas are immunized to obtain immunized alpaca PBMC cells. RNA is extracted from the PBMC cells, reverse transcribed into cDNA, and antibody gene fragments are obtained by PCR. These fragments are then constructed into a phage expression vector. The obtained vector containing the antibody gene fragment is electroporated into SS320 electrocompetent cells to obtain an NKG2D immune antibody library. After two rounds of NKG2D protein panning and Clone ELISA detection, antibodies binding to NKG2D protein are obtained. After sequencing analysis, unique sequences are selected and expressed in a mammalian cell high-throughput expression system to obtain antibody proteins. The antibodies are then detected using an NKG2D high-expression cell line via FACS, ultimately yielding antibodies binding to NKG2D. The method of this invention is simple and rapid; the mammalian cell expression system induces highly efficient antibody expression, and the translated antibodies can be further modified, resulting in activity closer to that of natural antibodies.