930 results about "Mammalian cell" patented technology

This document relates to materials and methods for modulating ligand gated ion channel (LGIC) activity. For example, modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD) are provided. Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC, as well as methods of modulating ion transport across the membrane of a cell of a mammal, methods of modulating the excitability of a cell in a mammal, and methods of treating a mammal having a channelopathy.

The invention belongs to the field of molecular biology and immunology, and particularly relates to an anti-CD3 nano antibody capable of activating the activity of T cells as well as a preparation method and application of the anti-CD3 nano antibody. The invention provides a preparation method of the anti-CD3 nano antibody. The preparation method comprises the following steps: constructing a CD3 plasmid and an overexpression cell line, and immunizing alpaca to obtain peripheral blood mononuclear cells (PBMC) of the alpaca; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription to obtain cDNA; carrying out PCR amplification on the antibody gene segment and cloning the antibody gene segment to a phage display vector; and electrically transforming the vector into SS320 competent cells to obtain the nano antibody library. After two rounds of panning, detecting and screening an antibody clone capable of being combined with the CD3 protein through ELISA (Enzyme-Linked Immunosorbent Assay); an antibody sequence is selected through sequencing, and an antibody protein is expressed by using a mammalian cell high-throughput expression system; and then detecting and screening the antibody which is only specifically combined with the CD3. The method disclosed by the invention is simple, convenient and rapid to operate, and can be used for preparing active antibodies for treating potential tumors and proliferative diseases.

The invention discloses a mammalian cell expression vector, an expression plasmid and an expression system. The expression vector sequentially comprises the following elements: (a) a promoter; (b) a Kozak sequence; (c) a polyadenine sequence, the polyadenine sequence sequentially comprises a bGH PolyA sequence and a 5 'UTR PolyA sequence, the nucleotide sequence of the bGH PolyA sequence is as shown in SEQ ID NO.5, and the nucleotide sequence of the 5' UTR PolyA sequence is as shown in SEQ ID NO.6; (d) copying the start site; and (e) screening marker genes. According to the present invention, the expression vector is optimized by the expression element, such that the exogenous gene can be conveniently, efficiently and flexibly inserted and edited by the expression vector so as to achieve the stable extracellular or intracellular expression of the exogenous protein, and the expression efficiency of the exogenous protein can be improved by carrying out the expression plasmid construction based on the expression vector; and more efficient expression and purification of the foreign protein are realized.

The invention relates to a biotechnology, and particularly discloses a plasmin degradation product D-dimer antibody and application thereof. According to the application, a single B cell antibody preparation technology and a flow cytometry sorting method are applied to the development of a blood coagulation in-vitro diagnosis fibrinolysis system detection antibody for the first time, and a mammalian cell expression system (293F) is adopted to successfully express and obtain three strains of D-dimer monoclonal antibodies (antibody numbers are DD-1, DD-2 and DD-3) with biological activity. Compared with the on-sale Roch D-dimer antibody, the three antibodies have the same affinity with the DD antigen, and can identify and detect the D-dimer in plasma with high specificity and high sensitivity; meanwhile, the probe has no cross reaction with fibrinogen, primary fibrinolytic substances (X, Y and D) and soluble fibrin (SF), and can be used for qualitative or quantitative detection of D-dimer.

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 present invention belongs to the technical field of bioengineering. Provided are an IscB mutein and the use thereof. Compared to the wild-type IscB protein, the IscB mutein comprises arginine at at least one of the following amino acid positions: position 84, position 96, position 102, position 111, position 159, position 368 and position 386; preferably, the IscB mutein further comprises arginine at position 401 and position 456. The relatively strong interaction between the IscB mutein and a double-stranded DNA target improves the editing activity for mammalian cell lines, which can greatly enhance the application potential of the IscB mutein as a new-generation underlying tool for gene editing, and promote the use of the IscB mutein in the aspects of precision medicine, animal model construction and crop breeding.

The invention features compounds (e.g., macrocyclic compounds) capable of modulating biological processes, for example through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein). These compounds bind endogenous intracellular presenter proteins, such as the FKBPs or cyclophilins, and the resulting binary complexes selectively bind and modulate the activity of intracellular target proteins. Formation of a tripartite complex among the presenter protein, the compound, and the target protein is driven by both protein-compound and protein-protein interactions, and both are required for modulation of the targeted protein's activity. In some embodiments, the compounds of the invention “re-program” the binding of the presenter proteins to protein targets that either do not normally bind to the presenter protein (e.g., do not show detectable binding in mammalian cells absent the compound). In some embodiments, provided compounds “re-program” presenter protein binding to greatly enhance interaction with a particular target with which it may have some interaction absent the compound. Interactions achieved through such reprogramming result in an ability to modulate the activity of these new targets.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and / or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs. In some embodiments, methods for targeted nucleic acid editing are provided.

Lipid nanoparticles (LNPs) encapsulating self-amplifying mRNA, compositions, and methods of use of the novel nucleic acid constructs and compositions are disclosed. The LNP constructs comprise a novel ionizable lipid. The novel sa-mRNA constructs encode a modified SARS-CoV-2 spike protein in which the polynucleotide has been truncated to comprise no nucleotides encoding SARS-CoV-2 transmembrane domain and short cytoplasmic domain amino acids, as well as immunomodulators. The sa-mRNA may be used as a therapeutic, diagnostic and / or prophylactic agent for mammalian cells or organs.

The invention relates to a triple-hole paper-based three-dimensional cell culture chip as well as a preparation method and application thereof. The chip comprises a paraffin hydrophobic region and a paper-based hydrophilic region. The preparation method mainly comprises the following steps: 1) designing a mask plate; 2) cutting the mask plate by laser; and 3) preparing the triple-hole paper-based chip by hot pressing. The thickness of the chip is 100-200 [mu] m, the porous fiber structure of the paper-based material allows transportation of oxygen and waste, and a three-dimensional microenvironment for in-vivo cell growth can be simulated. Meanwhile, a transparent and liquid-tight supporting layer is fused on the lower layer of the paper-based chip, so that leakage of cells inoculated on the upper layer of the paper-based chip is avoided. The paper-based chip is embedded into a 6-pore plate, and bladder cancer cells (5637), mouse astrocytoma (U87) and human breast cancer cells (MCF-7) are inoculated in a paper-based hydrophilic region respectively. The cell compatibility of the paper-based chip and the growth behaviors of three different types of cells in three-dimensional paper fibers are investigated. It is proved that the chip can be used for researching three-dimensional construction and growth of human and mammalian cells, and the difference between three-dimensional culture and two-dimensional culture is revealed by detecting the proliferation capacity and survival rate of different cells.

The invention provides recombinant human III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant human III-type collagen is shown as SEQ ID No.1, and the nucleotide sequence of a gene for coding the recombinant human III-type collagen is shown as SEQ ID No.2; the invention also provides a method for expressing the recombinant human III type collagen in mammalian cells. The recombinant human III-type collagen provided by the invention is high in purity, has triple helix characteristic circular dichroism, has the molecular weight of 21.8 kDa, is far smaller than that of natural full-length collagen, has the effects of promoting cell proliferation and cell migration, and reduces obstacles for the collagen to penetrate through a skin barrier to exert biological functions.

Reported herein is, inter alia, a method for producing a trivalent bispecific antibody, the method comprising the steps of culturing a mammalian cell comprising a deoxyribonucleic acid encoding the trivalent bispecific antibody, and recovering the trivalent bispecific antibody from the cell or culture medium, wherein the deoxyribonucleic acid encoding the trivalent bispecific antibody is stably integrated into the genome of the mammalian cell and comprises in the 5' to 3' direction a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the first light chain or the second heavy chain or the second light chain, and a seventh expression cassette encoding the second light chain, wherein the first heavy chain The chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, a peptide linker, a second heavy chain variable domain and a CL domain, the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain and a CH1 domain, and the second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain and a CL domain, wherein the second heavy chain variable domain and the first light chain variable domain form a first binding site, and the first heavy chain variable domain and the second light chain variable domain form a second binding site.

The invention relates to the technical fields of gene engineering, antibody engineering and immunodiagnosis. The invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody. The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the three complementary determining regions are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the heavy chain variable region comprises three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the three complementary determining regions are shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively. The invention also provides a method for preparing the monoclonal antibody. The recombinant humanized anti-Cpn IgM monoclonal antibody provided by the invention is expressed in mammalian cells by utilizing a gene recombination technology, has the characteristics of high purity, small batch difference and the like, and can be applied to a Cpn IgM antibody detection kit as a quality control product; as a quality control product, the recombinant humanized anti-Cpn IgM monoclonal antibody has the advantages of high sensitivity, high safety, inter-batch stability and the like.

The invention relates to an application of bifidobacterium breve i1088 or a postbiotic thereof in improvement of cognition. The bifidobacterium breve i1088 or the afterbiogen of the bifidobacterium breve i1088 is used for preparing a product for enhancing the oxidative stress capacity of mammalian cells, improving the in-vitro free radical scavenging level of mammals and changing at least one of the expression levels of mammalian cells Nrf2 and anti-oxidation related genes. The invention aims to explore the potential of the bifidobacterium breve in the aspect of improving the Alzheimer's disease and provide a safer and more effective nutritional supplement scheme for middle-aged and elderly people.

The invention belongs to the technical field of gene engineering, and particularly relates to a biotin ligase mutant as well as a coding gene, an expression vector, a recombinant cell and application thereof. The amino acid sequence of the biotin ligase mutant is as shown in SEQ ID NO. 2. The biotin ligase (Bar A) mutant provided by the invention can be efficiently expressed in eukaryotes, the expression level of the biotin ligase (Bar A) mutant is obviously improved in mammalian cells compared with a wild type, and the biotin ligase (Bar A) mutant has good catalytic activity after expression, can effectively catalyze biotinylation of protein containing a biotin receptor sequence, and has good application prospects. The method has a wide application prospect in the fields of biotinylated protein preparation, and protein labeling, positioning, immunodetection, affinity purification, function research and the like based on a biotin-avidin system (BAS).

This invention provides a nucleic acid construct comprising; (i) a first nucleotide sequence for expression, (ii) a first promoter operably linked to the first nucleotide sequence, said first promoter being inducible, (iii) a first insulating sequence located upstream of the first nucleotide sequence and first promoter, (iv) a second insulating sequence located downstream of the first nucleotide sequence and first promoter, (v) a second nucleotide sequence encoding a transcription regulator protein that induces expression from the first promoter; and (vi) a second promoter operably linked to the second nucleotide sequence. The second nucleotide sequence and second promoter may be located either (a) upstream of the first insulating sequence or (b) downstream of the second insulating sequence. Also provided are vectors and mammalian cells comprising the construct, methods of inducibly expressing a nucleotide sequence in the construct, and methods of producing a mammalian cell by inducing expression from the construct.

A Cas12 protein and a use thereof. The amino acid sequence of the Cas12 protein comprises or is a sequence having at least 50% sequence identity to SEQ ID NO: 1, and the amino acid sequence of the Cas12 protein comprises or is a sequence different from SEQ ID NO: 1 in amino acids at one, two or more positions. The amino acid sequence of the Cas12 protein comprises or is an amino acid sequence having at least 50% identity to SEQ ID NO: 18, and a PAM sequence identified by the Cas12 protein is A. By carrying out rational and non-rational mutation on the amino acid sequence of a natural Cas12 protein, the gene editing efficiency of the natural Cas12 protein in mammalian cells is improved. The Cas12 protein achieves higher editing efficiency and / or a lower off-target rate, and the PAM sequence is a single A base, so that many target sequences which are previously difficult to edit can be edited, thereby greatly expanding the editing range.

The invention discloses a decafluorobiphenyl-based Tat47-57 binding peptide, the amino acid sequence of the binding peptide is shown as SEQ ID NO.1 or SEQ ID NO.2, the fourth amino acid and the eighth amino acid of the SEQ ID NO.1 are bound through decafluorobiphenyl, and the fifth amino acid and the twelfth amino acid of the SEQ ID NO.2 are bound through decafluorobiphenyl. In particular, the decafluorobiphenyl-based Tat47-57 binding peptides (P3 and P6) are efficient CPPs, capable of delivering a variety of cargo molecules into the cytoplasm of mammalian cells. Meanwhile, decafluorobiphenyl cyclization plays a key role in cellular uptake and endosome escape. The two peptides P3 and P6 show remarkable cell permeability, and are speculated to enter cells through gridding protein dependent endocytosis. They also show efficient endosome escape ability, and have successfully applied P3 and P6 as CPPs to deliver phospholipid peptides and avidin.

The present invention belongs to the technical field of monoclonal antibody preparation, and specifically relates to a method for preparing a universal whole-pig monoclonal antibody. The present invention is based on single B cell antibody preparation technology, and antigen-specific single B lymphocytes are separated from the peripheral blood of immune pigs or clinically recovered pigs by flow cytometry sorting technology; according to the pig immunoglobulin IgG antibody gene sequence in the database, primers are designed to specifically amplify the complete coding genes of antibody heavy and light chains in pig single B cells and add His tags, and pCDNA3.1-Leader is used as a eukaryotic expression vector to construct a recombinant vector of complete coding genes for antibody light and heavy chains, and natural whole-pig monoclonal antibodies are expressed by mammalian cells. The present invention establishes a universal whole-pig monoclonal antibody preparation method for the first time, which can be used for the preparation and screening of pig monoclonal antibody libraries of different pathogens. Compared with other antibody preparation methods, it has the advantages of high throughput, whole pig source, good diversity, complete antibodies and natural pairing, etc., which provides technical support for the rapid preparation and screening of pig monoclonal antibodies and has broad application prospects.

The disclosure provides for cryopreservation formulations, cryopreservation media and method of cryopreserving mammalian cells or tissues. The cryopreservation formulations and media contain a combination of cryoprotective agents selected from the group consisting of; (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine: and (d) glycerol, trehalose, ascorbic acid, and taurine. In certain embodiments, the cryopreservation formulations and media are free of dimethyl sulfoxide.

The invention relates to a biotechnology, and particularly discloses a soluble fibrous protein (SF) antibody and an application of the soluble fibrous protein antibody. According to the present invention, the single B cell antibody preparation technology and the flow cytometry sorting method are firstly applied to the blood coagulation in vitro diagnosis fibrinolysis system detection antibody development, and the mammalian cell expression system (293F) is adopted to successfully express and obtain the three strains of the SF monoclonal antibodies (the antibody numbers are SF-1, SF-2 and SF-3) with the biological activity; the three antibodies have specific reactions with SF, have no cross reactions with primary fibrinolytic substances (X, Y and D), can be applied to the fields of SF indirect ELISA and Western blotting detection and screening, make up the vacancy of domestic SF antibodies in the market, and realize fibrinolytic system detection integrity.

The disclosure features novel methods of producing nucleic acid lipid nanoparticle (LNP) compositions employing a modifying agent after formation of a precursor nucleic acid lipid nanoparticle, the produced compositions thereof, and methods involving the nucleic acid lipid nanoparticles useful in the delivery of therapeutics and / or prophylactics, such as a nucleic acid, to mammalian cells or organs to, for example, to regulate polypeptide, protein, or gene expression.

The invention relates to a biotechnology, and particularly discloses a plasmin degradation product FDP antibody and application thereof. According to the present invention, the single B cell antibody preparation technology and the flow cytometry sorting method are firstly applied to the blood coagulation in vitro diagnosis fibrinolysis system detection antibody development, and the mammalian cell expression system is adopted to successfully express and obtain the three FDP monoclonal antibody strains (the numbers are FDP-1, FDP-2 and FDP-3) with the biological activity; on an ELISA (enzyme-linked immuno sorbent assay) platform, the three FDP recombinant rabbit monoclonal antibodies have specific reaction with primary fibrinolytic substances (X and Y) and secondary fibrinolytic substances (DD); on an immunoturbidimetric platform, the FDP-1 has specific reaction with the primary fibrinolytic substance and the secondary fibrinolytic substance, and the FDP-2 only has specific reaction with the primary fibrinolytic substance on the immunoturbidimetric platform. The three FDP antibodies provided by the invention can make up the vacancy of domestic FDP antibodies on the market, ensure the joint monitoring suitability of D-dimer and FDP, and realize the detection integrity of fibrinolytic system.

The invention relates to the technical field of gene editing, in particular to a Cas9 protein mutant and application thereof. According to the present invention, the mutation is introduced at the position far away from the catalytic site to induce the Cas9 protein conformation change so as to obtain the new Cas9 protein mutant, only the Cas9-sgRNA target chain is cut, and the Cas9-sgRNA non-target chain is not cut, such that the new Cas9 protein mutant can be used for constructing the novel base editor, and the high-efficiency base editing can be performed on the mammalian cell genome. The Cas9 protein mutant disclosed by the invention is fused with a mutant TX with DNA binding deletion of 3 '-> 5' exonuclease TREX2, so that the gene editing efficiency of a 3'terminal generated by paired single-nick enzyme cutting can be greatly improved, and the safety of the Cas9 protein mutant is maintained.

The invention provides a method for obtaining engineered recombinant mammalian cells and application of the engineered recombinant mammalian cells, and relates to the technical field of biology. The method comprises introducing into a mammalian cell a minicircle DNA comprising a transposon and a polynucleotide encoding a transposase, the transposon comprising a fragment encoding an antigen binding domain. The method has the advantages of shortening the preparation period of recombinant mammalian cells, simplifying the process and reducing the cost.

Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations containing them. Also provided are methods of preparing and using the disclosed capsid-protein-mutated rAAV constructs in a variety of diagnostic and therapeutic modalities, including, inter alia, as mammalian cell-targeting delivery agents, and as human gene therapy vectors. Also disclosed are large-scale production methods for capsid-modified rAAV expression vectors, viral particles, and infectious virions having improved transduction efficiencies over those of the corresponding, un-modified, rAAV vectors, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and / or in vivo applications.

The disclosure features novel lipids and compositions involving the same. Nanoparticle compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Nanoparticle compositions further including therapeutic and / or prophylactics such as RNA are useful in the delivery of therapeutic and / or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.

The utility model discloses a bioreactor bubble-free air interchanger which comprises a shell, an air inlet pipe extends into the shell, an air outlet port of the air inlet pipe extends into the shell, the top of the shell is connected with an exhaust pipe, an air inlet port of the exhaust pipe is located at the top of the shell, a liquid passing hole is formed in the cylinder wall of the shell, and the liquid passing hole is communicated with the air inlet port of the air inlet pipe. A filtering body for liquid to pass through is arranged on the periphery of the shell and is of a porous net structure. Compared with the prior art, the bubble-free air interchanger of the bioreactor has the advantages that the bubble-free air interchanger of the bioreactor is made of 316 stainless steel materials, is small in occupied space and easy to install and operate, can achieve bubble-free air interchange in the bioreactor, eliminates bubble damage to cells while meeting the requirement of a culture system for oxygen concentration, and improves the air interchange efficiency of the bioreactor. The device is suitable for the culture process of mammalian cells sensitive to shear force. The device is flexible in application, can be adapted to bioreactors of different specifications, and is easy for process development and amplification.

To provide: recombinant Cpf1 nucleic acids and polypeptides for use in CRISPR / Cpf1 endonuclease systems; and mammalian cell lines encoding the recombinant Cpf1 polypeptides.SOLUTION: The invention includes utilizing the Cpf1 systems from Acidaminococcus sp., where CRISPR / Cpf1 endonuclease systems having a suitable AsCpf1 crRNA are selected from a length-truncated AsCpf1 crRNA, a chemically-modified AsCpf1 crRNA, and an AsCpf1 crRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these systems and reagents are also provided.SELECTED DRAWING: Figure 1

The invention relates to the field of preparation of materials for articular cartilage repair, in particular to collagen and chitosan cross-linked temperature-sensitive gel for articular cartilage repair and a preparation method of the collagen and chitosan cross-linked temperature-sensitive gel. The cartilage repair material prepared by the invention adopts the recombinant human collagen with a triple helix structure as a main raw material. The raw material is prepared by using a mammalian cell expression system, is close to protein folding and polymerization of natural protein, and has a spatial structure and modification necessary for active protein. The recombinant human collagen with the triple-helix structure is good in biocompatibility and free of rejection reaction and anaphylactic reaction, and the risk of disease transmission possibly caused by animal-derived collagen can be avoided. The injection type temperature-sensitive gel is liquid at low temperature and is converted into hydrogel at 37 DEG C, so that the injection type temperature-sensitive gel is suitable for being used as a transfer carrier for in-vivo injection treatment, secondary injury of an organism is effectively avoided, and the injection type temperature-sensitive gel has a wide clinical application prospect.