52 results about "Light chain gene" patented technology

The invention relates to transgenic animals bearing one or more human λ light chain loci. The invention also relates to methods and compositions for making transgenic animals that have incorporated human λ light chain loci. The invention further relates to methods of using and compositions derived from the transgenic animals that have incorporated human λ light chain loci.

Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a rodent (e.g., a mouse), wherein the non-human animals rearrange human immunoglobulin light chain gene segments in the context of heavy chain constant regions and express immunoglobulin-like molecules comprising human immunoglobulin light chain variable domains fused to heavy chain constant domains that are cognate with human immunoglobulin light chain variable domains fused to light chain constant domains.

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 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 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.

The present invention is characterized by that making the cysteine of 112th site of enterokinase light chain gene produce site-specific mutation, using colibacillus and microzyme to express enterokinase light chain variant, and utilizing Zn-Sepharose and STI-Sepharose affinity chromatography so as to obtain high-purity enterokinase light chain variant protein. As compared with wild enterokinase light chain said varient has higher stability and enzyme activity.

The invention relates to a synthetic method of an enterokinase light chain gene and a preparation method of an expression product of the enterokinase light chain gene, in particular to cow enterokinase light chain protein produced by adopting a DNA (Deoxyribonucleic Acid) recombination technology. The encoding gene of the protein is shown in a sequence (3) of a sequence list. The invention further discloses a fermentation and purification process of the cow enterokinase light chain protein. The optimized fermentation and purification method of the invention is utilized to successfully express the cow enterokinase light chain protein with high biological activity in a secretory expression mode. The cow enterokinase light chain protein is used as a tool protease for the specific cutting of fusion protein and is particularly suitable for researching the biological engineering pharmaceutical industry and gene engineering, biochemistry, molecular biology, and the like.

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 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.

Belonging to the technical field of genetic engineering antibodies, the invention specifically discloses a vascular endothelial growth factor receptor 2 (VEGFR2 or KDR) targeted fusion antibody JZA01of antibody JZB00 and interferon alpha (IFN alpha), a preparation method and application thereof. The invention also discloses the amino acid sequences of heavy chain and light chain immunoglobulin molecules of JZA01. The invention also provides a construction method of JZA01 heavy chain and light chain gene, CHO cells are transfected, monoclone is picked by limited dilution method, then by means of eukaryotic cell secretory expression and affinity chromatography purification, the fusion antibody can be obtained. The fusion antibody provided by the invention can specifically bind VEGFR2 and inhibit tumor angiogenesis, the coupled interferon part also can also play a direct tumor killing and immunoregulation role, and no toxic or side effect is generated at the same time, thereby better inhibiting tumor growth. The fusion antibody can inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and some tumor cells in vitro.

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.

A fusion gene encoding the fusion protein LhFVII-LDP is formed by linking the gene encoding the light chain of human blood coagulation factor VII and the gene encoding lidamycin prosthetic protein, and its nucleotide sequence is shown in SEQ ID NO. 3. A recombinant plasmid expressing the fusion protein LhFVII-LDP contains a fusion gene encoding the fusion protein LhFVII-LDP. The fusion protein LhFVII-LDP is expressed from the above-mentioned recombinant plasmid, and its amino acid sequence is shown in SEQ ID NO.4 in the sequence listing. The enhanced fusion protein LhFVII-LDP-AE is composed of the fusion protein LhFVII-LDP whose amino acid sequence is shown in SEQ ID NO.4 in the sequence listing and the enediyne chromophore AE. The fusion protein LhFVII-LDP and the strengthened fusion protein LhFVII-LDP-AE can be used in the preparation of medicines for treating tumors.

The invention discloses an anti-p185<erbB2> human-mouse chimeric antibody ChAb26. The anti-p185<erbB2> human-mouse chimeric antibody ChAb26 is mainly composed of a heavy chain gene H and a light chain gene L, and the heavy chain gene H and the light chain gene L are respectively connected to a mammary gland specific expression plasmid pBC1 in order to obtain p185<erbB2> human-mouse chimeric antibody ChAb26 mammary gland specific expression vectors pBC1-H and pBC1-L. The pBC1-H and pBC1-L are linearized, the linearized pBC1-H and pBC1-L are introduced to FVB mouse zygotes to produce a transgenic FVB mouse, and the anti-p185<erbB2> human-mouse chimeric antibody ChAb26 can be obtained through mammary gland specific expression with the transgenic FVB mouse as a mammary gland bioreactor. The antibody reduces the immunogenicity of the v monoclonal antibody, and a medicinal recombinant protein produced through the transgenic FVB mouse has the advantages of high output, low cost, high activity, easy large-scale formation, shortening of the manufacturing period of new medicines, and huge market potential.

The invention discloses a gene fragment sequence and application thereof for controlling silking of a lepidoptera-a major cotton leafroller (Syleptaderogata). According to silk fibroin light chain gene sequences of reported Corcyracephalonica and Galleriamellonella, a primer is designed by an RACE method and a target gene fragment is amplified. The full length of the gene fragment cDNA is 1087bp; a region from 46bp to 849bp is an open reading frame of the gene fragment, 267 amino acids are encoded; a 5' non-coding region has 45 basic groups; and a 3' non-coding region has 238 base groups. The gene fragment has a significant impact on the silking of the Syleptaderogata; and successful cloning of the gene fragment and detection on the gene sequence lay an important foundation for further researching and controlling the silking and leaf-rolling damage of the leafroller.

The invention discloses a Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and a method thereof. The plasmid takes a piggyBac transposon as a basis, carries an Amp resistant gene and contains a T4 ligase gene serving as an exogenous gene and a function expression box of a green fluorescent EGFP gene serving as a marker gene. The plasmid is constructed by using a molecular biology method, and two restriction enzyme cutting sites unique to ApaL and NheI are contained between DDDDK and a fibroin light chain gene poly. After the ApaL and NheI double restriction enzyme cutting universal plasmid is connected with the T4 ligase gene, the plasmid and an auxiliary plasmid are jointly injected into Bombyx mori zygote, and characteristics of the transposon are utilized to guide the green fluorescent protein gene and the T4 ligase gene into a Bombyx mori genome and to be stably inherited and expressed to obtain transgenic Bombyx mori. The transgenic Bombyx mori is screened with the help of the fluorescent marker gene, and a Bombyx mori silk gland cell is utilized to specifically synthesize T4 ligase protein.

The invention relates to a single chain antibody of human anti-placenta growth factor, which is encoded by a heavy-chain gene with an SEQ NO.1 sequence and light-chain gene with an SEQ NO.2 sequence.The invention also provides a preparation method thereof, comprising the following steps in sequence: amplifying human total RNA heavy-chain variable region, light-chain variable region and constant region gene of the whole set by a PCR technique, splicing and constructing an overall-length single-chain antibody cDNA library; expressing the cDNA library by an externally-coupled transcription/translation system to obtain an antibody-ribosome-mRNA compound; performing affiliation selection and clution on the compound by a magnetic bead coated by specific antigen; amplifying selected destinationantibody gene by the PCR technique; and expressing the amplified antibody gene using the externally-coupled transcription/translation system repeatedly to obtain the single-chain antibody. The antibody is adapted to prepare medicament for treating ovarian cancer.

A human artificial chromosome capable of being efficiently transferred to offspring (next generation) and a method of use thereof. That is, a human artificial chromosome in which a region of about 3.5 Mb to about 1 Mb containing the antibody lambda light chain gene derived from human chromosome 22 is linked to a chromosomal segment derived from another human chromosome, which can be transmitted through the reproductive system of non-human animals to offspring; a non-human animal having the artificial human chromosome and its offspring; a method for constructing the non-human animal; a method for constructing a human antibody using the non-human animal or its offspring; and a production human having the above-mentioned human artificial chromosome antibody mice.

The present invention relates to cell capture assay for the selection of a high producer cell line expressing anti-CD34 antibodies that recognize the CD34 membrane-protein in the cell membrane. The monoclonal antibody secreted by the hybridoma cell line 9C5/9069 binds to human CD34 and is used to isolate stem cells. The DNA sequences encoding for the antibody heavy and light chain have been identified, isolated from the hybridoma cells and cloned into appropriate expression vectors. After co-transfection of the heavy and light chain genes into HEK293T or in CHO cells either conditioned medium or purified antibody were assessed for binding to CD34 protein located in the cell membrane in different cell capture assays. The binding of the antibody to CD34-positive cells could be shown with these assays for several cell lines.