473 results about "Lentivirus" patented technology

The invention provides a lentivirus for treating cancer as well as a preparation method, application and a corresponding pharmaceutical composition of the lentivirus. According to the invention, an hTERT promoter and a CDC6shRNA gene sequence are inserted behind an enzyme cutting site of a vector, an obtained plasmid is transferred into an expression strain, the expression strain is cultured and screened to obtain a cloned strain, and the cloned strain is shaken and packaged to obtain the hTERT-CDC6shRNA lentivirus; when the lentivirus disclosed by the invention is used for infecting cancer cells, the experimental result shows that the proliferation of the cancer cells can be effectively inhibited.

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method of viroid particles displaying scFv antibodies, a product of the viroid particles and application of the viroid particles. The preparation method provided by the invention comprises the following steps: constructing a specific targeting recombinant plasmid, wherein the specific targeting recombinant plasmid is obtained by connecting a vector for expressing virus capsid protein and a coding gene for expressing an scFv antibody display structure through homologous recombination. The vector for expressing the virus capsid protein comprises a coding gene of VSV-G signal peptide, a transmembrane region and an intracellular region, and is as shown in SEQ ID NO. 2. And the coding gene for expressing the scFv antibody display structure is shown as SEQ ID NO. 3-5. According to the preparation method disclosed by the invention, the virus-like particles of which the capsid is a modular component of a specific targeting immune cell are constructed based on a lentivirus system, and 50-90% of infection efficiency can be realized in the immune cell.

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 belongs to the technical field of biology, and particularly relates to an immortalized alpaca kidney cell line and a construction method and application thereof. The immortalized alpaca kidney cell line disclosed by the invention is named as immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0, and the preservation number of the immortalized alpaca kidney cell line is CGMCC (China General Microbiological Culture Collection Center) No.46058. The construction method of the immortalized alpaca kidney cell line comprises the following steps: extracting and culturing alpaca primary kidney cells; infecting alpaca primary kidney cells by using SV40 large T antigen immortalized lentivirus; carrying out resistance screening on the immortalized alpaca kidney cells; carrying out alpaca kidney cell immortalized cell detection and identification; and obtaining the immortalized alpaca kidney cell line. The immortalized alpaca kidney cell constructed by the invention has the advantages of simple culture, repeated passage, good homogeneity, high expression quantity of constructed exogenous genes and the like, and provides a more powerful tool cell for research on alpaca immunity; the immortalized alpaca kidney cell line provides a new tool for virus infection mechanism, drug in-vitro function detection and antibody drug development and screening.

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 provides a lentivirus for treating craniopharyngeal tubuloma as well as a preparation method, application and a corresponding pharmaceutical composition of the lentivirus. The lentivirus carries an hTERT (human telomerase reverse transcriptase) promoter and a CDC6shRNA (complementary deoxyribonucleic acid) gene. According to the invention, an hTERT promoter and a CDC6shRNA gene sequence are inserted behind an enzyme cutting site of a vector, an obtained plasmid is transferred into an expression strain, the expression strain is cultured and screened to obtain a cloned strain, and the cloned strain is shaken and packaged to obtain the hTERT-CDC6shRNA lentivirus; when the lentivirus disclosed by the invention is inoculated into a tumor body of a patient with craniopharyngeal tubuloma, the result shows that the lentivirus can effectively inhibit proliferation of craniopharyngeal tubuloma cells and can induce self apoptosis of the craniopharyngeal tubuloma cells.

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.

A preparation method for a micro-vehicle formed by enveloping siRNA-loaded polypeptide dendrimer nanogel with a cell membrane of engineered MSCs, and the use of same. The method comprises: using lentivirus-mediated gene transfection technology for constructing MSCs that highly express CXCR4 and PSGL-1; using cytokinin B for stimulating a cell to generate vesicles, and then performing centrifugation to collect the vesicles; by means of the electrostatic attraction between polypeptide dendrimer nanoparticles and siRNAs, attaching the siRNAs of IL-6 and HIF-1α into the polypeptide dendrimer nanoparticles; and by means of using a membrane extruder, enveloping the siRNA-loaded polypeptide dendrimer nanogel with the cell membrane. The prepared micro-vehicle exhibits a good property of aggregating at inflammatory blood vessel sites and a targeting function for joint cavities with rheumatoid arthritis. In addition, the loaded siRNAs can inhibit the expressions of IL-6 and HIF-1α in the joint cavities and inhibit the cell migration and angiogenesis. The micro-vehicle targeting inflammation sites has the advantages of excellent biocompatibility, ideal siRNA loading ratio, good inflammation targeting function, etc.

The invention belongs to the technical field of gene engineering, and particularly relates to application of a TRIM3 gene or protein serving as a target spot in screening of drugs for inhibiting foot and mouth disease virus replication. The method specifically comprises the following steps: firstly, constructing a TRIM3 knockout cell line by utilizing a CRISPR / Cas9 system, wherein the cell line obviously inhibits the replication of FMDV; secondly, according to the invention, a TRIM3 stable expression cell line is constructed by using a lentivirus packaging system, and the cell line promotes the replication of FMDV. Therefore, the inhibition or silencing of the TRIM3 gene / protein can inhibit the replication of the foot-and-mouth disease virus, can be used as a target spot for screening drugs for inhibiting the replication of the foot-and-mouth disease virus, provides some theoretical bases for preventing or inhibiting FMDV infection in the future, and has a wide application prospect.

The invention belongs to the field of immunology, and particularly relates to amplification of NK cells, in particular to a method for amplifying NK cells by using NK92 cells as trophoblast cells, which specifically uses NK92 cells secreting IL-21, IL-15 and IL-18 as trophoblast cells and PBMC as seed cells to directionally amplify and activate natural killer cells, and comprises the following steps: transfecting an NK92 cell strain through lentivirus; the NK92 cell strain capable of stably expressing IL-21, IL-15 and IL-18 is obtained; then obtaining monoclonal cells of NK92 cells through flow sorting and limit dilution methods, finally culturing and amplifying, preparing NK92 trophoblasts through irradiation, and mixing the NK92 trophoblasts with PBMC (peripheral blood mononuclear cells) for culturing so as to prepare the NK cells. According to the method, the NK cells can be efficiently amplified and activated, and the activated NK cells can improve the immunity of the organism, so that tumor and virus infection is resisted. The invention has wide application in medicine.

The invention discloses a virus-like plasmid and application thereof. The virus-like plasmid is characterized in that mIL-21 and 4-1BBL coding genes are inserted into a lentivirus plasmid; the amino acid sequences of the coding genes of the mIL-21 and the 4-1BBL are respectively as shown in SEQ ID NO. 1 and SEQ ID NO. 2. Virus-like particles (VLPs) prepared from the virus-like plasmids are added into an NK cell culture solution, and the VLPs can synergistically stimulate the NK cells, promote the rapid growth of the NK cells (the amplification multiple reaches 1850 times after 18 days of culture), and can improve the purity (about 92%) and the killing rate (when the efficiency-target ratio is 2: 1, up to 80%) of the NK cells.

The invention belongs to the technical field of biological detection, and relates to a method for detecting lentivirus titer by a double-standard-curve RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) method. Infecting the target cells by adopting a to-be-detected sample containing a recombinant lentivirus, and collecting and extracting genome DNA of the infected target cells; carrying out a viral genome RT-qPCR amplification reaction and an internal reference genome RT-qPCR amplification reaction by respectively taking the original virus standard substance and the internal reference standard substance which are diluted in a gradient manner and the extracted target cell genome DNA as templates; and calculating the lentivirus titer corresponding to the sample according to data obtained by the RT-qPCR amplification reaction. The method can be used for absolute quantitative qPCR detection of lentivirus titer, can accurately determine lentivirus successfully integrated into a cell genome, and reflects the real infection ability of the virus; in addition, the technology has high sensitivity and specificity, is not influenced by the extraction efficiency of the cell genome DNA, and ensures the reliability of the detection result.

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 present disclosure describes a next-generation antisense transfer vector along with methods for a high titer lentivirus production, allowing efficient transduction of T cells with a constitutively expressed tumor-targeting receptor along with the activation-induced expression of various gene cargos. The disclosed antisense transfer vector and the methods can reduce virus production costs as well as enhance the efficacy and safety of next-generation CAR- or TCR-T cells reaching the clinic.

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 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 discloses an avian flavivirus NS1 trans-complementary pseudovirus system and application thereof. According to the pseudovirus system disclosed by the invention, a BHK21 cell line for stably expressing USUV-NS1 protein carrying a V5 label, namely a BHK21-NS1 stably transfected cell line, is constructed by utilizing a lentivirus packaging method; meanwhile, deletion mutation is introduced into the NS1 protein of USUV, infectious clone plasmids are constructed, and the 157th-251st amino acid of the NS1 protein is deleted by the deletion mutation. The infectious clone plasmid is transfected with a BHK21-NS1 stably transfected cell line, so that the trans-complementary virus USUV-delta NS1 which can be rescued and proliferated only in the BHK21-NS1 stably transfected cell line is obtained. The USUV-deltaNS1 trans-complementary virus is subjected to continuous passage on a BHK21-NS1 cell for 10 generations, so that the TMN-deltaNS1 virus with high yield is obtained, and complete live virus is not generated by recombination in the passage process. Therefore, the avian flavivirus NS1 trans-complementary pseudovirus system disclosed by the invention can be used as a tool to be applied to USUV vaccine preparation and related antibody evaluation.

The invention discloses an Nfkb1 gene overexpression cell line and a construction method thereof, and relates to the field of gene engineering. The constructed recombinant lentiviral expression vector comprises a first LTR, a CMV promoter, an Nfkb1 gene, a Flag polypeptide tag, an EF-1a promoter, a green fluorescent protein gene, a shearing element, a screening gene and a nucleic acid fragment of a second LTR which are sequentially arranged from the 5'end to the 3 'end; co-transfecting a host cell by using the recombinant lentivirus expression vector and lentivirus packaging helper plasmid, and carrying out virus packaging to obtain an Nfkb1-overexpressed recombinant lentivirus; the HT22 cell line overexpressing the Nfkb1 is obtained by infecting the HT22 cell with the recombinant lentivirus overexpressing the Nfkb1, and screening the HT22 cell line overexpressing the Nfkb1 by using a resistance gene. Recombinant lentivirus is used as a vector, HT22 cells are infected with the recombinant lentivirus vector, a cell line capable of stably overexpressing the Nfkb1 gene is obtained through screening, and the effect of the Nfkb1 in neuroinflammation is conveniently researched.

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 present invention relates to the field of molecular biology, and in particular to an SLC17A5 gene inhibitor and a use thereof. The present invention provides a use of the SLC17A5 gene inhibitor in preparation of a kidney cancer treatment product. According to the SLC17A5 gene inhibitor provided by the present invention, a proper RNAi target sequence and oligo DNA double-stranded sequence are designed for a target gene, and a lentiviral vector plasmid containing the oligo DNA double-stranded sequence and a finally formed lentivirus are constructed; the resistance gene carried in the lentivirus can effectively reduce the mRNA expression of the target gene SLC17A5, and the lentivirus has an very obvious knockdown effect, has high inhibitory efficiency on cell proliferation and cell migration of kidney cancer cells, and can be used in drugs for treating kidney cancer; moreover, the present invention can be operated with high throughput and has high repeatability.

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.