141 results about "Variable region gene" patented technology

The present invention relates to transgenic non-human animals that are engineered to contain human immunoglobulin gene loci. In particular, animals in accordance with the invention possess human Ig loci that include plural variable (VH and Vκ) gene regions. Advantageously, the inclusion of plural variable region genes enhances the specificity and diversity of human antibodies produced by the animal. Further, the inclusion of such regions enhances and reconstitutes B-cell development to the animals, such that the animals possess abundant mature B-cells secreting extremely high affinity antibodies.

The present invention provides camel antibody libraries that maintain in vivo diversity of camelid antibody variable region genes. The in vivo diversity of antibody variable region genes can be accomplished by, for example, mixing genes derived from a plurality of animals or modifying gene amplification conditions. Conventional methods yield only VHHs with limited repertoire diversity. However, the present invention provides libraries comprising genes encoding functional VHHs with sufficient repertoire size. According to the present invention, libraries that enable to freely obtain VHHs against arbitrary antigens are provided. VHHs have excellent solubility and stability, and show a reactivity that usually cannot be expected from tetrameric IgGs.

The invention discloses an anti-human Tim-3 neutralized monoclonal antibody L3D and an application thereof. Antibody light and heavy chain variable region genes are cloned from a prepared anti-human Tim-3 neutralized monoclonal antibody L3D hybridoma cell; and the obtained light chain and heavy chain variable region genes can be used for encoding a monoclonal antibody variable region. Based on the light and heavy chain variable region genes of the monoclonal antibody, a plurality of small molecular genetic engineering antibodies can be constructed and expressed. Polypeptides or proteins encoded on the basis of the genes can be cross-linked with a plurality of bioactive molecules, so that a Tim-3 expression level detection reagent is prepared for diagnosing and treating diseases caused by abnormal expression of Tim-3.

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses a single light chain variable domain derived from a single rearranged light chain variable region gene in the germline of the non-human animal, wherein the single rearranged light chain variable region gene comprises a substitution of at least one non-histidine encoding codon with a histidine encoding codon. Methods of making non-human animals that express antibodies comprising a histidine-containing universal light chain are provided.

The invention discloses a variable region of heavy and light chains of an anti-human IL-13R alpha 2 single clone antibody. The invention uses recombinant human IL-13R alpha 2 to immunize a BALB / c rat to prepare a group of rate anti-human IL-13R alpha 2 single clone antibody and screen anti-human IL-13R alpha 2 single clone antibody FMMU-IL-13R alpha 2-7 with high affinity. The variable region genes of heavy and light chains of the single clone antibody are cloned to obtain the sequences of genes and amino acid in the variable region of the heavy and light chains of the single clone antibody, and confirm the uniqueness of the gene and protein sequences. The amino acid sequence of the variable region and the gene sequences encoding the variable zones have great potential application value in single chain antibody, chimeric antibody, humanized antibody or vaccine for treating malignancy with human IL-13R alpha 2 as the target.

The invention provides a human anti-vascular endothelial cell growth factor (VEGF) antibody as well as a coding gene and an application thereof. According to the invention, by virtue of genetic engineering means and a phage surface display technology, an anti-VEGF genetic engineering single-chain antibody is screened from a complete synthesis single-chain human antibody library, a variable region gene sequence of the anti-VEGF genetic engineering single-chain antibody is obtained, amino acids in a CDR (complementary determining region) of the anti-VEGF genetic engineering single-chain antibody are transformed by virtue of bioinformatics means, and finally a series of high-affinity anti-VEGF antibodies are obtained by constructing and screening mutation libraries and utilizing a chain exchange method, wherein affinity of the anti-VEGF antibodies with human VEGF is 10<-9>-10<-11>M. According to the invention, identification on immunocompetence and biological activity of the antibody is completed, the anti-VEGF antibody is verified to have the characteristic of being antagonistic to combination of VEGF and VEGFR2 and can effectively inhibit proliferation of vascular endothelial cells induced by VEGF. The invention provides a new specific candidate antibody molecule for resisting tumour and senile macular degeneration targeted by VEGF in future.

The invention discloses a process for preparing variable zone genes of high affinity tumor necrosis factor (TNF) resistant monoclonal antibody (F6 mAb) comprising, using recombinated human TNF immune BALB / c mouse to prepare a plurality of mouse Anti-TNF monoclonal antibody, screening high affinity F6 mAb using indirect ELISA method. By cloning the monoclonal antibody light chain and heavy chain variable zone genes, the monoclonal antibody light chain and heavy chain variable zone gene sequence and amino acid sequence can be obtained, and the unicity of the gene sequence and protein sequence can be confirmed.

Described herein are antibodies that bind to human tumor necrosis factor alpha (hTNFα). And list the nucleotide sequence of antibody heavy chain and light chain variable region and its derived amino acid sequence. The heavy chain and light chain variable region genes are respectively connected with the human immunoglobulin gamma 1 (hIgG1) heavy chain constant region and human kappa (k) light chain constant region genes to form a chimeric gene. Then, the vector containing the chimeric gene is introduced into the host cell line to express the antibody protein. This recombinant chimeric antibody protein can neutralize the activity of hTNFα in vitro, and is suitable for treating patients with excessive secretion of harmful hTNFα, including certain inflammations, such as rheumatoid arthritis and Crohn's disease.

The present invention relates to the anti-human hepatocarcinoma McAb HAb18 heavy chain and light chain variable region genes, the polypeptides encoded by the same, as well as to their use in the preparation of a medicament for diagnosing and treating tumors or inflammation diseases. Based on the heavy chain and light chain variable region genes, various novel small molecule genetic engineering antibodies, including single chain antibodies, chimeric antibodies, Fab antibodies and the like can be constructed and expressed for the diagnosis and treatment of hepatoma.

The invention relates to gene engineering and antibody preparation and specifically discloses a preparation method and application of a high-throughput fully-humanized antibody. The preparation methodcomprises the following steps: separating peripheral blood monouclear cells; separating single cells of plasma cells or antigen-specificity memory B blymphocytes; amplifying the heavy-chain and light-chain gene variable regions of single B cell antibody by use of a primer provided by the inventor; establishing an expression system containing antibody heavy-chain and light-chain genes by use of alinear expression system containing a heavy-chain fragment or light-chain fragment constant area; finally, separating and purifying a fully humanized monoclonal antibody. By adopting the primer combination provided by the invention to amplify the genes in the antibody heavy-chain and light-chain variable areas, natural pairing of the light-chain and heavy-chain variable areas can be maintained, and the preparation method has the advantages of high gene diversity, high titer, full humanization, high antibody affinity, strong specificity, no heterologous serum reaction, no risk of propagating other infectious diseases, etc.

A genetically modified mouse is provided, wherein the mouse is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa (K) constant gene at the endogenous mouse K locus, wherein the mouse expresses a reverse chimeric antibody having a light chain variable domain derived from one of only two human light chain variable region gene segments and a mouse K constant domain,. and a human heavy chain variable domain and a mouse heavy chain constant domain, from an endogenous mouse heavy chain locus. Bispecific epitope-binding proteins that are fully human are provided, comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments.

The invention relates to gene engineering and antibody preparation and specifically discloses a preparation method and application of a high-throughput fully-humanized antibody. The preparation methodcomprises the following steps: separating peripheral blood monouclear cells; separating single cells of plasma cells or antigen-specificity memory B blymphocytes; amplifying the heavy-chain and light-chain gene variable regions of single B cell antibody by use of a primer provided by the inventor; establishing an expression system containing antibody heavy-chain and light-chain genes by use of alinear expression system containing a heavy-chain fragment or light-chain fragment constant area; finally, separating and purifying a fully humanized monoclonal antibody. By adopting the primer combination provided by the invention to amplify the genes in the antibody heavy-chain and light-chain variable areas, natural pairing of the light-chain and heavy-chain variable areas can be maintained, and the preparation method has the advantages of high gene diversity, high titer, full humanization, high antibody affinity, strong specificity, no heterologous serum reaction, no risk of propagating other infectious diseases, etc.

The invention relates to a gene assembly method of phage genetically engineered antibody library, belonging to the field of biomedical study and clinical applications. The method aims to overcome the problems of the prior art, including poor stability, low efficiency, high mutation probability and low fidelity, and indirect application to humanized single chain antibody study. The technical proposal adopted by the invention comprises the following steps of: obtaining heavy chain variable region gene and light chain variable region gene of a humanized antibody by RT-PCR, and ligating the antibody heavy chain variable region, a linker and the antibody light chain variable region with the pre-designed restriction enzyme cutting sites on two ends of primers using PCR and restriction enzyme method at the same time.

The invention discloses an anti-CD19 engineering antibody and its application which is used in target direction combining lymphocytic leukemia cell. It lays a foundation for the next trituration genetic engineering medicine of the target direction therapy leukemia. It relates to anti-CD19 monoclonal antibody HI19a heavy and light chain variable region gene, and application of the gene code polypeptide, the gene carrier, and using the gene and polypeptide to make leukemia therapy medicine. The heavy and light chain variable region gene is come from the anti-CD19 monoclonal antibody HI19a. The invention successfully adopts gene engineering technique to make anti-CD19 gene engineering antibody, and lays a foundation for the target direction therapy of the leukemia.

The invention relates to an anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and a preparation and an application thereof. The antibody is VH-connecting peptide-VK of a three-structural single-stranded antibody comprising a variable region of heavy chain and a variable region of light chain by using 12 sequences of amino acid of the 1 (CH1) 5' end of a constant region of a heavy chain as the connecting peptide AKTTAPSVYPL, and the antibody realizes efficient expression in a prokaryotic system. The biological characteristic studies show that the RD9 is an anthropogenic genetic engineering antibody which has high appetency and better stability and can specially centralize rabies virus as well as genes and gene products of the antibody can be used for preparing clinical drugs for preventing and treating the rabies; the preparation method of the antibody is easy for massive industrial production. The invention solves the problem in the other technology of the application of genes of the variable region of the heavy chain and the variable region of a light chain of the antibody and polypeptide coded by the genes in the drugs for preventing and treating the rabies.

The invention provides a human McAb to tetanus toxin, which is a human antibody comprising a variable region of the antibody. The antibody consists of or not consists of a constant region of the antibody and has the ability of neutralizing the tetanus toxin, with the variable region gene and protein sequence indicated as the sequence table 1. The antibody is characterized in that the antibody can not induce obvious allergic reaction, has higher titer and longer effect without any animal virus pollution, and can be produced in an unlimited quantity. The antibody also provides the relating biological functions, the testing methods, the manufacturing method and the application.

The invention discloses a B7.1-CD19scFv merge gene engineering protein and usage to treat B lymphocyte leukosis, lymph tumor, which comprises the following parts: human B7.1 external cell area, antihuman CD19 monoclonal antibody heavy chain and light chain variable area gene.

The present invention relates to a humanized single anti-Hu-ScFv18 light and heavy chain variable region gene for resisting HAb18G / CD147 and A coding polypeptide thereof and application thereof. The invention uses a set of designed primers to humanizedly reconstruct the anti-HAb18G / CD147 single light and heavy chain variable region gene excreted from HAb18 hybridoma, and the humanized single anti-Hu-ScFv18 heavy chain and light chain variable region gene may encode a correct antibody variable region. Based on the cloned anti-HAb18G / CD 147 humanized single anti-Hu-ScFv18 light and heavy chain variable region gene, it is capable of constructing and expressing various small molecule genetic engineering antibodies such as single-chain antibody, jogged antibody, Fab antibody and the like against the HAb18G / CD 147 molecule by a genetic engineering method, so as to be used for diagnosis and treatment of hepatocarcinoma.

The invention relates to a preparation method of the completely humanized antibody of a infectious disease pathogen, which mainly comprises the following steps of: preparing an infectious disease pathogen antigen; fluorescently labeling an infectious disease pathogen antibody; concentrating and purifying the peripheral B cells of an infectious disease patient; combining the labeled pathogen antigen and the antigenic-specificity B cells; screening antigenic-specificity single B cells through a flow cytometer; carrying out single cell RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on genes positioned in the heavy chain and light chain variable region of the infectious disease pathogen antibody; connecting with a heavy chain and light chain constant region to construct the eukaryotic expression vector of the humanized antibody; and carrying out the high-efficiency expression and purification of a recombined antibody in an eukaryotic cell. The humanized monoclonal antibody prepared through the method disclosed by the invention can be used for the infectious disease diagnosis and the infectious disease treatment.

The invention discloses an anti-human PCSK9 (pro-protein convertase subtilisin / kexin 9) chimeric antibody, and preparation and application thereof. The preparation method comprises the following steps: respectively amplifying mouse light chain and heavy chain variable region genes from mouse hybridoma cells, respectively carrying out chimerism with light chain and heavy chain constant region genes of human IgG, and carrying out recombination expression to obtain the human-mouse chimeric antibody. The human-mouse chimeric antibody has favorable affinity with human PCSK9, obviously inhibits the degradation activity of the PCSK9 for liver cell low-density lipoprotein receptors (LDLR), enhances the ingestion of liver cells for LDL-cholesterol (LDL-C), and lowers the cholesterol level in blood.

The invention discloses a single-chain antibody of broad-spectrum anti-p21ras protein and a preparation method thereof. The gene segment of the single-chain antibody is constructed by connecting light and heavy chain variable region gene segments of monoclonal antibody hybridoma cell strains of the broad-spectrum anti-p21ras protein through flexible oligonucleotide linkers; the gene segment of the single-chain antibody is connected with a phagemid expression vector to construct recombinant phagemid; the recombinant phagemid is converted into amber inhibiting colon bacillus TG1 to perform fusion expression; the positive recombinant phagemid is identified by using indirect ELISA screening through phage library exhibition; the positive recombinant phagemid is converted into non-amber inhibiting colon bacillus BL21 (DE3) to perform soluble expression of the single-chain antibody; indirect enzyme-linked immunosorbent assay and immune cell and tissue chemical experiments prove that the single-chain antibody for the soluble expression has strong binding specificity with three p21ras proteins of H-ras, K-ras and N-ras and high affinity; and the single-chain antibody is used for constructing small molecular antibodies such as intracellular antigens and the like and used for diagnosis and research of ras gene related tumors.

The invention relates to an anti-DON single-chain antibody ScFv and a preparing method and application thereof, belonging to biological product field. In the invention, variable region gene Fab section of heavy chain and light chain of antibody is cloned through a PCR method in a hybridoma cell strain McGill078 which secretes anti-DON monoclonal antibody; two genes are recombined through a gene engineering method; a prokaryotic expression plasmid vector is constructed and is transformed to Escherichia coli to express an ScFv antibody active fragment which can specifically recognize DON; and then the recombined single chain antibody ScFv is purified and renatured. The anti-DON recombined single-chain antibody ScFv has an activity for binding with DON and has the advantage of residual detection for DON owing to recognition and binding activity to DON and having antibody medicament prospect for treating DON poisoning after humanized modification; and the constructed transformation vector plasmid is easy to preserve and produce in a great quantity.

The invention relates to a preparation method for a fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses, belonging to the technical field of preparation of antibodies. The preparation method comprises the following steps: 1) performing primary, secondary and tertiary immunization on cattle by using O-type foot-and-mouth disease viruses; 2) screening antigen-specific individual B cells of the O-type foot-and-mouth disease viruses; 3) amplifying heavy-chain and light-chain variable genes of a bovine antibody; 4) acquiring the heavy-chain and light-chain constant region sequences of the bovine antibody; 5) preparing a full-length heavy-chain vector of a fully-bovine-derived monoclonal antibody and a full-length light-chain vector of the fully-bovine-derived monoclonal antibody; and 6) applying the full-length heavy-chain vector and the full-length light-chain vector of the fully-bovine-derived monoclonal antibody to cotransfection of cells, taking a cell culture supernatant, and purifying the supernatant so as to obtain the fully-bovine-derived broad-spectrum neutralizing antibody against O-type foot-and-mouth disease viruses. The preparation method of the invention utilizes different foot-and-mouth disease virus strains for infecting cattle and carries out screening to obtain the neutralizing antibody capable of neutralizing O-type foot-and-mouth disease viruses of three pedigrees; and the preparation method can prepare the fully-bovine-derived broad-spectrum neutralizing antibody.

The invention discloses an antibody combined with human tumor necrosis factor Alpha (hTNF a), juxtaposing out antibody heavy chain and nucleotide sequence of light chain variable region, and the amino acid sequence derived from the nucleotide sequence. The heavy chain and the light china are connected into chimeric genes with the genes in the human immunoglobulin gamma 1(hIgG1) heavy china constant region and in the kappa (k) light chain constant region respectively, then the carrier with the chimeric genes is introduced into the host cell line expressing antibodies protein. The restructuringchimeric antibody protein is tested outside human body and can neutralize the hTNF Alpha activity. The invention is suitable for treating disease of excessive harmful hTNF Alpha secretion, comprisinga plurality of inflammations such as rheumatoid arthritis and regional enteritis.

The invention discloses a preparation method for a monoclonal antibody. According to the method, magnetic granular microballoon spheres of an immobilized antigen are added into B lymphocyte culture micropores, magnetic separation is carried out on the magnetic granular microballoon spheres so as to allow the spheres to enter into corresponding microplates, a single B lymphocyte which secretes a specific antibody is detected by using the method of immunochemiluminescence or immunofluorescence, a light chain variable region gene and a heavy chain variable region gene of the antibody are amplified by using the method of single-cell RT-PCR and nested RT-PCR and are recombined to expression plasmid containing a constant domain of the antibody, and a host cell is transfected to express the monoclonal antibody.

The invention relates to heavy chain and light chain variable region genes of a monoclonal antibody of a human vascular endothelial growth factor, a polypeptide that is encoded by the genes, a carrier that contains the genes, and application of the genes and the polypeptide in the preparation of a clinical testing agent of VEGF-related tumors. The invention utilizes gene engineering means to firstly obtain the heavy chain and light chain variable region genes of the monoclonal antibody from a hybridoma cell strain of the monoclonal antibody with VEGF165 secretion, the heavy chain variable region gene has the nucleotide sequence with SEQ ID NO of 1, the light chain variable region gene has the nucleotide sequence with SEQ ID NO of 3, and the genes are combined with scFv small molecule antibody and cloned into a pET28a expression carrier, have high-efficiency expression in colon bacillus and generate protein that has reserved antibody combination capability and VEGF165 action function. Through the modification with gene engineering techniques, the invention establishes the foundation for biological missile construction, has ultra-convenient antibody production and greatly lowered cost, and is favorable for industrial production.

The invention relates to a heavy and light chain variable region gene of monoclonal antibody resisting human amyloid and coded polypeptide thereof. The invention also relates to the application of the gene and the polypeptide in preparing reagents and drugs for diagnosing Alzheimer's disease, and clones the light and heavy chain variable region genes of the antibody from cultured oligomer high affinity antibody A8 hybridoma. The obtained gene can correctly code a mouse antibody variable region. Based on the cloned A8 antibody light and heavy chain variable region genes, a gene recombination method can be adopted to construct and express the chimeric antibody, single chain antibody, Fab and other genetic engineering anbodies of a plurality of micromolecules in the hope of being used for diagnosing and treating Alzheimer's disease.

The invention discloses a porcinized single-chain antibody resisting a porcine epidemic diarrhea virus and a preparation method of the porcinized single-chain antibody, and belongs to the field of biotechnology. A gene sequence of a heavy-chain variable region of the single-chain antibody is as shown in SEQ ID NO.1; the gene sequence of a light-chain variable region is as shown in SEQ ID NO.2. The single-chain antibody also comprises the gene sequence of a porcine anti-body constant region Fc segment as shown in SEQ ID NO.3, wherein the gene sequence of the heavy-chain variable region is connected with the gene sequence of the light-chain variable region through a linker sequence as shown in SEQ ID NO.4. The length of scFv-Fc is 1128bp; the molecular weight translated into a polypeptide chain is about 40kDa and is 1.4 that of natural immune globulin; the penetrability between tissues can be improved by reduction of the molecular weight; and the Elisa result shows that PEDV can be identified by the scFv-Fc.