71 results about "Antibody expression" patented technology

The invention relates to the technical field of biological medicines, and particularly discloses an anti-human CD3 rabbit recombinant monoclonal antibody as well as a preparation method and application thereof. The antibody obtains anti-CD3 immune response by immunizing a rabbit, is obtained by combining single B cell sorting, gene cloning and a mammalian cell expression system, and has high affinity and good specificity. The antibody can effectively recognize CD3 positive T cells in human peripheral blood and can be applied to the fields of flow cytometry, immunohistochemistry, T cell function research and the like. Experimental results show that the antibody has the advantages of high titer, strong fluorescence signal, low background and the like, and is superior to the traditional murine anti-CD3 antibody. Amino acid sequences of light chain and heavy chain variable regions of the antibody are further obtained, and controllability and repeatability of antibody expression are achieved. The antibody has important scientific research value and industrial application prospect, and is suitable for various scenes such as T cell marker detection, immune state evaluation, immunotherapy auxiliary research and the like.

The invention belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

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.

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 invention discloses an autoantibody reagent for screening early-stage lung squamous cell carcinoma. The autoantibody comprises autoantibodies of tumor-associated antigens: RNF43; or, RNF43 and KLF4; or, RNF43, KLF4, and P53; or, RNF43, KLF4, P53 and NCOR1, or, RNF43, KLF4, P53 and NCOR1; or, RNF43, KLF4, P53, NCOR1, and CCND1 are selected from the group consisting of: RNF43, KLF4, P53, the reagent provided by the invention is used for detecting the expression level of the antibody in a human body, can effectively detect the lung squamous cell carcinoma, especially early lung squamous cell carcinoma, has high sensitivity and specificity, and can enable the sensitivity to be up to 62.4% and the specificity to be up to 82.3% when the combination of RNF43, KLF4, P53, NCOR1 and CCND1 is used for diagnosing the early lung squamous cell carcinoma. Meanwhile, compared with other combinations, the Youden index is closer to 1, which shows that the combination has higher diagnostic value and higher application value.

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 provides polynucleotide for coding a three-chain antibody, a vector, a recombinant cell as well as a preparation method and application of the polynucleotide, and relates to the technical field of biology. The three-chain antibody comprises a heavy chain, a light chain and a recombinant heavy chain; the heavy chain and the light chain form a half antibody, and the recombinant heavy chain contains a heavy chain Fc region and a single-chain antibody scFv; the polynucleotide for coding the bispecific antibody contains a coding gene of a light chain, a coding gene of a heavy chain, a coding gene of a recombinant heavy chain and a coding gene of the recombinant heavy chain which are arranged in sequence, so that the problems of low expression quantity and high mismatch rate of a host cell for expressing a three-chain antibody are solved.

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 provides polynucleotide for coding a three-chain antibody, a vector, a recombinant cell as well as a preparation method and application of the polynucleotide, and relates to the technical field of biology. The three-chain antibody comprises a heavy chain, a light chain and a recombinant heavy chain; the heavy chain and the light chain form a half antibody, and the recombinant heavy chain contains a heavy chain Fc region and a single-chain antibody scFv; the polynucleotide for coding the bispecific antibody contains a coding gene of a recombinant heavy chain, a coding gene of a light chain, a coding gene of the heavy chain and a coding gene of the light chain which are sequentially arranged, so that the problems of low expression quantity and high mismatch rate of a host cell for expressing a three-chain antibody are solved.

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 invention discloses a detection kit and a detection method for chlorate in dairy products and water, chlorate is converted into 2, 4-dichloroaniline, the chlorate detection kit is prepared by constructing a 2, 4-dichloroaniline antigen-antibody expression system and utilizing an immunodetection technology, and further, colloidal gold test paper is prepared. The detection time of the kit is only 5-10 minutes, the sensitivity can reach 50-100 mu g / kg (0.05-0.1 ppm), on this basis, a method for detecting chlorate in dairy products and water is further provided, the method is simple in operation step, high in detection speed, high in sensitivity and small in relative error, and compared with the national standard, the method has the advantages that a detection instrument is simple and easy to operate, the detection cost is low, and the like. The method is suitable for rapid detection of large-batch samples.

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 invention provides a CD93 monoclonal antibody as well as a preparation method and application thereof. Heavy chain and light chain variable region nucleotide sequences of the CD93 monoclonal antibody are obtained by constructing a rabbit phage 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 a CD93 monoclonal antibody; three CD93 monoclonal antibodies with the strongest affinity are screened out through ELISA (enzyme-linked immuno sorbent assay) detection. According to the invention, the three screened monoclonal antibodies are pairwise paired to screen antibody pairs with strong antigen binding capacity, and the antibody pairs are used for preparing a CD93 ELISA kit which is used for quantitatively detecting the content of CD93 in a human biological sample and assisting in diagnosis and prognosis evaluation of hepatocellular carcinoma.

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 pertains to a Lonicera japonica flower water extract composition for preventing or treating Helicobacter pylori infection, the composition containing secoxyloganin as an active ingredient. The extract contains a specific amount of secoxyloganin, and thus exhibited excellent antibacterial effects when used on Helicobacter pylori bacteria, and exhibited excellent effects in terms of reducing Helicobacter pylori IgG antibody expression in the blood, alleviating histopathological lesions, and reducing cytokine expression when used on Helicobacter pylori-infected mice. Thus, the Lonicera japonica flower water extract of the present invention can be usefully used as a composition for preventing or treating Helicobacter pylori infection.

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.