72 results about "Heavy chain gene" patented technology

The present invention relates to a method for the generation of VH heavy chain-only antibodies in a transgenic non-human mammal. In particular, the present invention relates to a method for the production of a VH heavy chain-only antibody in a transgenic non-human mammal comprising the step of expressing more than one heterologous VH heavy chain locus in that mammal.

A non-human mammal containing an endogenous lambda light chain gene locus, an endogenous kappa light chain gene locus and an endogenous heavy chain gene locus, each of which can re-arrange so that immunoglobulin heavy and light chain genes are formed and expressed in B-cells following antigen challenge but said loci have been mutated so that the ability to form functional immunoglobulin tetramers comprising re-arranged heavy and light chains produced from said mutated loci has been substantially reduced or eliminated.

The invention discloses a bombyx mori silk fibroin heavy chain gene mutant obtained by utilizing CRISPR / Cas technology and a mutation method and application. The mutation method includes: mixing cas9mRNA with sgRNA, micro-injecting a mixture into a bombyx mori egg, identifying genotype through PCR (polymerase chain reaction), screening out heterozygotes, and mutually mating the heterozygotes to obtain F1 generation in which pure heterozygotes appear; performing genotype detection on bombyx mori with phenotype, mating the bombyx mori with same genotype, screening out homozygotes which can be stably inherited to next generation, and then screening out the bombyx mori silk fibroin heavy chain gene mutant, wherein a core sequence of sgRNA is designed aiming at 1213-1236, 1274-1297 or 1349-1372 loci of bombyx mori silk fibroin heavy chain gene. By the method, an experiment material having economic value and scientific value is obtained quickly and efficiently, and a new material is provided for large-scale production of bombyx mori sericine cocoon and enabling the bombyx mori to serve as a bioreactor to express foreign protein.

A human artificial chromosome vector comprising a human antibody heavy chain gene, a human antibody light chain gene, and a human antibody surrogate light chain gene.

A transgenic non-human mammal containing a heterologous heavy chain gene locus that is capable of producing soluble heavy chain only antibodies and antigen-binding fragments thereof following immunization.

The invention relates to a silkworm fibroin heavy-chain gene mutation method, which specifically comprises the step of: acting mRNA (Messenger Ribonucleic Acid) of a coded zinc-finger nuclease sequence on loci 1325-1362 of a silkworm fibroin heavy-chain gene shown as SEQ ID NO:1 to form target positions for recognizing zinc-finger nuclease, thereby obtaining a series of silkworm fibroin heavy-chain mutated genes; and the mutated sequence can be applied to preparation of sericin and extrinsic proteins. Mutants provided by the invention have the following advantages that: (1) a posterior division of silkgland of a fibroin heavy-chain gene mutant provided by the invention serious degrades, a cocoon shell only contains the sericin synthesized and secreted by a middle division of silkgland, if the mutated strains are utilized to transgenically express the extrinsic proteins, the expressed amount and the purity of the extrinsic proteins can be greatly increased, and thus, a brand-new useful genetic material is provided for the development of a silkworm fibroin bioreactor; and (2) the cocoon shell of the mutated strain provided by the invention only contains the sericin, and thus, a new source is provided for the large-scale production of the sericin.

The invention relates to a preparation method of a mouse capable of producing a human antibody. The preparation method of the mouse capable of producing the human antibody comprises the step of carrying out hybridization on a transgenic mouse carrying a fragment containing a human antibody heavy chain gene locus part and a mouse carrying a fragment which is inserted into mouse genomic DNA (deoxyribonucleic acid) and contains an non-rearranged human antibody light chain gene locus part, wherein gene rearrangement and gene conversion can be carried out in the transgenic mouse by virtue of the human antibody heavy chain gene locus and the human antibody light chain gene locus, so as to produce various human immune globulins, and a mouse endogenous antibody heavy chain gene locus, a mouse endogenous antibody kappa light chain gene locus and a Lamda light chain gene locus are inactivated.

Belonging to the field of biotechnology, the invention relates to fully human monoclonal antibodies against Zika virus, antigen binding fragments and application thereof. The antibodies provided by the invention are determined by complementary determining region (CDR) specific gene sequences in antibody light chain and heavy chain gene variable regions and are antibodies specifically bound to Zikavirus envelope glycoprotein E (protein E) and effectively expressed in prokaryotic and eukaryotic cells. The antibody CDR region or partial or whole genes can be utilized to transform and produce genetic engineering antibodies of different forms in prokaryotic and eukaryotic cells and any expression system, and can prevent or treat Zika virus related diseases clinically.

The invention discloses a staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use. The staphylococcal enterotoxin gene engineering reshaped antibody has an amino acid sequence shown in the formula of SEQ ID No.1 in the sequence table. Through construction of light and heavy chain eukaryotic co-expression vectors of the staphylococcal enterotoxin monoclonal antibody, a high-efficiency expression and stable-secretion mammalian cell line and the gene engineering reshaped antibody having high singularity and strong affinity are obtained. The staphylococcal enterotoxin gene engineering reshaped antibody can be used in staphylococcal enterotoxin detection, cell indirect immunofluorescence detection and flow cytometry detection.

The invention relates to a human body beta-amyloid protein detection kit and application thereof. The human body beta-amyloid protein detection kit is prepared by the following steps: firstly, immunizing a mouse by use of antigen beta-amyloid precursor protein, separating out obvious lymph nodes in the spleen and the body of the mouse after confirming successful immunization, extracting total RNAs (Ribose Nucleic Acids), performing reverse transcription on the total RNAs to obtain cDNAs (complementary Desoxvribose Nucleic Acids), and amplifying the light and heavy chain genes of an antibody by virtue of PCR (Polymerase Chain Reaction); secondly, assembling and amplifying scFvDNA and constructing pCANTAB5E-scFv recombinant plasmid; thirdly, converting the plasmid to obtain a single-chain antibody phage library, and screening out to obtain target antibodies; and finally, assembling the target antibodies to form the human body beta-amyloid protein detection kit. The detection range of the screened antibodies is relatively wide from 0 to 1,000pg / ml, the linear correlation coefficient of the kit is high, the detection sensitivity of the kit can be 3.5pg / ml, and the kit achieves high stability; during detection, the reaction patterns of a two-step method and a room temperature oscillating method can be adopted, with low requirements on the laboratory conditions and the equipment.

The invention discloses a bombyx mori fibroin heavy chain expression system for fibroin and sericin expressing a target protein, and a preparation method and application of the bombyx mori fibroin heavy chain expression system. The expression system contains a fibroin heavy chain promoter 3 at the 5' end and an expression cassette of a heavy chain gene poly(A) sequence at the 3' end. The system isused to express a foreign protein, through the identification and detection analysis of transgenic bombyx mori, it is found that the presence of EGFP is detected in both the silk gland and the silk,and EGFP is also distributed in the sericin layer in the silk, so that the result indicates that in the absence of the C-terminal, the N-terminal is not an essential factor for the formation of the silk, and the absence of the C-terminal can make the protein be distributed in the sericin layer. The analysis of the N-terminal of fibroin provides an experimental and theoretical basis for the silk formation mechanism of the silk, and is conducive to creating a really practical bombyx mori silk gland bioreactor, maintaining the sustainable development of the silk industry, and promoting the long-term development of sericulture and insect science in China.

The invention belongs to the biotechnological field and particularly relates to a fully humanized single-domain antibody for CD16, an antigen combining fragment of the antibody and applications of theantibody and the antigen combining fragment in preparation of preparations for disease diagnosis and treatment. The invention discloses the fully humanized single-domain antibody capable of specifically combining CD16 or the antigen combining fragment of the antibody. The single-domain antibody is mainly characterized by being determined by a CDR (complementarity-determining region) specific genesequence existing in an antibody heavy chain gene variable region, and the effectively expressed antibody specifically combined with the CD16 is obtained from prokaryotic and eukaryotic cells. Different forms of genetically engineered antibodies can be modified and produced in prokaryotic and eukaryotic cells and any expression system by use of the CDR or partial or full gene of the body, and theantibody can be used for preparing preparations for clinical diagnosis or treatment of related diseases.

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 recombinant oncolytic vaccinia virus, a preparation method and application thereof. A thymidine kinase TK region of a genome of a recombinant oncolytic vaccinia virus comprises an anti-mouse / human TIGIT antibody genetic sequence, and the thymidine kinase TK region of the genome of the recombinant oncolytic vaccinia virus can express an anti-mouse / human TIGIT antibody. Theanti-mouse / human TIGIT antibody genetic sequence is formed by connecting an antibody heavy chain gene, 2A peptide and an antibody light chain gene in series, and the nucleotide sequence of the anti-mouse / human TIGIT antibody gene is shown as SEQ ID NO. 1. The recombinant oncolytic vaccinia virus oncolytic virus kills tumors through direct oncolysis and activation of a human immune system. The oncolytic vaccinia virus can effectively activate the immune response of T cells to tumor cells so as to exert multiple anti-tumor effects.

Provided is a method for Bombyx mori silk fibroin heavy chain mutation including causing the mRNA of a coded zinc-finger nucleases sequence to act upon a target point such as positions 1325-1362 of Bombyx mori silk fibroin heavy chain gene as expressed in SEQ ID NO:1 so as to obtain a series of Bombyx mori silk fibroin heavy chain mutated genes. Also provided is a mutation sequence prepared according to the method. Also provided is the use of the mutation sequence in the preparation of sericin and foreign proteins. Also provided is a Bombyx mori mutant including the described mutation sequence as a novel Bombyx mori salivary glands bioreactor, the posterior salivary glands of the mutants being severely degraded and the cocoon layer thereof including sericin synthesized and excreted by the middle salivary glands.

The invention discloses an efficient expression vector of an antibody. The efficient expression vector contains resistance genes and two target gene binding sites for binding the genes, the two target gene binding sites can be respectively combined with heavy chain genes and light chain genes of antibody molecules, the two target gene binding sites are respectively positioned in two expression units in the efficient expression vector and respectively form a heavy chain expression unit and a light chain expression unit of the antibody, and each expression unit also comprises a strong expression inductivity promoter positioned on the upstream of each target gene binding site. The efficient expression vector can simultaneously express the heavy chain and the light chain of the antibody, solve the problem about imbalanced expression of the heavy chain and the light chain of the antibody in the prior art, increase the expression rate of antibody protein and improve the screening effectiveness of subsequent monoclonal antibody cell strains.

The invention discloses a bombyx mori silk heart protein heavy-chain expression system with expression protein distributed on a bombyx mori silk glue layer and a preparation method and an applicationthereof, the bombyx mori silk heart protein heavy-chain expression system contains a target gene expression cassette, and the 5'end of the expression cassette is a signal peptide sequence of a bombyxmori silk heart protein heavy chain promoter 3; the 3'end is a heavy chain gene poly (A) sequence; when the system is used for expressing the target protein, the target protein only exists in a sericin layer and is not expressed in a silk fibroin layer, so that the system can be used for expressing the target protein in the sericin layer, the creation of a real and practical bombyx mori silk glandbioreactor is facilitated, and the long-term development of silkworm science and insect discipline in China is promoted; and experimental and theoretical bases are provided for the silk forming mechanism of the silk.

Provided is a monoclonal antibody, effective in diagnosing IgA nephropathy, that specifically recognizes and bonds to the hinge region of a polypeptide coded for by the immunoglobulin A1 heavy chain gene, which contains a serine / threonine-linked sugar chain with no galactose bound thereto. Also provided are a fragment of said antibody, a diagnostic agent using the provided antibody or antibody fragment, and a therapeutic agent containing the provided antibody or antibody fragment as an active ingredient.

The invention discloses an anti-paralichthys olivaceus immunoglobulin D monoclonal antibody. The anti-paralichthys olivaceus immunoglobulin D monoclonal antibody is secreted by hybridoma cells of a hybridoma cell strain JF-IgD, with the collection No.: CCTCCNO: C2014159, the collection unit: China Center for Type Culture Collection, the collection address: Wuhan University in Wuhan, Hubei of China, and the collection date: September 2, 2014. Indirect enzyme-linked immune reaction experiment results show that the anti-paralichthys olivaceus immunoglobulin D monoclonal antibody can be specifically bound with recombinant flounder IgD heavy chain proteins; immunoblotting results show that the anti-paralichthys olivaceus immunoglobulin D monoclonal antibody can specifically recognize recombinant protein encoded by flounder IgD heavy chain genes delta1-delta 4 and flounder natural IgD heavy chain proteins with the molecular weight of 120kDa. The anti-paralichthys olivaceus IgD monoclonal antibody is prepared, and an important tool is provided for detecting flounder IgD protein molecules and IgD surface positive (IgD +) lymphocytes.

The present invention provides a recombinant humanized anti-hepatitis B surface antigen (HBsAg) Fab antibody as human hepatitis B surface antibody and its preparation method. The preparation method includes the following steps: utilizing phage display technique to obtain the gene of a humanized anti-HBsAg Fab fragment, making expression in prokaryotic expression system, on this basis utilizing PCK technique to amplifying the light-heavy chain gene of Fab, constructing it into the methyl alcohol yeast expression vector, using two-step process to convert yeast, constructing Fab engineering bacterium, secreting, expressing and producing Fab antbody fragment. The Fab antibody has strong HBsAG combination activity and antigen specificity, and the expression product of said gene has high application value in clinical therapy of hepatic disease.

The invention discloses a single-chain antibody of an anti-schistosome monoclonal NP11-4 as well as a preparation method and application thereof, and relates to the field of gene engineering and the field of therapeutic drugs against schistosomiasis. The invention is to use a monoclonal antibody NP11-4 located at adult membrane, cercaria memberane, schistosomulum membrane, egg shell and miracidium in egg of Schistosoma japonicum, extract total RNA of hybridoma cells through a gene engineering technology, adopt RT-PCR for proliferation of genes VH and VL, and use overlap-extension PCR to connect the genes VH and VL into a single-chain antibody gene in the form of VH<-linker-VL, which is subsequently connected with a pBAD / gIIIA carrier and cloned into Top10F'of Escherichia Coli to induce a soluble expression of the target gene. The zone from amino acid No.1 to No. 122 of the expressed single-chain antibody of the anti-schistosome monoclonal NP11-4 is a heavy chain gene repertoire of the single-chain antibody (VH), and the zone from the amino acid No.141 to No.254 is a light chain gene repertoire (VL) of the single-chain antibody, wherein the linker between the heavy and light chain gene repertoires consists of 18 amino acids including repeated glycines and serines, and the single-chain antibody has 254 amino acids in full length.

The invention relates to a method for quantitative detection of the light and heavy chain gene copy numbers of antibody molecules in a transgenic animal cell CHO. An SYBR-Green quantitative PCR method is adopted to realize quantitative, accurate and high-flux detection of the light chain gene copy number and the heavy chain gene copy number of the antibody molecules. The method has the advantages of high detection sensitivity, wide linear range and reliable result, can be used for researching the stability of antibody cell strains and screening stable high-expression engineering cell strains.

The invention discloses a method for rapidly obtaining the coding region sequence of a goose ferritin heavy-chain gene and quantitatively detecting the expression of the gene, and primers thereof. The method comprises the following steps: extracting the total RNA from goose tissues, carrying out inverse transcription, and designing an FTH coding region sequence primer FTH-L (SEQ ID NO:1-2) and an FTH expression rule detection primer FTH-S (SEQ ID NO:3-4); and carrying out RT-PCR amplification, recovering and purifying the obtained product, carrying out target gene connection and conversion, and screening and sequencing a positive bacterial colony to obtain the FTH coding region sequence and the sequencing identification fluorescence quantitative PCR primers. The method has the advantages of low cost, high efficiency, obtaining of the complete coding region sequence only through one-time cloning, and rapid and accurate detection of the expression rule of the FTH in samples of different tissues and different growth periods, and lays a theoretic foundation for the clarification of the FTH structure and function, and the researches of the iron metabolism mechanism in the goose.

According to the invention, based on the reported light and heavy chain amino acid sequences of the anti-NKG2A antibody in the prior art, the method comprises the steps of: constructing a light and heavy chain mutation antibody library for affinity maturation to obtain the mutant antibody with improved affinity and / or improved dissociation constant; and constructing a human Fab heavy chain gene expression vector and a mammalian cell expression vector containing a human kappa subtype light chain constant region gene in a CDRs region of a mutant antibody, carrying out cross pairing on a heavy chain vector and a light chain vector of an antibody with mature affinity, screening to obtain an anti-NKG2A mutant Fab antibody, and connecting an Fc segment of the human antibody; and connecting a second antigen binding molecule (such as an anti-PD-L1 antibody) to the C end of the Fc segment through linker to obtain the bispecific antibody.

The present invention provides a producing method of streptococcus specific phage lyase. The lyase is composed of a heavy chain (about 50kDa / per chain) )PlyCA and a light chain (about 8kDa / per chain) according to the ratio of 1:8, its gene name is PlyC, having following nucleotide sequences: DNA sequences of PlyCA(sequence 1) and PlyCB(sequence 3) in sequence table code amino acid sequence (sequence 2) of PlyCA and amino acid sequence (sequence 4) of PlyCB in the sequence table. The invention adopts coli expression system, constructing recombinant plasmids containing lyase heavy chain gene PlyCA and light chain PlyCB respectively, transforming to BL21(DE3) cell, fermenting culture engineering bacteria, making PlyCA and PlyCB highly express. Streptococcus phage lyase PlyC is obtained by purifying and renaturing expression production. Compared with the conventional method, the method of the invention has low production cost, high product renaturation yield, strength product activity, safety production method, meeting mass production.

The invention relates to an antibody library. An immune globulin light chain variable region VL gene sequence is inserted between Xba and Sac restriction enzyme cutting sites of a Pcomb3 vector. An immune globulin heavy chain variable region VH gene sequence is inserted between Spe and Xho endo restriction enzyme cutting sites. The antibody library is characterized in that two gene fragments comprise the possible combination of antibody variable region light chain and heavy chain, and the antibody library can realize affinity enrichment with an avian influenza virus antigen, and can be applied to avian influenza immunodetection. The antibody library has the advantages that a high affinity antibody can be acquired without animal immunity; a preparation period is short; the antibody library is a phage antibody library which is constructed by taking mice natural immune globulin variable region light chain and heavy chain genes as sources, theoretically contains all possible antibodies in a mice, and is a specificity antibody library which can directly acquire different avian influenza viruses through screening; and the screening has the advantages of high efficiency, high flux, large selection base and high specificity, thus the phage antibody library has a wide application prospect in the aspect of the disease detection of avian influenza and other animals.

The invention discloses a cell strain constructing method for preparing an anti-EGFR completely-humanized monoclonal antibody. The method comprises the following steps: in a POOL screening step aftercells of Chinese hamster ovary cell dihydrofolate reductase deficient strains CHO / DHFR- complete a transfection step, screening cell strains integrated with a heavy chain gene by using an SFM4CHO culture medium without containing HT in the first stage, and screening out the cell strains integrated with a light chain gene by using Blasticidin in the culture medium at the same time; and in the second stage, adding MTX to increase copy numbers of the light and heavy chain genes integrated into cell strain genomes, as to improve an expression quantity. An efficient stable cell strain, of which theexpression quantity reaches 2.0-3.0 g / L, suitable for a large trial production process is firstly cloned and screened out, product SEC, pI-cIEF, a de-sugaring molecular weight, and antibody activitybased on the cells are remarkably improved, so possibility is provided for realizing industrialization of recombining the anti-EGFR completely-humanized monoclonal antibody in China.

The invention belongs to the biotechnological field and particularly relates to a fully humanized single-domain antibody for human TIM-3 (T cell immunoglobulin and mucin-3), an antigen combing fragment of the antibody and applications of the antibody and the antigen combining fragment. The invention discloses the fully humanized single-domain antibody for the immune checkpoint molecule TIM-3 and the antigen combing fragment of the antibody. The single-domain antibody is mainly characterized by being determined by a CDR (complementarity-determining region) specific gene sequence existing in anantibody heavy chain gene variable region, and the effectively expressed antibody specifically combined with the TIM-3 is obtained from prokaryotic and eukaryotic cells. Different forms of geneticallyengineered antibodies can be modified and produced in prokaryotic and eukaryotic cells and any expression system by use of the CDR or partial or full gene of the body, and the antibody can be used for preparing preparations for clinical diagnosis or treatment of autoimmune diseases, chromic virus infection, tumor and other diseases.

The invention discloses a human-derived anti-amylin antibody and a preparation method thereof, and belongs to the field of immune globulin. The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO:1, the light chain variable region amino acid sequence of the antibody is shown as SEQ ID NO:2, the heavy chain variable region DNA sequence of the antibody is shown as SEQ ID NO:3, and the light chain variable region DNA sequence of the antibody is shown as SEQ ID NO:4. The heavy chain gene is an IgG1 subclass VH3 gene family, and the light chain gene is a Lambda gene family. The human-derived amylin antibody is screened out by establishing a high-capacity phage antibacterial library, the antibody has good antigen specificity and antigen combination activity, production of an amylin detecting kit is guaranteed, and wide application prospects are achieved.

High expression locus vectors based, in part, on the ferritin heavy chain locus are disclosed. The vectors include distal 5′ flanking sequences and / or proximal 5′ regulatory sequences derived from ferritin heavy chain locus. The vectors include a site for insertion of heterologous sequences and proximal 3′ regulatory and distal 3+ flanking sequences. The proximal 3′ regulatory and distal 3′ flanking sequences are optionally derived from the ferritin heavy chain locus. Cells transformed with the vectors, and methods of producing heterologous proteins encoded by the vectors, are also disclosed.