119 results about "293t cell" patented technology

The invention relates to a CHO-S cell strain capable of stably expressing H5N1 hemagglutinin protein and a construction method of the CHO-S cell strain, and belongs to the field of bioengineering.H5N1 hemagglutinin protein expression plasmids are obtained in an in-vitro synthesis and seamless cloning mode, 293T cells are transfected through the H5N1 hemagglutinin protein expression plasmids, and it is proved that the H5N1 hemagglutinin protein is expressed; the CHO-S cell strain capable of stably expressing the H5N1 hemagglutinin protein is obtained by transfecting CHO-S cells by using the H5N1 hemagglutinin protein expression plasmids and carrying out multiple rounds of cloning and screening, so that a basis is provided for obtaining H5N1 recombinant protein influenza vaccines.

The invention relates to the technical field of molecular biology, in particular to a codon optimization method. The invention provides a codon optimization method, the codon is replaced according to parameters such as codon type and quantity, local GC content, local repetitive sequence, mRNA secondary structure, mRNA free energy and the like, and the optimized sequence can be subjected to experimental screening to obtain a DNA sequence of high-expression protein. The downstream of the method is mainly the application in the IVT mRNA direction: the optimized DNA sequence is constructed into a vector, the plasmid is linearized to serve as a template for in-vitro mRNA transcription, the mRNA is encapsulated into LNP or LNP coupled with other molecules, and the protein expression in 293T cells (not limited to 293T cells) or mice is obviously improved.

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The invention discloses a recombinant I-type collagen, a preparation method and application, and belongs to the technical field of binding tissue peptides, the recombinant I-type collagen is formed by sequentially connecting the following modules in series from the N terminal to the C terminal: a signal peptide, a truncated I-type collagen and a His tag; the nucleotide sequence of the truncated I type collagen is as shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequence of the truncated I type collagen is as shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; according to the invention, a mammalian expression system and a 293T cell line are used for expressing recombinant I-type collagen, so that collagen with natural biological activity is obtained, and the proliferation efficiency and repair capacity of the collagen for promoting HSF cells can be improved.

The invention provides brain cell specific targeting delivery based on exosome, and the exosome secreted by 293T cells is used for engineering transformation, so that the capabilities of penetrating BBB and specifically targeting neurons are achieved. According to the present invention, the L1CAM is loaded on the exosome, the exosome is obtained by transfecting a 293T cell of a Lamp2b-L1CAM PCDH-HygroHA plasmid through a 293T-Lamp2b-L1CAM cell line, and the BBB cell-penetrating peptide 4F-T7 and the neuronal targeting protein L1CAM are loaded on the surface of the exosome so as to treat the neurodegenerative diseases such as the Alzheimer's disease and the Parkinson's disease.

The invention relates to an oncolytic virus VSV-aPDL1 / aCD137 for expressing a bispecific antibody as well as a construction method and application of the oncolytic virus VSV-aPDL1 / aCD137. The preparation method comprises the following steps: firstly designing aPDL1 / aCD137, then synthesizing an aPDL1 / aCD137 gene in vitro, amplifying the aPDL1 / aCD137 gene, connecting the aPDL1 / aCD137 gene to a pT7-VSV whole genome plasmid to obtain a virus whole genome plasmid pT7-VSV-aPDL1 / aCD137, and then co-transfecting the plasmid and a helper plasmid to a 293T cell to obtain the VSV-aPDL1 / aCD137 oncolytic virus. According to the present invention, the local anti-tumor immune response of the tumor is activated through the virus, the infiltration of the lymphocyte to the tumor tissue is recruited, the anti-PD-L1 / anti-CD137 bispecific antibody is expressed, the inhibition of the PD-1 / PD-L1 pathway on the T cell is blocked, and the inhibition signal of the PD-1 / PD-L1 pathway on the tumor cell surface is reversed into the CD137 signal of the activated T cell, such that the functional failure of the T cell caused by the PD-1 / PD-L1 pathway can be blocked, and the anti-tumor immune response of the tumor can be inhibited; the function and viability of T cells can be further improved, long-term immune surveillance on tumors is realized, and long-acting anti-tumor immune response is generated. Experiments show that the oncolytic virus can significantly prolong the lifetime of tumor-bearing mice, and a new strategy is provided for development of solid tumor drugs.

The invention relates to a construction method and application of a recombinant oncolytic virus VSV-sPD1IL15RaIL15. The recombinant oncolytic virus VSV-sPD1IL15RaIL15 is constructed by using homologous recombination, and the recombinant oncolytic virus VSV-sPD1IL15RaIL15 can be used for preparing the recombinant oncolytic virus. The preparation method comprises the following steps: firstly designing sPD1 and IL15RaIL15, then fusing the sPD1 and the IL15RaIL15, synthesizing a sPD1IL15RaIL15 gene, amplifying the sPD1IL15RaIL15 gene, inserting the sPD1IL15RaIL15 gene into an ATU region of a VSV whole genome cDNA cloning plasmid pT7-VSV by using a homologous recombination method to obtain a virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15, and co-transfecting the virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15 and an auxiliary plasmid to a 293T cell to prepare the VSV-sPD1IL15RaIL15 oncolytic virus. According to the oncolytic virus, specific anti-tumor immunity is activated, and sPD1IL15RaIL15 is locally expressed in a tumor, so that a long-acting anti-tumor immune response is generated. When the oncolytic virus is used for treating solid tumors, the functions of tumor specific immune cells in a tumor microenvironment can be improved, infiltration of CD8 + T cells and NK cells can be promoted, and the lifetime of tumor-bearing mice can be remarkably prolonged.

The invention belongs to the technical field of biology, and particularly relates to sgRNA of a targeted ASCT2 gene and application of the sgRNA. Two efficient knockout targets are provided for the human ASCT2 gene, and knockout effect detection and identification show that the two sgRNAs can effectively knock out the ASCT2 gene on a 293T cell. The 293T cells without the ASCT2 gene are high in stability, formation of syncytial bodies induced by BaEV envelope glycoprotein can be remarkably inhibited, cell source impurities in lentivirus preparation liquid are greatly reduced, the titer of BaEV envelope lentivirus is increased by 5 times, and the method has practical application value.

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for efficiently producing BaEV enveloped lentivirus. Comprising the following steps: inoculating 293T cells into a culture dish for culturing; the preparation method comprises the following steps: uniformly mixing plasmids pGFP, pLP1, pLP2 and plp-BaEV with a DMEM complete medium, and incubating at room temperature to obtain a plasmid medium mixture; uniformly mixing the PEI solution with the DMEM complete culture medium to obtain a PEI culture medium mixture; adding the PEI culture medium mixture into the plasmid culture medium mixture, and uniformly mixing to obtain a plasmid PEI mixture; adding the plasmid PEI mixture into a culture dish for culturing, and transfecting 293T cells; after 5-6 hours of transfection, replacing the culture medium with an equivalent DMEM complete culture medium for continuous culture; and after transfecting for 36-72 hours, harvesting a 293T cell culture solution, and filtering the 293T cell culture solution to obtain the lentivirus. The lentivirus yield is effectively increased.

The invention discloses a polyketone compound derived from actinomycetes as well as a preparation method and application thereof, and relates to the technical field of microbial natural product mining and biological medicine, and the key point of the technical scheme is that the invention discloses a novel polyketone compound Strepactone derived from actinomycetes Streptomyces sp. DP0001, and the molecular formula of the novel polyketone compound Strepactone is C25H38O5. The compound is obtained through strain fermentation, ethyl acetate extraction and multi-step chromatographic purification, and the structure is identified through ESI-MS, 1H NMR and 13C NMR. Experiments show that Strepactone has an inhibition effect on staphylococcus aureus and methicillin-resistant staphylococcus aureus and has remarkable inhibition activity on human non-small cell lung cancer A549 cells, the half inhibitory concentration IC50 of the Strepactone is 5.36 mu M, and the Strepactone has no obvious cytotoxic activity on human embryo kidney cells 293T cells at the concentration of 30 mu M. The compound provided by the invention can be used for preparing anti-drug-resistant bacteria drugs and anti-human non-small cell lung cancer drugs, and also provides a structural basis for the development of novel antibacterial and anti-tumor leading drugs.

The invention discloses a reverse transcriptase, wherein a coding sequence of the reverse transcriptase is derived from a Rattus norvegicus genome. The protein sequence of the protein comprises a protein sequence of a wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus. Furthermore, the protein sequence of the gene is as shown in any one of SEQ No.1-55. In a rice genome site and a human HEK 293T cell genome site, the system shows efficient editing efficiency of a guide editor. The invention provides a series of efficient reverse transcriptase components which can be carried on a guide editing system, important bottom-layer technical support is provided for research of point mutation in a genome, creation of a special site disease model and correction of genetic mutation sites, and the reverse transcriptase component has a wide application prospect in the field of precision medicine and precision agriculture breeding.

The present application relates to the technical field of genetic engineering, in particular to a T7-293T cell and application of the T7-293T cell in bovine parainfluenza virus type 3 infectious clone rescued virus, and the T7-293T cell provided by the present application has the preservation number of CGMCC No.46335.The T7-293T cell provided by the present application is a 293T cell stably expressing T7 RNA polymerase, and has the advantages of high transfection efficiency and cheap transfection reagent.Further, the present application establishes a method for bovine parainfluenza virus type 3 infectious clone rescued virus based on the T7-293T cell, constructs three genes of NP, P and L of bovine parainfluenza virus type 3 into the same helper plasmid, and co-transfects the T7-293T cell with the full-length gene plasmid of the virus, so as to avoid the low efficiency of co-transfecting four plasmids, and further improve the success rate of bovine parainfluenza virus type 3 infectious clone rescued virus.

The invention belongs to the technical field of exosomes, and particularly relates to an engineered exosome for overexpressing EGF and FGF as well as a preparation method, a product and application of the engineered exosome. An LAMP2B fusion expression technology is adopted, EGF and FGF are directionally anchored to the surface of a 293T cell source exosome membrane, free factor degradation is avoided, and the problem that the carrying amount of natural exosome factors is insufficient is solved. Through the synergistic effect of two factors, the exosome can reverse proliferation inhibition of DHT on hair follicle hair papilla cells, improve the cell migration ability, promote AGA mouse hair follicles to recover from a resting period to a growing period and block hair follicle microminiaturization, and finally reconstruct an AGA hair follicle microenvironment to achieve functional hair growth. Cell experiments confirm that the compound can repair hair follicle cells damaged by DHT, animal experiments show that the AGA mouse hair coverage rate is increased, the safety is good, the primary clinical effect is achieved, an efficient, safe and flexible new scheme is provided for AGA treatment, and the compound has good clinical transformation potential.

The application discloses a kind of immortalization human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line construction method and its application in liver differentiation, it is related to immortalized cell technical field.The application first connects target gene to vector, and the slow virus plasmid containing target gene is constructed, the slow virus plasmid containing target gene is introduced into 293T cell, and high titer slow virus containing target gene is generated;Immortalized human umbilical cord mesenchymal stem cell is infected with slow virus, and mixed clone cell strain is screened, and the immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line is obtained.The application proves that the differentiation efficiency of immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression can effectively improve liver-like cell to glycogen staining and Western blotting experiment.This research result is expected to provide new technical thought for the next step development to improve the yield of MSCs liver differentiation.

The present application relates to a porcine reproductive and respiratory syndrome virus infectious clone construction and rescue method, and belongs to the technical field of bioengineering. The porcine reproductive and respiratory syndrome virus infectious clone contains a full genome sequence of the porcine reproductive and respiratory syndrome virus, and the nucleotide sequence of the full genome sequence is the 26th to 15384th nucleotide of SEQ ID NO: 1 or a fragment having more than 80% identity with SEQ ID NO: 1 and having the same function. The present application also provides a plasmid pCAGGS-HA-CD163 expressing a porcine CD163 gene. By co-transfecting the aforementioned two plasmids into HEK-293T cells, the virus is rescued in vitro, a first porcine reproductive and respiratory syndrome virus infectious clone rescue platform based on HEK-293T cells is successfully built, and the porcine reproductive and respiratory syndrome virus is successfully rescued.

The invention discloses a method for drawing a human full-coverage protein interaction network based on digital PCR next-generation sequencing. After the advanced pedestrian 293T cell line obtains mRNA, a modified random primer or an oligo-dT primer is adopted to obtain a cDNA library; carrying out homogenization treatment on the human cDNA library; the method comprises the following steps: transforming plasmids of a BACTH bacteria double-hybrid system to obtain transformed plasmids; carrying out homologous recombination on the transformed plasmids of the sample library and the double-impurity system, and introducing into escherichia coli for screening to obtain positive PPI clones of the library with interaction; and carrying out digital PCR-based next-generation sequencing on the screened clones to draw the human full-coverage protein interaction network. The method is suitable for performing high-throughput screening after thousands of positive PPI combinatorial clones are obtained by'library-to-library 'bacteria or yeast double hybrids, identifying the same cell bar code cDNA combinatorial pairs, and realizing PPI network identification in different species, among species and in hybridization technology system extensive scenes.

The invention relates to a method for efficiently establishing a stably transfected cell strain for knocking down and overexpressing a dyrk1b gene. Comprising the following steps: designing a specific RNA (Ribonucleic Acid) sequence to knock down a dyrk1b gene, constructing a DNA (Deoxyribose Nucleic Acid) sequence for highly expressing dyrk1b, and carrying out virus packaging and predetermined cell infection by utilizing a three-generation lentivirus system. A positive clone cell strain is obtained through Puromycin screening, OVCAR3 and 293T cells are infected by adopting a specific plasmid proportion, and knock-down and overexpression efficiency is verified. According to the method, the function of the dyrk1b gene is evaluated by observing the morphological change of OVCAR3 and COV362 cells, such as cell body elongation and antenna growth. The innovation point of the invention is that an efficient virus packaging system and precise gene expression regulation and control ensure the stability of the cell strain and the reliability of the experiment. In addition, the invention also relates to application of the stably transfected cell strains in comprehensive treatment of ovarian cancer magnetic hyperthermia, small molecule inhibitor and chemotherapy, and provides a new research tool and strategy for cancer treatment.

The invention relates to the technical field of virus packaging. The invention provides a packaging method of a CAR (chimeric antigen receptor) virus. The packaging method comprises the following steps: mixing a plasmid pCMX-dR8.91, a plasmid pCMV-BaEV, a plasmid pAdVAnti and an opti-MEM culture medium, standing, mixing with a PEI transfection reagent, standing, mixing with a 293T cell, culturing, replacing the culture medium, continuously culturing, and harvesting the virus. A second-generation lentivirus packaging system is adopted, transfer plasmids are second-generation transfer plasmids needing to be activated by a Tat element, packaging plasmids are second-generation packaging plasmids pCMV-dR8.91 containing ag, pol, rev and tat, enveloping plasmids are second-generation enveloping plasmids pCMV-BaEV, plasmids pAdVAnti beneficial to improvement of transfection efficiency are added, and the packaging efficiency of the CAR virus is improved through the system.

The invention belongs to the field of gene engineering, and particularly relates to application of chikungunya virus structural protein in improvement of pseudovirus stability, a construction method of the chikungunya virus structural protein and vaccines, antibody evaluation and in-vivo gene therapy products. The method comprises the following steps: constructing a human codon optimized Asian strain CHIKV-E3 + E2 + 6K + E1 structural protein eukaryotic vector, and designing two types of binder expression plasmids of targeted T cells; the vector, a psPAX2 helper plasmid and a lentivirus target vector containing an SFFV promoter and a ZsGreen reporter gene (or a CAR therapeutic gene) are co-transformed into a 293T cell, and the lentivirus is prepared through culture, filtration and centrifugation. The lentivirus can efficiently infect Jurkat cells and activate human T cells, obviously reduces the infection efficiency on 293T cells so as to reduce the off-target effect, has excellent stability in human serum, and provides a safe and efficient gene delivery tool for in-vivo CAR-T therapy.

The invention discloses an FUT2 high-expression cell strain as well as application and a method of the FUT2 high-expression cell strain in vaccine quality control, and belongs to the technical field of vaccines. The technical problem to be solved is that a simple and easy-to-operate cell model is lacked in the prior art, and the cell model is used for evaluating the effectiveness of NoV vaccines on the cellular level. According to the key points of the technical scheme, the FUT2 high-expression cell strain is provided, the cell strain is an HEK 293T cell strain (HEK 293T-FUT2-EGFP) with EGFP fluorescence for overexpressing human alpha 1, 2-fucosyltransferase 2 (FUT2), and the cell strain stably expresses H, Lewis b and Lewis y type HBGA antigens; meanwhile, an alternative neutralization experiment of the cell level of the norovirus vaccine based on the cell strain is developed, a basis is provided for interaction research of the norovirus and host cells, and the cell strain has high practical application value.

The invention discloses a DNA (Deoxyribonucleic Acid) molecule for promoting mRNA (Messenger Ribonucleic Acid) expression and 3'UTR (Untranslated Region) in the mRNA molecule obtained by transcription of the DNA molecule, and the DNA molecule is derived from COX7B of a cytochrome C oxidase family. The invention also discloses an application of the composition containing the 3 'UTR in preparation of RNA (Ribonucleic Acid) therapeutic drugs or mRNA (Messenger Ribonucleic Acid) vaccines. Compared with a control group alpha-globin, the insertion of the 3'UTR fragment provided by the invention has the advantages that the expression quantity of renilla luciferase in Lent-X 293T cells is increased by 2.03 times, and the expression quantity of renilla luciferase in HeLa cells is increased by 3.62 times. The invention shows that the 3 'UTR has an excellent prospect of being applied to RNA (Ribonucleic Acid) therapeutic drugs or mRNA vaccines.

The invention discloses an AQP4-IgG (immunoglobulin G) detection method based on the combination of AQP4-M1 and M23 subtypes. According to the invention, two protein subtypes of AQP4-M1 and M23 are mixed in proportion by simulating physiological conditions and co-transfected to 293T cells, so that heterotetramers are formed and further assembled into a complete orthogonal array (OAP), and more antigen epitopes are exposed, thereby remarkably improving the recognition specificity and detection sensitivity of the AQP4-IgG antibody. Compared with traditional single subtype detection, the system can more truly simulate abundance and distribution of the AQP4 subtype in vivo, reduces false positive and false negative risks, and has a great value of rapidly and accurately screening AQP4-IgG related diseases clinically.

The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a single-chain fragment variable (scFv) targeting human platelet-derived growth factor receptor (PDGFR)-β and use thereof in chimeric antigen receptor (CAR)-T cell immunotherapy. In the present invention, a scFv sequence targeting a human-derived PDGFRβ antigen is first obtained by immunizing a mouse, and then a second-generation CAR is constructed based on this, and additionally a CAR-T cell is obtained via lentivirus infection. The CAR-T cell can effectively kill a PDGFRβ antigen-positive 293T cell. The present invention provides a brand-new idea for eliminating PDGFRβ-positive cells to treat chronic kidney diseases, chronic liver diseases, cardiovascular diseases and various tumor diseases including various organ fibrosis, and has extremely attractive further development value and application prospects.

Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.

The application discloses a method for constructing a cell membrane fusion model induced by a novel coronavirus-like particle, and belongs to the technical field of biology. The method comprises the following steps: constructing a virus-like particle containing a SARS-CoV-2 spike protein gene and an RNA packaging sequence PS9, using HEK-293T cells as packaging cells, co-expressing structural proteins M, E, N and S protein, and realizing assembly of VLPs without replication ability. Subsequently, the VLPs are used to infect immortalized type II alveolar epithelial cells, S protein expression is induced and cell-cell membrane fusion is mediated without the need of BSL-3 laboratory conditions. The formation of fused giant cells can be directly observed through ZO-1 protein immunofluorescence staining, the model is stable, has high biological safety, and is repeatable in vitro. The application overcomes the safety risk of live virus infection and the authenticity problem of a transient transfection model, can be used for research on a SARS-CoV-2 virus membrane fusion mechanism, and has a good application prospect.

The present invention discloses a RAW 264.7 cell line stably overexpressing SRSF3, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. The present invention constructs a Lenti-Flag-hyg-SRSF3 plasmid overexpressing SRSF3, which forms a three-plasmid expression system with the psPAX2 and pMD2.G packaging vectors, co-transfects 293T cells, collects lentivirus, and then transfects RAW 264.7 macrophages with the lentivirus to obtain a stable cell line overexpressing the SRSF3 gene. The stable cell line overexpressing the SRSF3 gene is detected with a fluorescent antibody against Flag, and an LPS-induced inflammation model is used. Through Western Blot and qPCR detection, the stable cell line overexpressing SRSF3 can inhibit autophagy and reduce the expression level of inflammatory factors, and has the development and application prospects of inhibiting inflammation.

This invention relates to a detection kit and preparation method for enhancing the detection efficacy of autoimmune encephalitis (CBA) antibodies. The kit comprises genetically engineered 293T cells stably integrating and co-expressing the eIF2α S52A mutant and HSP70, and transiently transfected with CBA-associated antigens. The preparation method includes constructing a co-expression vector, establishing a stable transfection cell line, stress preconditioning treatment, antigen transfection, and kit assembly. A method for assessing antibody affinity maturation using this kit is also provided. By synergistically modifying detection cells at three levels—translational regulation, protein folding, and anti-apoptosis—the invention significantly increases antigen expression, reduces apoptosis, and improves detection sensitivity and specificity. Furthermore, it integrates antibody affinity assessment into CBA detection for the first time, providing auxiliary information for clinical staging.

The invention provides a preparation method and application of fibronectin, and relates to the technical field of biology. The preparation method comprises the following steps: expressing fibronectin by using HEK-293T cells, and taking AGRN protein signal peptide as the signal peptide of the fibronectin. According to the preparation method, the HEK-293T cells can secrete and express fibronectin with high yield and high biological activity, and the application of the fibronectin in the aspects of tissue regeneration, wound healing and the like is facilitated.

The invention discloses a chimeric antigen receptor NK cell overexpressing a GPR133 gene, and a preparation method and application thereof, and belongs to the technical field of biological medicine. The preparation method comprises the following steps: (1) respectively mixing a pLL3.7-GPR133 plasmid with a packaging plasmid psPAX2, mixing a pLL3.7-NKG2D-CAR plasmid with a packaging plasmid psPAX2, mixing a pLL3.7-NKG2D-CAR plasmid with a packaging plasmid pMD2. G with a proper amount of an Opti-MEM culture medium; (2) mixing PEI with a proper amount of Opti-MEM culture medium, and incubating at room temperature; (3) adding the PEI-Opti-MEM solution in the step (2) into the Opti-MEM culture medium containing the plasmids in the step (1), and standing at room temperature; (4) adding the mixture obtained in the step (3) into 293T cells, uniformly mixing, and incubating at 37 DEG C; (5) collecting viruses from the cell supernatant, so as to respectively obtain a pLL3.7-NKG2D-CAR virus and a pLL3.7-GPR133 virus; and (6) co-transfecting an NK92 cell with the pLL3.7-NKG2D-CAR virus and the pLL3.7-GPR133 virus, so as to obtain the chimeric antigen receptor NK cell of the over-expressed GPR133 gene. According to the present invention, the GPR133 gene is subjected to overexpression in the CAR-NK cell for the first time, such that the biological function of the CAR-NK cell is significantly enhanced so as to improve the tumor cell killing ability of the CAR-NK cell.

The application belongs to the technical field of antibodies, and particularly relates to an antibody for specifically treating orf of milk goat kids and application thereof. Virus liquid of a diseased goat is collected from a lesion site, and ORFV virus strains are obtained through subculture and amplification of goat skin fibroblasts; milk goats are immunized with a weak vaccine to prepare high-titer immune serum; flow sorting technology is used to separate ORFV-specific B cells from PBMC (peripheral blood mononuclear cells); antibody genes are amplified through nested PCR, expression vectors are constructed, and 293T cells are transfected to express monoclonal antibodies. The monoclonal antibody No. 2 obtained in the application has high affinity and specificity, the heavy chain CDR1-3 region sequence is SEQ ID NO: 1-3, and the light chain CDR1-3 region sequence is SEQ ID NO: 4-6. The antibody can be used for diagnosis, treatment and prevention of orf of goats, and has the characteristics of high efficiency, specificity and stability, and provides a new biological preparation for prevention and control of orf of goats.