357 results about "Lentivirus" patented technology

The invention relates to the technical field of gene engineering. The invention provides a functional sequence shRNA1 (short hairpin ribonucleic acid 1) for silencing a circular RNAh-hsacirc005838, and further provides a lentiviral vector of the functional sequence shRNA1 and a construction method of the lentiviral vector. The invention also provides a lentivirus packaging titer kit containing the lentiviral vector with the functional sequence shRNA1, and also provides an application of the lentiviral vector with the functional sequence shRNA1 or the lentiviral vector with the functional sequence shRNA1 in preparation of a kit for breast cancer diagnosis. According to the invention, an interference target sequence siRNA1 is obtained by aiming at the annular target gene RNA h-hsacirc005838, then the finally obtained functional sequence shRNA1 can directly and efficiently infect cells and silence the annular RNAh-hsacirc005838 sequence, and the transfection effect is stable.

The invention relates to the technical field of stem cells, in particular to a lentivirus envelope plasmid combination and application thereof, a lentivirus and a packaging method thereof and a method for transduction of hematopoietic stem cells. The invention provides an envelope plasmid combination for lentivirus packaging, which is composed of lentivirus packaging plasmids containing VSVG glycoprotein and lentivirus packaging plasmids containing BaEV glycoprotein in a ratio of 3: 7. The invention further provides a method for transduction of the hematopoietic stem cells by the lentivirus, the method is simple and convenient to operate, the long-term dryness of the hematopoietic stem cells in vitro can be maintained, efficient and stable transduction of the hematopoietic stem cells can be realized, and the transduction rate is greater than 90%. The lentivirus transduction method hematopoietic stem cells can be used for hematopoietic stem cell gene therapy hematopoietic system genetic diseases, such as severe combined immunodeficiency, beta-thalassemia and sickle cell disease, Wiskott-Aldrich syndrome and the like, and the application prospect is good.

The application discloses a kind of carrier, kit and application for long-acting gene knockdown of animal parasitic nematode, belong to the field of animal parasitic disease prevention and control.The lentivirus vector in the application integrates specific coding sequence to the genome of the infected nematode, stably and continuously expresses short hairpin RNA (shRNA) in the nematode, efficiently produces primary single-stranded small RNA (siRNA), these siRNAs target homologous target gene mRNA to cause its degradation, realize stable and continuous gene silencing effect.The application breaks through the bottleneck of traditional animal parasitic nematode RNA interference (RNAi) technology with low efficiency and instability, significantly improves the timeliness of conventional RNAi, can be used to establish long-acting RNAi technology system of animal parasitic nematode, has application value in the research of host in vivo process such as nematode infection, parasitism and pathogenicity and animal parasitic nematode disease prevention and control.

The invention relates to the technical field of cell engineering, and particularly discloses a construction method and application of a sika deer immortalized renal epithelial cell line, and the construction method comprises the following steps: taking renal epithelial primary cells from healthy sika deer renal cortex tissues to obtain primary renal epithelial cells; treating the primary renal epithelial cells with trypsin to obtain a cell suspension; inoculating the cell suspension into a complete culture medium, and transfecting with lentivirus loaded with SV40 large T antigen genes to obtain transfected cells; 3 [mu] g / mL puromycin is applied to the transfected cells for selective culture, so that non-transfected cells are eliminated, and drug-resistant cells are obtained; carrying out continuous passage on the drug-resistant cells for at least 30 generations to obtain the immortalized renal epithelial cell line of the sika deer; according to the invention, through a specific action mechanism (inhibiting a p53 / pRb pathway and blocking cell cycle exit) of the SV40 large T antigen, a proliferation limit caused by inhibition of telomerase activity of primary cells of the cervidae animals is overcome, and a cell resource library capable of realizing continuous passage is established.

The invention belongs to the technical field of biological medicine, and particularly relates to an engineered cell microvesicle and a delivery system based on the engineered cell microvesicle, the system realizes efficient preparation of 1-5 [mu] m cell microvesicles, and the cell microvesicles have a large space volume and can be used for preparing the cell microvesicles. The carrier can be used for loading and delivery of target protein, polypeptide and recombinase which are specifically expressed in mother cells. The system transfects mother cells through lentivirus transfection or plasmid transfection to further produce cell microvesicles, and the microvesicles can load more goods and inherit membrane proteins of the mother cells, and can also effectively load intracellular proteins to realize effective delivery. By virtue of good structural stability, high immunogenicity and excellent biocompatibility, the cell microvesicle reduces systematic toxic and side effects of a traditional carrier, is expected to become an effective drug delivery system, and has great application potential in the field of gene therapy.

Among other things, provided herein are systems that replace the natural random integration activity of a retrovirus with site-specific integration machinery. This approach allows for a more precise targeting of a gene of interest into a human genome, e.g., for therapeutic purposes. The system may include integration-deficient retrovirus (e.g., lentivirus) (IDLV), in which the natural integration activity has been reduced (e.g., by mutation to the viral integrase polypeptide). Instead, the system may comprise a site-specific recombinase (e.g., a serine recombinase, e.g., a serine integrase) capable of directing insertion of a template DNA, or portion thereof, into a desired site in the human genome.

The invention relates to an engineered exosome loaded with specific cytokines as well as a preparation method and application of the engineered exosome, and belongs to a biopharmaceutical technology. The engineered exosome is prepared by the following steps: (1) constructing an expression vector or lentivirus carrying an artificially optimized coding gene of a hepatocyte growth factor; (2) transfecting host cells, and screening successfully transfected host cells; (3) carrying out amplification culture on the selected transfected host cells, and separating and collecting exosomes of the transfected host cells; the expression vector is a transient expression vector or a lentiviral vector; an ExoBoost cell is adopted as the host cell. Experimental data show that the engineered exosome provided by the invention can promote hair follicle growth and has the application potential of improving AGA symptoms.

The invention provides a construction method and application of a gene modified K562 cell line, and belongs to the technical field of biological medicines. According to the invention, two viruses CD86-4-1BBL and mbIL-21 are respectively subjected to gene transduction packaging by adopting a third-generation lentivirus four-plasmid packaging system, a K562 cell line is infected by the lentiviruses CD86-4-1BBL and mbIL-21, and a monoclonal cell line capable of stably expressing CD86, 4-1BBL and mbIL-21 proteins is obtained through screening. According to the method, cell lines with the cloning numbers of V5-E9, V10-A5, V5-D6 and V6-D7 are obtained through screening, the four cloning cell lines can serve as feeder cells for stimulating NK cell amplification after being amplified and subjected to irradiation treatment, NK cell amplification can be effectively promoted, the amplification multiple of the NK cells reaches up to 6000-8000, and large-scale preparation and production of the NK cells for treatment are met.

The invention relates to the field of biomedicine, and discloses a method for screening KRAS mutant interacting proteins, an HEK293T cell line for continuously expressing an HA-Turbo ID-KRAS variant is established by utilizing lentivirus transduction, the stability of transgene expression is ensured through puromycin screening, and variation caused by transient transfection is reduced as much as possible. After a Turbo ID mediated proximity marker is activated by biotin treatment, KRAS proximity binding proteins are covalently biotinylated, captured by NeutrAvidin bead purification, and analyzed by quantitative mass spectrometry. According to the invention, two potential KRAS mutation treatment target proteins of LZTR1 and LAMTOR1 can simultaneously inhibit activation and efficacy exertion of three different mutants of G12C, G12D and G12V of the G12 mutant, compared with drugs on the market that a single target of G12C directly targets the KRAS mutant, a brand new KRAS mutation targeting intervention strategy is provided, the clinical curative effect can be further improved, and the occurrence rate of secondary drug resistance can be effectively reduced.

The invention provides a chimeric envelope glycoprotein, a preparation method thereof and an envelope plasmid, a packaging method and a kit of the chimeric envelope glycoprotein, and particularly relates to a preparation method of the chimeric envelope glycoprotein, the chimeric envelope glycoprotein and a lentivirus envelope plasmid. The invention discloses a pseudotyped packaging method of lentivirus and a kit. The amino acid sequence of the wild type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of other envelope glycoproteins, and compared with the initial wild type envelope glycoprotein, the obtained chimeric envelope glycoprotein can effectively improve the lentivirus packaging efficiency, biological activity and other properties.

The invention provides BaEV-G envelope glycoprotein, an optimization method of the BaEV-G envelope glycoprotein and an envelope plasmid, a packaging method and a kit which relate to the BaEV-G envelope glycoprotein, and particularly relates to the optimization method of the BaEV-G envelope glycoprotein, chimeric BaEV-G envelope glycoprotein, a lentivirus envelope plasmid, a pseudotyped packaging method of lentivirus and the kit. The BaEV-G envelope glycoprotein and the VSV-G envelope glycoprotein are chimeric to obtain the chimeric BaEV-G envelope glycoprotein, and the properties of lentivirus packaging efficiency, biological activity and the like can be effectively improved.

The invention discloses application of circFAM126A in preparation of a medicine for reversing oxaliplatin resistance of gastric cancer, and belongs to the technical field of molecular biology. According to the application disclosed by the invention, by constructing a lentivirus for targeted interference of novel circFAM126A and transfecting gastric cancer cells, it is proved that the lentivirus carrying a specific interference sequence can stably knock down circFAM126A expression in the gastric cancer cells, the drug resistance of the gastric cancer cells to oxaliplatin is effectively reversed, the tumor cell death effect induced by chemotherapeutic drugs is remarkably enhanced, and the tumor cell death effect is remarkably improved. Meanwhile, the proliferation and clone formation capability of gastric cancer cells is inhibited. Meanwhile, it is found that circFAM126A is highly expressed in oxaliplatin-resistant gastric cancer cells and is in positive correlation with poor prognosis of gastric cancer patients, a detection reagent aiming at circFAM126A can be used for auxiliary diagnosis of gastric cancer prognosis conditions, and the drug resistance degree of the gastric cancer cells to oxaliplatin can be evaluated by detecting the expression level of circFAM126A.

The invention relates to a recombinant human 2Ig-B7-H3 protein coding gene, a recombinant vector, a host cell, a pharmaceutical composition and application of the recombinant human 2Ig-B7-H3 protein coding gene. The recombinant human 2Ig-B7-H3 protein coding gene comprises a nucleotide sequence with the length of 3095bp, wherein a basic group C at the 1722nd site is replaced by T at a fixed point. The medicine prepared by combining the recombinant human 2Ig-B7-H3 protein coding gene packaged by the lentiviral vector and the wild type human 2Ig-B7-H3 protein coding gene packaged by the lentiviral vector according to a certain proportion has an unexpected effect in the aspect of treating or preventing cancers.

The invention provides a recombinant lentivirus capable of indicating bovine spermatogonial stem cell differentiation, a spermatogonial stem cell line and application, and belongs to the technical field of cell biology. The invention provides a method for constructing a spermatogonial stem cell line for indicating bovine sperm differentiation on the basis of a lentivirus transduction technology, and a visual detection system for differentiating spermatogonial stem cells into spermatoblasts is constructed by regulating expression of mCherry protein by utilizing a PRM1 promoter. According to the method, the expression of PRM1 is indicated by fluorescent protein mCherry, so that the differentiation process of the bovine spermatogonial stem cells is indicated, and a good tool is provided for the induction of differentiation of the bovine spermatogonial stem cells to sperms, the research of a development mechanism and the optimization of a differentiation system of the bovine spermatogonial stem cells. The method is a new way for solving the neck clamping problem that good-variety bovine sperms are difficult to obtain in scientific research and production, can also be used for optimization of a spermatogonial stem cell differentiation system, sorting and purification of sperms, even downstream research of drug screening and the like, and has a good application prospect.

The present application relates to an anti-CD3 antibody and its application. The present application develops a specific antibody molecule targeting CD3 epsilon chain, which can activate T cells by combining with CD3 epsilon on T cells. A T cell binding protein containing the CD3 antibody is further designed, which can specifically target and bind to CD3 epsilon on the surface of T cells in vivo and in vitro, so as to achieve moderate activation of T cells. The CAR lentivirus containing the T cell binding protein of the present application can induce the generation of CAR-T cells in vitro and in vivo, and the CAR-T cells have good tumor cell killing efficiency.

The present disclosure relates to methods for producing lentiviral vector-producing cells. Specifically the methods utilize two plasmids, rather than four, to provide the required packaging elements and transfer vector to a cell, allowing for the production of a large number of lentiviral producer cells, including suspension-based cells, and the production of high amounts of lentivirus. These methods allow for the production of cells that can be later induced to produce lentivirus, and can be tailored to include a specific gene of interest.

The invention provides an application of a human C1orf186 gene or a human C1orf186 protein in preparation of a targeted therapeutic drug for ovarian cancer, and relates to the technical field of biomedical engineering. The invention relates to an application of a human C1orf186 gene or a human C1orf186 protein in preparation of an ovarian cancer targeted therapy drug. Experiments prove that C1orf186 is an ovarian cancer cell surface specific membrane protein molecule, a C1orf186 positive cell population has tumor stem cell characteristics, and prepared human C1orf186 lentivirus packaging plasmids and other targeted therapeutic drugs have an obvious inhibition effect on the growth of ovarian cancer. Therefore, the invention provides a specific target C1orf186 for molecular therapy of the ovarian cancer, also provides several targeted drug types, and more importantly provides a thought for treating the ovarian cancer by using the anti-C1orf186 targeted drug, thereby laying a foundation for deeply researching the function of the C1orf186. Meanwhile, the invention provides reliable theoretical basis and experimental data for clinical treatment of ovarian cancer, and has important significance and wide prospects.

The invention discloses an HER2-CAR lentivirus and application thereof, and belongs to the technical field of lentiviruses. The invention provides the CAR with a brand new structure, the expression level, the stability and the targeting property of the CAR protein in immune cells such as T cells and NK cells can be remarkably improved, the survival time of engineered immune cells in vivo can be prolonged, the lasting anti-tumor capacity of the engineered immune cells can be prolonged, and the tumor cell removing capacity can be improved; the lentiviral vector provided by the invention is high in infection efficiency and wide in host range, provides a reliable platform for large-scale preparation of clinical-grade CAR-T / CAR-NK cell products, and has huge development potential and application value in the field of immunotherapy of HER2 positive solid tumors.

The application discloses application of a reagent for detecting LRRN3 expression level in preparation of a benign prostatic hyperplasia diagnosis product. It is proved through cell function experiments and animal experiments that overexpression of the LRRN3 gene can inhibit the proliferation of prostate cells, block the cell cycle and promote cell apoptosis, thereby effectively inhibiting the prostatic hyperplasia. The discovery provides a new target and treatment strategy for the treatment of the prostatic hyperplasia, and provides a brand-new thought and method for the treatment of the prostatic hyperplasia. Based on the research results, a gene therapy drug taking the LRRN3 gene as a target can be further developed, for example, a lentivirus vector carrying the LRRN3 gene or other gene delivery systems are constructed and used for the treatment of the prostatic hyperplasia. In addition, the LRRN3 gene can be combined with other treatment methods to improve the treatment effect.

The invention belongs to the technical field of biological medicines, and particularly relates to application and a method of PHF20 in preparation of a medicine for treating systemic lupus erythematosus and the medicine. The invention discloses an application of PHF20 in treatment of systemic lupus erythematosus, a lentivirus with murine PHF20 is selected as a gene therapy tool, an R848 induced lupus mouse model is taken as an experimental subject for gene therapy, and results show that injection of the overexpressed PHF20 lentivirus in a lupus mouse body has the following effects: (1) spleen swelling is improved, and spleen index is significantly reduced; (2) reducing urine protein, urea nitrogen level, anti-dsDNA antibody level and IgG antibody level of the model mouse; and (3) the pathological damage to the kidney of the model mouse is relieved. The research results show that the PHF20 serving as the target provides important reference value for potential application prospects in systemic lupus erythematosus gene therapy and even autoimmune disease gene therapy.

The disclosure provides novel virus packaging elements and cells transduced by such elements to enable large-scale library screens with combinatorial elements that heretofore were impractical due to the presence of recombination between library elements that occurs during conventional lentivirus production and transduction of target cells. The present disclosure overcomes these problems by generating clonal virus packaging cells that each produce a genetically homologous virus.

The invention discloses liver-responsive synthetic macrophages and a construction method and tumor immunotherapy application thereof, and belongs to the technical field of biological medicines. The system is an IBMDM cell line of a stable expression alpha SLC17A2-P65 / shSIRP alpha-synM system, and comprises an extracellular domain and an intracellular domain, the extracellular domain is composed of a single-chain antibody scFv for recognizing an antigen SLC17A2 and a transcription factor GAL4-VP16 (GV), and the intracellular domain is composed of a UAS promoter sequence recognized by the GV and P65 / shSIRP alpha. When the antibody responds to the liver, GV is released through the self-cleavage effect of synNotch, and the GV recognizes a UAS promoter sequence after entering a nucleus to activate downstream gene expression. The invention also provides a construction method of the system. The construction method comprises the following three steps: constructing a vector plasmid, packaging lentivirus and constructing a target cell line. The system can be applied to screening anti-liver cancer immunotherapy drugs, and a new research tool is provided for liver cancer treatment.

The embodiment of the invention discloses a heavy chain variable region and a light chain variable region of an anti-GPC3 monoclonal antibody and application of the heavy chain variable region and the light chain variable region. The high-affinity anti-GPC3 sequence is obtained through screening, the binding specificity of the high-affinity anti-GPC3 sequence and the GPC3 antigen is high, and dissociation is slow. The sequence is used as an extracellular targeting domain to construct GPC3-CAR, after macrophages are transduced through lentivirus, CAR-M can specifically recognize GPC3 positive target cells, activate intracellular signal channels and remarkably improve in-vitro phagocytosis and killing activity, the hepatoma cell lysis effect is better, and technical support is provided for anti-hepatoma application.

The invention discloses an application of Notch2 overexpression lentivirus in promoting aTreg cell differentiation, and relates to the field of immune cell culture, and the application comprises the following steps: suspending PBMC in a T cell culture medium, laying a plate, adding Notch2 overexpression lentivirus, and culturing at 36.8-37.2 DEG C. The invention also discloses a preparation method of the Notch2 overexpression lentivirus. According to the application, the Notch2 overexpression lentivirus and the PBMC (peripheral blood mononuclear cell) are co-cultured on a culture medium, and the PBMC (peripheral blood mononuclear cell) is transfected by utilizing the Notch2 overexpression lentivirus, so that Notch2 protein molecules are overexpressed, and the number of Treg cells and aTreg cells in lymphocytes is increased.

The invention provides a chimeric envelope glycoprotein, a preparation method thereof and an envelope plasmid, a packaging method and a kit of the chimeric envelope glycoprotein, and particularly relates to a preparation method of the chimeric envelope glycoprotein, the chimeric envelope glycoprotein and a lentivirus envelope plasmid. The invention discloses a pseudotyped packaging method of lentivirus and a kit. The amino acid sequence of the wild type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of other envelope glycoproteins, and compared with the initial wild type envelope glycoprotein, the obtained chimeric envelope glycoprotein can effectively improve the lentivirus packaging efficiency, biological activity and other properties.

The invention discloses a liver cancer cell line with LAPTM5 gene stable overexpression as well as a construction method and application of the liver cancer cell line. According to the invention, a human liver cancer cell line Huh7-LAPTM5 overexpression cell line is successfully constructed through a lentivirus transfection technology. Protein expression is detected through Western blot, and it is proved that overexpression cells are successfully constructed. Meanwhile, experiments prove that under the normal culture condition, LAPTM5 overexpression has no obvious influence on the processes of cell proliferation, apoptosis and the like. The Huh7-LAPTM5 overexpression cell line constructed by the invention plays a biological role by promoting a cell autophagy process, and the cell line is suitable for basic clinical cross correlation researches based on autophagy, drug resistance and the like.

The application discloses a method for detecting residual fragment distribution of recombinant lentivirus host cell DNA, which uses real-time fluorescent quantitative PCR to accurately detect HEK293T host cell DNA residual in 122bp, 244bp and 562bp three fragment length distribution in a recombinant lentivirus sample. The method of the application adopts a novel prepGEM enzyme to crack the rLVV virus, and the virus is cracked within 5-15 minutes, the enzyme is inactivated within 2-10 minutes, the virus sample amount is small, and the virus cracking solution can be directly applied to a real-time fluorescent quantitative PCR reaction. Compared with the magnetic bead method, the prepGEM enzyme cracking method is more stable and reasonable, and the variance and CV are smaller.

The application provides a quality control for detecting respiratory pathogen nucleic acid, which comprises any one or more of the following respiratory pathogens: coronavirus, influenza virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, rhinovirus or / and Legionella pneumophila. The full-length genome sequence of the coronavirus is divided into 6 target fragments, each with a length of 4000-5500 bp. The quality control of the application covers all detection target sequences (or target points) of the coronavirus, has a wide coverage, comprehensively detects target points, and does not have the phenomenon of missed detection. The quality control of the application contains main respiratory pathogen infections, has a wide range, and uses real virus samples and lentivirus samples as raw materials, and has more accurate detection target points. The preparation of the coronavirus quality control of the application integrates the target gene sequence into the host genome through the lentivirus vector, knocks out the self-replication gene in the preparation process, and has a "self-inactivation" ability, so that the recombinant lentivirus cannot replicate in the target cells and infect other cells.

An apparatus and method for T-cell separation, activation, transduction and expansion. Three-dimensional (3D) bioreactors may be employed that include antibody coatings. Such 3D bioreactors can be employed for T-cell separation from peripheral blood mononuclear cells including attachment of T-cells to the 3D bioreactor surface for activation and transduction by lentivirus vectors to produce CAR T-cells. The CAR T-cells can then be expanded in a separate downstream bioreactor therein providing a scalable automated system.

The invention discloses a high-yield lentivirus cell line and a preparation method thereof, and belongs to the technical field of molecular biology and cell biology. In order to improve the yield of the lentivirus, the invention provides a recombinant cell which is a cell without a specific gene. The specific genes are any one, two, three, four, five, six or seven of an LDAH protein coding gene, a GBP3 protein coding gene, a BPIFC protein coding gene, an NHLRC1 protein coding gene, a ZNF425 protein coding gene, an NHLRC3 protein coding gene and an SKIC3 protein coding gene. A CBE base editor and a specific gRNA sequence are jointly transfected into an HEK293T cell, the editing efficiency is detected through sequencing, and a stable recombinant cell line for efficiently producing the lentivirus is successfully established and applied to lentivirus production.