4160 results about "Cell strain" patented technology

The invention discloses a human SARS-CoV-2 monoclonal antibody. The preparation method of the human SARS-CoV-2 monoclonal antibody comprises the steps: adopting SARS-CoV Nucleocapsid recombinant protein as immunogen, immunizing BALB/c mice, performing fusion and subcloning on spleen cells and myeloma cells of mice, then performing a large amount of repeated screening and domestication of cell lines through commercialized products SARS-CoV-2 Nucleocapsid and MERS Nucleocapsid so as to obtain a hybridoma cell line capable of secreting the SARS-CoV-2-resistant N monoclonal antibody with high affinity and high specificity finally and successfully, and finally performing ascites preparation and purification so as to obtain the monoclonal antibody, wherein the amino acid sequence of the SARS-CoVNucleocapsid recombinant protein is shown in SEQ ID No. 1. The invention also discloses application of the monoclonal antibody in preparation of SARS-CoV-2 virus detection products and preparation ofdrugs for inhibiting the SARS-CoV-2 viruses. The monoclonal antibody can be used for detecting the SARS-CoV-2 in human throat swabs/pulmonary secretions and other samples by using a double-antibody sandwich method, and can be applied to diagnosis and prevention and control of SARS-CoV-2 virus infection and scientific researches of viruses and other study.

The invention relates to a mammal cell high-efficiency expression vector pSNEO. The vector is constructed on the basis of neomycin resistance screening gene NEO by combined strategy of weakening screening gene expression and reinforcing target gene expression. The screening gene weakening comprises the following two sides: introducing deletion mutant into the promoter to implement transcription weakening of the screening gene, and introducing a hairpin structure sequence before the translation initial site of the screening gene to implement the translation weakening of the screening gene. The expression reinforcement of the target gene is implemented by adding a transcription control element WPRE sequence between polyclone site and polyA tail of the target gene expression frame. The pSNEO vector can conveniently and quickly complete the construction of the stable high-expression cell strain of the target gene, and provides a new tool for preparing the high-polymer recombinant protein.

The present invention relates to a pentacyclic triterpanoid derivative of multiple-oxide substitution of the A ring and the C ring and the medicine salt or solvate of the derivative, and the present invention also relates to the preparation method, the drug combination, and medical use of the derivative. The compound of the present invention has the functions of inhibiting the activity of six human tumor cell strains in vitro, such as human prostate cancer cell (PC-3), nasopharyngeal carcinoma cells (CNE), oral squamous carcinoma cell(KB), human lung cancer cell (A549), human hepatoma cell (BEL-7404), and human cervix cancer cell (Hela), and the function of the invention is at the same magnitude of the positive control of cisplatin, thereby the compound can be used as expected antitumor drug. The compound of the present invention also inhibits the alpha glucosidase strongly, and the inhibiting effect is greater than the positive control of acarbose, thereby the compound can be used as expected medicine for preventing and treating diabetes and the treatment of the virus diseases.

The invention provides a hybridoma cell line 1C11 and an anti-aflatoxin general monoclonal antibody secreted by the same as well as the applications thereof. The hybridoma cell line 1C11 can be used for preparing a high-titer aflatoxin antibody, and a mouse hydroperitoneum antibody is measured to reach 5.12*106 by using an ELISA (Enzyme-Linked Immunosorbent Assay). The anti-aflatoxin general monoclonal antibody has high sensitivity, respectively reaches the IC50 (50% inhibiting concentration) of aflatoxin B1, B2, G1 and G2 to be 1.2, 1.3, 2.2 and 18.0 pg/mL, is the antibody with highest sensitivity among currently reported four aflatoxin antibodies, is used for measuring the total aflatoxin amounts, i.e. the total amounts of the aflatoxin B1, B2, G1 and G2 and has great practical application values.

The invention discloses a method and application for screening an ultralow fucose cell line. The invention provides a method for constructing fucose deficit type host cells capable of expressing an antibody and an IgG-FC fusion protein, a detection method for the fucose activity of the antibody and the IgG-Fc fusion protein, and concrete application of the cell lines. The method provided by the invention is realized by highly-efficient knockout of fucose-based transferase (FUT8) gene in an engineering cell producing the antibodies and the IgG-Fc fusion protein through a TALEN (and/or CRISPR) technology; and through lens culinaris lectin (LCA) pressurizing, gene sequencing and a flow cytometry screening process, the host cells with highly-efficiently knocked-out fucose is obtained. Meanwhile, fucose deficit CHOK1 host cell lines are constructed into stable engineering cell lines capable of expressing antibody proteins; and after the antibody proteins are obtained, glycoform analysis is performed; and results show that fucose knockout efficiency reaches to 99% or above.

The invention discloses a miR-205 gene knockout kit based on CRISPR-Cas9 gene knockout technology. According to the invention, an optimal CRISPR-Cas9 target sequence of a certain amount of miR-205 is obtained through target design software and an sgRNA single chain is synthesized in vitro; an insertion fragment is obtained through processing; then sgRNA is inoculated into a plasmid vector by using T4 ligase; and a miR-205 gene knockout cell strain is obtained through transfection of an LNCap cell strain, continuous drug screening and fluorescence detection. Heterogenous hybridization double strands are obtained by extracting DNAs of a cell, PCR amplification of miR-205, purification, denaturation of a PCR product and annealing; T7E1 enzyme digestion test is employed to determining shearing efficiency of a CRISPR-Cas9 system on miR-205; a verified optimized a miR-205 gene knockout CRISPR-Cas9 target sequence is obtained; and the kit is constructed on the basis of the target sequence and can be used for directional knockout of miR-205 genes. The kit has the characteristics of high gene knockout efficiency, fast speed, easiness and economic performance and has wide prospects in the aspects of construction of animal models and clinical research of medical science.

The invention belongs to the technical field of the molecular biology and the biomedicine, and particularly relates to application of a gRNA sequence based on a CRISPR/Cas9 system to cause tumor cell apoptosis. According to the design principle of CRISPR/Cas9, two target points are designed on a human genome, and corresponding oligos is synthesized and established on a px458 carrier. The CRISPR/Cas9 system guided by the gRNA is established in a human hepatoma cell line (HepG2) according to the design of the two target points, a human TCAB1 gene can be effectively knocked out, the system is easy to operate, and the knockout efficiency of the human TCAB1 gene is high. The CRISPR/Cas9 system guided by the gRNA can be expected to be applied in novel tumor treating medicine.

The invention provides a serum-free cell culture fluid suitable for enriching and culturing tumour stem cells. DMEM/f12 serves as the basic culture fluid of the serum-free cell culture fluid and transferrin, insulin and other substances are also added as the components of the serum-free cell culture fluid. The serum-free cell culture fluid provided by the invention can efficiently enrich tumour stem cells from malignant tumour cell strains and tumour tissues. In addition, the serum-free cell culture fluid can promote stable growth of the tumour stem cells and maintain fine cell activity and physiological properties of the tumour stem cells, thus being very suitable for research fields related to tumour cells and tumour stem cells.

The invention relates to an aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and an application thereof. The kit comprises a fluorescent test strip and a sample reaction bottle containing an europium-labeled anti-aflatoxin B1 monoclonal antibody lyophilized product, wherein the fluorescent test strip comprises a cardboard, a water absorption pad, a detection pad and a sample pad are sequentially pasted on one surface of the cardboard from top to bottom, adjacent pads are connected at the connection in an overlapping manner, the detection pad treats a cellulose nitrate membrane as a base pad, a transverse quality control line and a detection line are arranged on the cellulose nitrate membrane from top to bottom, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the detection line is coated with an aflatoxin B1 bovine serum albumin conjugate; and the anti-aflatoxin B1 monoclonal antibody is secreted by a hybridoma cell strain having a preservation number of CCTCC NO.C201015. The kit can be used for the quantitative determination of the content of the aflatoxin B1, and has the advantages of simple operation, rapidness and high accuracy.

The invention discloses a targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats)/Cas9 slow virus system and application thereof. The sgRNA is selected from FTOsgRNAsp2 or FTOsgRNAsp3 with the following DNA sequence. The sgRNA has high cutting efficiency for the FTO gene. When the sgRNA-containing CRISPR/Cas9 slow virus system is used for transfecting SV-HUC-1, the FTO protein expression level of the obtained cell strain is obviously lowered. Therefore, the sgRNA disclosed by the invention can effectively implement targeted FTO gene knockout; and after the sgRNA is established into the CRISPR/Cas9 slow virus system, the system can knock out the FTO gene to obtain the FTO-gene-knockout cell strain, thereby being beneficial to researching the action mechanism of FTO in the cell strain.

The present invention provides an sgRNA sequence for knocking out the human CYP2E1 gene. The target DNA sequence of the sgRNA is at least one of the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. The present invention also provides a method for knocking out CYP2E1 gene of human embryonic kidney cells, which is adapted to transform CYP2E1 gene in human embryonic kidney cells by using CRISPR/Cas system. The invention also provides a CYP2E1 knockout cell strain. CYP2E1 involves in the important metabolism function of a human body. The CYP2E1 gene knock-out cell strain provided by the invention provides an effective platform for studying the metabolism function of exogenous chemical or exogenous poison in the body, and a powerful tool for studying chronic diseases (such as alcoholic liver disease and diabetes) and tumor-related diseases.

The invention provides an sg RNA sequence used for knocking out a human ALDH2 gene;,a target DNA sequence of the sg RNA is at least one of sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. The invention further provides a method for knocking out a human hepatoma carcinoma cell ALDH2 gene; the method utilizes a CRISPR/Cas system to modify the ALDH2 gene in a human hepatoma carcinoma cell. The invention further provides two ALDH2 gene deletion cell strains; ALDH2 participates in an important metabolic function of a body. The ALDH2 gene deletion cell strains provided by the invention provide an effective platform for metabolism study, in vivo, of exogenous chemicals or exogenous poisons, so that powerful means are provided for researching chronic diseases (such as alcoholic liver diseases and diabetes) as well as tumor-associated diseases.

The invention provides a method for knocking out a KDM2A gene of HEK293T cells with a CRISPR-CAS9 technology. The method comprises the steps as follows: (1), cell transfection; (2), cell genome extraction; (3), PCR identification; (4), gene level verification; (5), protein level verification: whether K3, K7-1 and K7-2 are KDM2A gene-deficient is further verified by Western blotting after a mutantcell line is verified at the gene level. The invention further provides a KDM2A knockout HEK293T cell line prepared with method and an application of the KDM2A knockout HEK293T cell line as a cell model in study of cell proliferation and apoptosis signaling pathways involved in KDM2A methylase.

The present invention discloses a nucleic acid aptamer, wherein the sequence of the nucleic acid aptamer comprises a DNA segment represented by any one sequence selected from a sequence 1, a sequence 2 and a sequence 3. The nucleic acid aptamer can further be various similar sequences with high homology or a derivative obtained from the sequence of the present invention. The invention further discloses a nucleic acid aptamer screening method, which comprises: synthesizing a random single-stranded DNA library and primers, carrying out SELEX screening, carrying out PCR amplification of library, preparing a DNA single strand library, and finally carrying out repeated screening, negative screening and multi-round screening to obtain the nucleic acid aptamer. The nucleic acid aptamer and the derivative thereof can be used in recognition of the prostate cancer cell strain PC-3 or preparation of kits, molecular probes and targeted mediums for prostate cancer detection, and can further be used in design and preparation of prostate cancer treatment drugs. Compared with the protein antibody, the nucleic acid aptamer of the present invention has advantages of high affinity, high specificity, no immunogenicity, capability of being chemically synthesized, small molecular weight, stability, easy storage, easy labeling and the like.

The invention discloses a CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene. Firstly, sgRNA for specific targeting of the SLC30A1 gene is obtained, and a base sequence of the sgRNA is shown in SEQ ID NO.1; secondly, the sgRNA of the SLC30A1 gene is constructed to a lentivirus vector system containing Cas9 proteins; finally, CRISPR/Cas9 lentivirus containing the sgRNA is infected with a human breast cancer cell MDA-MB-231 to obtain a cell strain of which the SLC30A1 protein expression level is obviously reduced. The CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene disclosed by the invention have the advantages of simple operation steps, good sgRNA targetability and high cuttingefficiency on the SLC30A1 gene; in addition, the constructed CRISPR/Cas9 lentivirus system has the advantage of high knockout efficiency and can specifically knock out the SLC30A1 gene so as to obtain the human breast cancer cell having the SLC30A1 gene knocked out; therefore, a powerful tool is provided for further researching an action mechanism of the SLC30A1 in breast cancer cells.

The invention discloses a CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and specific sgRNA thereof. The CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and the specific sgRNA thereof are characterized in that: firstly, sgRNA of a second exon of the specific targeted DEAF1 gene is obtained and the base sequence of the sgRNA is shown as SEQ ID NO.1; secondly, the sgRNA of the DEAF1 gene is constructed into a lentiviral vector system, which contains Cas9 protein; finally, the CRISPR/Cas9 lentivirus containing the sgRNA is infected with human colorectal carcinoma cell HT-29 cell, so that a cell strain of which DEAF1 protein expression level is obviously reduced is obtained. The CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene disclosed by the invention has the advantages of simple operation steps, good sgRNA target ability and high cutting efficiency for the DEAF1 gene; in addition, the constructed CRISPR/Cas9 lentiviral vector system has the advantage of high knockout efficiency and can specifically knock out the DEAF1 gene to obtain the human colorectal carcinoma cells knocking out the DEAF1 gene, and therebya powerful tool is provided for further studying an action mechanism of DEAF1 in the colorectal carcinoma cells.

The invention provides a genetic engineering cell strain which is constructed on the basis of CRISPR-Cas9 systems and is capable of secreting mouse interleukin-6. Target sequences of sgRNA for specifically targeting mouse ROSA26 genes are shown as SEQ ID NO.1, and site-specific cleavage vectors capable of simultaneously expressing the sgRNA and cas9 proteins are further constructed; donor vectorswith mouse interleukin-6 and ROSA gene homogenous sequences are further constructed, site-specific integration is carried out on the mouse interleukin-6 at ROSA26 sites of mouse cells SP2/0, selectionis carried out by the aid of selection markers to obtain homozygous cell strains, and the genetic engineering cell strain with a mouse interleukin-6 high-expression function can be obtained. The genetic engineering cell strain has the advantage that the genetic engineering cell strain can be used for producing hybridoma cells with high-proportion secretion capacity and has a broad application prospect in the field of monoclonal antibody preparation.

Strains of human-derived bacteria have been obtained from complex fecal samples and shown to induce accumulation of Th17 cells in the intestine and promote immune functions. Pharmaceutical compositions containing these bacteria can be used as anti-infectives and as adjuvants in mucosal vaccines.

The invention discloses a monoclonal antibody 64360-52D1 capable of specifically combining with SARS-Cov-2 virus structural nucleocapsid protein (N protein), a preparation method thereof, and 6 complementarity-determining regions (CDR) of heavy chain and light chain variable regions; and more specifically, the monoclonal antibody is secreted by a hybridoma strain 64360#52D1, and can specifically recognize the SARS-Cov-2 virus N protein rather than SARS virus N protein. Thus, the monoclonal antibody can be used for identifying the two coronaviruses with high similarity. The invention further provides an enzyme-linked immunosorbent assay (ELISA) and an immune colloidal gold test strip detection method for specifically detecting the SARS-Cov-2 virus N protein by preparing the 64360#52D1 antibody. The antigen of the antibody is SARS-Cov-2 virus N protein subjected to heat treatment and expressed in mammalian cells; the finally obtained antibody belongs to an IgG1 subtype; and a sequence for encoding the variable region of the antibody is obtained in a gene cloning mode.

The invention discloses a method for completing site-specific integration of a goat VEGF gene through mediation of a CRISPER/Cas9 system. In the method, according to a goat FGF5 gene sequence, a CRISPER/Cas9 system-based Cas9-gRNA expression vector and a CRISPER/Cas9 system-based VEGF targeting vector are constructed; then, the optimized Cas9-gRNA expression vector and the optimized VEGF targetingvector are co-transfected into a goat fetal fibroblast to obtain a cell strain in which the VEGF gene is integrated at a specific FGF5 site. The constructed CRISPER/Cas9-based targeting vector provides a simple, fast and safe way for the site-specific integration of the goat VEGF gene and targeted knockout of an FGF5 gene. By the method, any screening marker gene is not involved in the cell strain screening process, so that the safety of a transgenic animal is greatly improved. Therefore, the method has an important value in genetic goat breeding and research on gene functions.

The invention discloses an anti-rabies virus monoclonal antibody and a preparation method and an application, belonging to the field of biomedicine and particularly relating to the preparation of a monoclonal antibody capable of identifying rabies virus and the application. The monoclonal antibody of the invention is screened by indirect Enzyme-linked immunosorbent assay (ELISA), and specificity and affinity thereof combined with antigen are identified by methods such as polyacrylamide gel electrophoresis analysis, speckle ELISA, immunoblot analysis and the like. The anti-rabies virus monoclonal antibody of the invention can be applied in multiple testing methods of antigen of rabies virus and can be also applied in the preparation of rabies virus detecting kit. The anti-rabies virus monoclonal antibody is secreted by anti-rabies virus monoclonal antibody hybridoma cell strain 2C5 with the preservation number being CGMCC No.3014.

An interleukin-15 (IL-15) gene modified natural killing cell strain for immunotherapy of tumor is prepared through inserting the cDNA coding region of IL-15 in pcDNA3 eucaryotic expression carrier, configuring secretion-type recombinant eucaryotic expression carrier pc DNA3/IL-15, using liposom to transfecte cell NK-92, adding Geneticin (G418) to screening culture medium, screening, limited dilution to cloned cells, detecting activity, choosing positive cell clones, and amplification.

The invention discloses a CRISPR-Cas9 targeted-knockout human colorectal cancer cell PPP1R1C gene and a specific sgRNA sequence. The invention comprises the following steps: firstly, acquiring sgRNA of a specific targeting PPP1R1C gene functional area, wherein a base sequence of the sgRNA is shown as SEQ ID NO.1; then, constructing an sgRNA-lentiviral vector system of the PPP1RQC gene, wherein thesystem contains Cas9 protein; and finally, infecting human colorectal cancer HT-29 cells with CRISPR/Cas9 lentivirus containing the sgRNA, so that a cell line that a PPP1R1C protein expression levelis obviously reduced is obtained. The invention is simple in operating steps, good in sgRNA targeting performance and high in cutting efficiency on the PPP1R1C gene; the constructed CRISPR/Cas9 lentiviral system is high in knockout efficiency and is capable of achieving specific knockout of the PPP1R1C gene, so that the human colorectal cancer cells that the PPP1R1C gene is knocked out is obtained; therefore, a powerful tool is provided for further researching an action mechanism of the PPP1R1C in the colorectal cancer cells.