22 results about "Eucaryotic cell" patented technology

The invention discloses a double-structural-domain recombinant human fibronectin, the sequence of which is shown as SEQ ID NO.3, and the double-structural-domain recombinant human fibronectin is formed by fusing a collagen binding structural domain and a cell binding structural domain of human fibronectin. The invention also provides a recombinant expression vector containing the protein coding gene and a host cell. The protein is efficiently expressed through eukaryotic cells and engineering bacteria escherichia coli, and a fermentation and purification process suitable for an industrial scale is established in a matched manner. Functional experiments prove that compared with commercially available fibronectin products with a single structural domain, the double-structural domain recombinant human chemical fibronectin disclosed by the invention can remarkably promote proliferation, migration, adhesion and differentiation activity of human skin fibroblasts, and has wide application potential in the fields of skin repair, skincare and biomedical materials.

This invention discloses a camel-derived CRP single-domain antibody, its preparation method, and its applications, relating to the biomedical field. The method involves enriching CRP antigen in alpaca leukocytes immunized with the antigen, followed by single-cell screening and sequencing. Through sequencing structural analysis, a recombinant plasmid of the camel-derived CRP antibody is constructed. This plasmid is cloned and transformed into eukaryotic cells to express related antibodies. Monoclonal antibodies with human CRP resistance are screened for expression. The VHH domain of the monoclonal antibody is cloned and expressed, and paired screening yields CRP-VHH monoclonal antibodies with antibody activity. This antibody can be used in ELISA kits, chemiluminescence kits, and latex kits, possessing the same functionality as existing kits. Furthermore, this antibody also has applications in the preparation of antitumor drugs.

Levels of expression of antibiotic resistance genes are increased up to six-fold by inserting a proteasome-targeting tag into transgenes expressed in eukaryotic cells. Various selectable marker proteins are combined with different destabilization domains, leading to up to 70% increase in transgene expression. The increase in expression varies highly depending on the engineered construct and the lines cells used. Increase in expression drives exosome loading of cargo proteins in some aspects. By increasing expression and by editing trafficking signals of cargo proteins, proteins that normally locate to the ER can be trafficked to exosomes. This disclosure discloses efficient exosome delivery of a wide variety of engineered proteins, including modified antigen proteins of SARS-CoV-2 and influenza, and other proteins such as a modified alpha galactosidase A, an extracellular domain of vascular endothelial growth factor fused to a constant region of a human immunoglobulin heavy chain, and modified trastuzumab heavy and light chains.

The application discloses a transient transfection method for improving recombinant protein expression of CHO cells, and relates to the technical fields of eukaryotic cell gene transfection and animal cell culture. A cell suspension is obtained by mixing a CHO cell and a serum-free CHO cell basic culture medium; DNA and a transfection reagent are added into the cell suspension to mix a transfection culture, 5-azadeoxycytidine acid solution is added into the transfection culture at 0h-24h to form a transfection system and continue the transfection culture; the transfection system is added with a feeding culture medium at 1d-4d after the transfection culture to carry out suspension culture; the suspension culture is centrifuged at 7d to separate and harvest cell culture supernatant to complete recombinant protein expression. According to the application, 5-azadeoxycytidine acid is added into the transfection culture at a specific concentration, so that the expression of recombinant protein of the CHO cell is significantly improved during the transfection expression.

The invention discloses an antibacterial peptide with multiple phytopathogen antibacterial activity, a biological antibacterial agent and application of the antibacterial peptide and the biological antibacterial agent, and the amino acid sequence of the antibacterial peptide is FKLIRNLIILRQAWKLIKRRR and is marked as RR-21. Experiments show that the antibacterial peptide disclosed by the invention has a similar alpha-helical structure with a known antibacterial peptide LL-37, and has antibacterial activity on plant pathogenic bacteria represented by pseudomonas syringae and plant pathogenic fungi represented by fusarium pseudograminearum and fusarium oxysporum; compared with the LL-37 polypeptide, the MIC of the RR-21 is 4-8 [mu] g / mL, and the RR-21 has a more significant bacteriostatic effect; meanwhile, under the conventional bacteriostatic concentration (less than or equal to 16 mu g / mL), the RR-21 shows no hemolytic activity; the above fully proves that the antibacterial peptide has high biological safety to eukaryotic cells while exerting multiple antibacterial effects, and can be used as an active component of a biological antibacterial agent, and the biological antibacterial agent has the potential of being developed into an efficient and safe plant disease resistant drug or a plant green pesticide.

The present invention relates to a method of screening for peptides capable of binding to a ubiquitin protein ligase (E3), successful binding being determined by detecting the amount of a test protein in the cell. The invention relates to a method for determining if a peptide binds or is capable of binding to a ubiquitin protein ligase (E3) and thereby leads to degradation of a test protein, wherein the peptide is between about 7 and 110 amino acids in length, the method comprising: providing in a eukaryotic cell a candidate peptide functionally linked to a test protein, under conditions enabling ubiquitination of proteins by an E3; and detecting the amount of test protein present in the cell; whereby, a reduced amount of the test protein determines the candidate peptide as a peptide that binds or is capable of binding to an E3 (an E3-binding peptide).

The present application relates to the technical field of novel coronavirus, and particularly relates to a novel coronavirus trivalent DNA vaccine and a preparation method. The trivalent DNA vaccine takes a recombinant plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 1 or 2 as an active ingredient. The trivalent DNA vaccine provided in the present application can express a fusion antigen protein of three kinds of antigen genes of the novel coronavirus in eukaryotic cells, and produce serum IgG antibodies against the three kinds of antigens of the novel coronavirus. In addition, the novel coronavirus trivalent DNA vaccine provided in the present application can immunize mice to produce T cells secreting various cytokines, and has a stronger T cell immune protection effect.

Here, the inventors developed a strategy to modify the chromatin status of HSPCs at the HBG promoters to reactivate HbF expression. In particular, the inventors used from 1 to 4 single guide RNA (sgRNA) molecules spanning from the -220 to the -20 region of the HBG promoters together with CRISPR-based epigenome editors, namely dCas9-CBPcore and Tet1-dCas9. The first editor is capable of inserting histone acetylation while the second editor is performing DNA demethylation. Both epigenetic marks are associated with active transcription and are present in fetal erythroid cells expressing HbF. This strategy allows the ex vivo modification of adult HSCs to recreate a fetal-like epigenetic context leading to HbF production. Moreover, it avoids the genotoxicity associated with classical genome editing tools relying on DNA cleavage. Thus, the present invention relates to a method of increasing the expression of gamma globin in a population of eukaryotic cells through epigenome editing.

This article describes a system comprising eukaryotic cells, wherein the eukaryotic cells include: a plasma membrane polypeptide coupled to a transcription factor via a linker, wherein the linker contains a protease-cleavable site; and a plasma membrane-anchored protease, wherein the plasma membrane-anchored protease is capable of cleaving the linker. The system may further include a reporter construct, wherein the transcription factor can bind to the promoter of the reporter construct to indicate the ability of the plasma membrane polypeptide to be transported to the plasma membrane.

The invention relates to a DHRS4 gene specific primer, a detection kit, an overexpression vector and application thereof, and belongs to the technical field of biological medicine. In order to overcome the defects in the prior art, the invention provides a specific primer, a detection kit and an overexpression vector of the DHRS4 gene, and the specific primer, the detection kit and the overexpression vector have remarkable beneficial effects and wide application prospects in the field of diagnosis and treatment of lung cancer and colorectal cancer. Researches prove that the expression level of the DHRS4 gene in tumor tissues of lung cancer patients and colorectal cancer patients is remarkably reduced, the expression condition of the DHRS4 gene can be rapidly detected by specific primers, and a reliable basis is provided for auxiliary diagnosis of lung cancer or colorectal cancer. The DHRS4 gene overexpression vector can effectively express DHRS4 protein in eukaryotic cells, proliferation, clone formation and migration of lung cancer and colorectal cancer cells are inhibited by restoring or enhancing expression of the DHRS4 gene, and a brand new strategy is provided for targeted therapy of lung cancer and colorectal cancer.

The application discloses a double-domain recombinant humanized fibronectin, the sequence of which is shown as SEQ ID NO. 3, which is composed of the collagen binding domain and the cell binding domain of human fibronectin. The application also provides a recombinant expression vector and a host cell containing the gene coding the protein. The protein is efficiently expressed by eukaryotic cells and engineered Escherichia coli, and a fermentation and purification process suitable for industrial scale is established. Functional experiments prove that, compared with the single-domain commercial fibronectin product, the double-domain recombinant humanized fibronectin of the application can significantly promote the proliferation, migration, adhesion and differentiation activity of human skin fibroblasts, and has wide application potential in the fields of skin repair, beauty and skin care and biomedical materials.

This invention relates to fusion proteins, immunogenic compositions, recombinant vaccines, and applications for the prevention of Toxoplasma gondii infection. From a reverse vaccinology perspective, this invention, through screening and optimization, provides a novel fusion molecular structure comprising a fusion protein encoded by two genes: the SPATR antigen and the MIC2 antigen. This fusion protein can be successfully translated, correctly folded, and secreted extracellularly in eukaryotic cells. Based on the aforementioned SPATR-MIC2 fusion mRNA vaccine, a 100% protective effect against Toxoplasma gondii infection can be achieved in a mouse model of Toxoplasma gondii infection, significantly superior to the inactivated vaccine group. The fusion protein of this invention exhibits good immunogenicity and can provide effective immune protection against Toxoplasma gondii infection, showing broad application prospects.

The invention discloses an anti-helicobacter pylori monoclonal antibody as well as preparation and application thereof, and belongs to the technical field of medical detection. The antibody is prepared by taking inactivated helicobacter pylori ATCC 43504 whole bacteria as an immunogen, immunizing a New Zealand white rabbit and then carrying out single B cell sorting, limited dilution, gene cloning, plasmid construction and eukaryotic cell expression, has a specific CDR core sequence, is high in specificity and affinity, can accurately recognize a helicobacter pylori specific antigen, and can be used for preparing a specific antibody. The cross reaction with other bacteria or tissue impurities is effectively avoided. The kit can be used for detecting the helicobacter pylori, and a new tool and method are provided for pathological diagnosis of helicobacter pylori infection and related digestive system diseases.

PI3Kδ implicates hematologic cancers and solid tumors. Alternative splicing is a post-transcriptional process for acquiring proteomic diversity in eukaryotic cells. Emerging evidence highlights the involvement of aberrant mRNA splicing in cancer development / progression. PI3Kδ-L and PI3Kδ-S are overexpressed in advanced solid tumors, such as prostate, breast, colon, lung and pancreatic cancers. Differential PI3Kδ and PI3Kδ-S expression profiles were identified in a panel of solid tumor cells. PI3Kδ inhibitor Idelalisib and SRPK1 / 2 inhibitor SRPIN340 were employed to assess their efficacies on inhibiting the PI3Kδ-expressing solid tumors. Idelalisib effectively inhibits PI3Kδ-L and its downstream signaling. Idelalisib fails to inhibit PI3Kδ-S activity and its downstream signaling. SRPIN340 reverses the aberrant mRNA splicing, thereby inhibiting the downstream AKT / mTOR signaling. In vitro functional assays further demonstrate that a combination of Idelalisib and SRPIN340 achieve a synergistic drug effect, with drastically reduced cell viabilities / growths of tumor spheroids, in inhibiting the advanced tumor cells.

The present invention relates to a method for producing N-acetylneuraminic acid, comprising the steps of: infecting eukaryotic cells with a baculovirus bearing an exogenous gene encoding an N-acetylglucosamine 2-epimerase and an N-acetylneuraminic acid aldolase, the N-acetylglucosamine 2-epimerase and the N-acetylneuraminic acid aldolase can be expressed by the aid of the N-acetylglucosamine 2-epimerase; and culturing the obtained eukaryotic cell infected by the baculovirus in the presence of N-acetyl glucosamine and pyruvic acid.

The application discloses a uORF element for regulating chicken insulin receptor expression and application thereof, belongs to the field of molecular biology and genetic engineering, and the nucleotide sequence of the uORF element is shown in any one of SEQ ID NO. 1-2; the application comprises any one of the following: (1) regulating the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) being applied to genetic breeding of chickens, and the genetic breeding is breeding of meat chickens with reduced insulin resistance; (3) application in preparation of a reagent for regulating the expression of chicken insulin receptors. The application can regulate the expression level of INSR, improve insulin sensitivity, and provide material support for research on the mechanism of insulin in regulating blood glucose.

This invention discloses a camel-derived CRP single-domain antibody, its preparation method, and its applications, relating to the biomedical field. The method involves enriching CRP antigen in alpaca leukocytes immunized with the antibody, followed by single-cell screening and sequencing. Through sequencing structural analysis, a recombinant plasmid of the camel-derived CRP antibody is constructed. This plasmid is cloned and transformed into eukaryotic cells to express related antibodies. Monoclonal antibodies with human CRP resistance are screened for expression. The VHH domain of the monoclonal antibody is cloned and expressed, and paired screening yields CRP-VHH monoclonal antibodies with antibody activity. This antibody can be used in ELISA kits, chemiluminescence kits, and latex kits, possessing the same functionality as existing kits. Furthermore, this antibody also has applications in the preparation of antitumor drugs.

The application discloses an anti-helicobacter pylori monoclonal antibody and a preparation and application thereof, and belongs to the technical field of medical detection. The antibody is prepared by using inactivated helicobacter pylori ATCC 43504 whole bacteria as an immunogen, immunizing a New Zealand white rabbit, and then performing single B cell sorting, limited dilution, gene cloning, plasmid construction and eukaryotic cell expression. The antibody has a specific CDR core sequence, high specificity and high affinity, can accurately recognize helicobacter pylori specific antigens, and effectively avoids cross reactions with other bacteria or tissue impurities. The antibody can be used for detecting helicobacter pylori, and provides a new tool and method for pathological diagnosis of helicobacter pylori infection and related digestive system diseases.

The invention provides a construction body for specifically inserting a DNA (Deoxyribonucleic Acid) fragment and a transgene site into a genome, wherein the construction body comprises recombinase. The enzyme includes a modified HIV-1 integrase (HIV IN) tetramer having a plurality of mutations to facilitate heterodimer construction of the heterodimer wherein a first outer monomer and a second outer monomer are bound to a first inner monomer and a second inner monomer, respectively. A transcriptional activator-like effector (TALE) binds to the N-terminal region of the outer monomer via a polypeptide linker. The two TALEs confer enzyme sequence specificity and prevent non-specific interactions with the target DNA structure. The modified HIV IN monomer ensures the binding of the TALE to the outermost layer monomer and the arrangement of the TALE relative to the catalytic region of the HIV IN. A polynucleotide is bound to an internal monomer of the HIV IN. Upon transport to the nucleus alone or within the lentiviral capsid, the TALE interacts with DNA to locate the HIV IN, which then integrates the polynucleotide.

The invention discloses a sheep echinococcosis genetic engineering subunit vaccine and a preparation method thereof, and belongs to the technical field of vaccines. The invention designs a recombinant protein which comprises an amino acid sequence as shown in SEQ ID NO. 1. The sheep echinococcosis EG95 recombinant antigen protein is mainly prepared by constructing a recombinant expression vector, transfecting an eukaryotic cell strain, screening a high-expression clone cell strain, expressing and purifying. The recombinant antigen protein disclosed by the invention is simple and stable in purification process, high in protein expression quantity and high in purity, and can be used for preparing the antigen protein of a sheep echinococcosis genetic engineering subunit vaccine. The sheep echinococcosis genetic engineering subunit vaccine prepared by selecting the biphasic adjuvant is used for preventing sheep from being infected with echinococcosis, so that disease transmission is blocked, economic loss is reduced, and epidemic of human echinococcosis is indirectly controlled. According to the established sheep echinococcosis ELISA antibody detection method, the detection positive rate of a clinical sample can be higher, and the effect of the prepared vaccine after sheep immunization and the specifically generated antibody titer can be more accurately verified.

The invention discloses a fluorinated cationic polymer, a preparation method and application of the fluorinated cationic polymer in gene transfection. According to the method, poly (2-hydroxypropyleneimine) is modified through an ethylene oxide ring-opening reaction, poly (2-hydroxypropyleneimine) is modified through fluorocarbon epoxides with different lengths, a fluorinated poly (2-hydroxypropyleneimine) material is obtained, and the material can serve as a transport carrier for eukaryotic cell transfection. The fluorinated cationic polymer gene vector disclosed by the invention can realize high-efficiency and low-toxicity transfection of various eukaryotic cells in vitro, can implement high-efficiency and low-toxicity local administration in vivo, and has a wide application prospect.