105 results about "Slow virus" patented technology

The present invention relates to the field of lentiviral delivery. Specifically provided are a mutated vesicular virus envelope protein and the use thereof in lentiviral vector packaging.

Provided herein are engineered nucleic acids (e.g., expression vectors, including viral vectors, such as lentiviral vectors, adenoviral vectors, AV vectors, herpes viral vectors, and retroviral vectors) that encode OCT4; KLF4; SOX2; or any combination thereof that are useful, for example, in inducing cellular reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, or any combination thereof. Also provided herein are recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) comprising the engineered nucleic acids (e.g., engineered nucleic acids), engineered cells, compositions comprising the engineered nucleic acids, the recombinant viruses, engineered cells, engineered proteins, chemical agents that are capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, an engineered protein selected from the group consisting of OCT4; KLF4; SOX2; or any combination thereof, an antibody capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, and methods of treating a (e.g., ocular disease), preventing a disease (e.g., ocular disease), regulating (e.g., inducing or inducing and then stopping) cellular reprogramming, regulating tissue repair, regulating tissue regeneration, or any combination thereof).

Provided herein are engineered nucleic acids (e.g., expression vectors, including viral vectors, such as lentiviral vectors, adenoviral vectors, AAV vectors, herpes viral vectors, and retroviral vectors) that encode OCT4; KLF4; SOX2; or any combination thereof that are useful, for example, in inducing cellular reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, or any combination thereof. Also provided herein are recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) comprising the engineered nucleic acids (e.g., engineered nucleic acids), engineered cells, compositions comprising the engineered nucleic acids, the recombinant viruses, engineered cells, engineered proteins, chemical agents that are capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, an engineered protein selected from the group consisting of OCT4; KLF4; SOX2; or any combination thereof, an antibody capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, and methods of treating a (e.g., ocular disease), preventing a disease (e.g., ocular disease), regulating (e.g., inducing or inducing and then stopping) cellular reprogramming, regulating tissue repair, regulating tissue regeneration, or any combination thereof).

Provided herein are engineered nucleic acids (e.g., expression vectors, including viral vectors, such as lentiviral vectors, adenoviral vectors, AAV vectors, herpes viral vectors, and retroviral vectors) that encode OCT4; KLF4; SOX2; or any combination thereof that are useful, for example, in inducing cellular reprogramming, tissue repair, tissue regeneration, organ regeneration, reversing aging, or any combination thereof in the central nervous system or ex vivo. Also provided herein are recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs) comprising the engineered nucleic acids (e.g., engineered nucleic acids), engineered cells, compositions comprising the engineered nucleic acids, the recombinant viruses, engineered cells, engineered proteins, chemical agents that are capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, an engineered protein selected from the group consisting of OCT4; KLF4; SOX2; or any combination thereof, an antibody capable of activating expression of OCT4; KLF4; SOX2; or any combination thereof, and methods of treating a disease (e.g., a neurological disease), preventing a disease (e.g., neurological disease), regulating (e.g., inducing or inducing and then stopping) cellular reprogramming, regulating tissue repair, regulating tissue regeneration, or any combination thereof.

The invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

The invention discloses an oncolytic virus and application of the oncolytic virus in preparation of tumor inhibition drugs. The oncolytic virus is a lentiviral vector, and the lentiviral vector comprises polynucleotide encoding p16 protein or a bioactive part of the p16 protein containing CDKN2A gene, and can effectively inhibit growth of cancer related to CDKN2A gene mutation, so that the problem that the existing oncolytic virus has biological safety risk in delivery of cancer suppressor genes is effectively solved.

The invention discloses a construction and verification method of an HEK293T cell line capable of stably overexpressing human ANT2, which comprises the following steps: cloning a human ANT2 gene, constructing the human ANT2 gene into a lentivirus expression plasmid pLVX-TRE3G, co-transfecting the lentivirus expression plasmid pLVX-TRE3G and a helper plasmid pLVX-Tet3G into an HEK293T-ACE2 cell, and carrying out puromycin and G418 pressurized screening and Western blot identification to obtain the HEK293T cell line capable of stably overexpressing human ANT2. A result shows that the expression quantity of the ANT2 is the highest when the HEK293T-ACE2-ANT2 cell is induced for 12 hours at the Doxycycline of 6mg / L; when the ANT2 is induced to express, the virus nucleic acid load and the N protein expression level after SARS-CoV-2 infection are obviously reduced, which indicates that the ANT2 has an inhibition effect on SARS-CoV-2 infection replication, and a cell model and an experimental basis are provided for exploring the effect of the ANT2 in virus infection resistance.

This invention provides a method for constructing and applying MDCK cells stably integrated with disialotyltransferase, relating to the field of biotechnology. The construction method includes: infecting MDCK cells with lentiviral particles, followed by culture and screening to obtain MDCK cells stably expressing porcine ST3GAL1 and porcine ST6GAL1 genes; the lentiviral particles carry porcine ST3GAL1 and porcine ST6GAL1 genes; the nucleic acid sequence of the porcine ST3GAL1 gene is shown in SEQ ID NO.1; the nucleic acid sequence of the porcine ST6GAL1 gene is shown in SEQ ID NO.2. This construction method is simple and convenient, and the constructed MDCK cells can stably express porcine ST3GAL1 and porcine ST6GAL1 genes, effectively improving the culture efficiency of swine influenza virus, and thus can be used in the preparation of swine influenza virus.

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.

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.

This invention relates to retroviral gene transfer vectors, particularly lentiviral vectors, pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, comprising a promoter and a transgene; and methods of making the same. The present invention also relates to the use of said vectors in gene therapy, particularly for the treatment of respiratory tract diseases such as Cystic Fibrosis (CF).

The invention relates to a seawater oryzias medaka stable expression cas9 cell line, a construction method and application, and belongs to the technical field of gene editing, the cell line takes seawater oryzias medaka cells as a main body, and a cas9-Blasticidin gene is inserted into a seawater oryzias medaka cell genome. The construction method of the cell line comprises the following steps: infecting a testis cell line of oryzias melastigma by lentivirus to realize stable insertion of a cas9 gene in an oryzias melastigma genome, and screening a monoclonal cell line to obviously improve the expression quantity of cas9 in the cell line. The invention also provides a method for constructing the oryzias melastigma cell line by utilizing the cell line to edit the mstnb gene of the oryzias melastigma cell, and the method for constructing the oryzias melastigma cell line. The mstnb gene is successfully mutated by transfecting sgRNAs in the cell line.

The invention relates to recombinant polynucleotides encoding at least a recombinant polynucleotide expressing at least a first fusion polypeptide that comprises MHC class I T-cell epitopes suitable to elicit a T cell immune response in a host in need thereof, wherein the MHC class I T-cell epitopes originate from a plurality of antigens wherein the antigens comprise at least non-structural antigens and are from at least one flavivirus selected from the group of 10 Dengue virus (DENV), ZIKA virus (ZIKV) and Yellow Fever virus (YFV). The invention also relates to the polypeptides comprising polyepitopes of said antigens encoded by the recombinant polynucleotides.

The invention discloses a replication-limited recombinant feline herpesvirus as well as a construction method and application thereof, and relates to the technical fields of genetic engineering and preventive veterinary medicine, exogenous feline parvovirus VP2 protein is expressed in the recombinant feline herpesvirus, and meanwhile, a gL gene in the feline herpesvirus is deleted. The construction method comprises the following steps: 1, constructing a QD-1 gL gene stable expression F81 cell line by using a lentivirus packaging three-plasmid system; 2, constructing a PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid, and carrying out GFP FPV VP2 recombinant plasmid construction; 3, an sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the gL gene is constructed, and the sgRNA expression vector lentiCRISPRv2-gL-sgRNA of the And 4, transfecting the recombinant vectors in the step 2 and the step 3 to an F81-gL cell line to obtain the QD-1 gL-FPV VP2 recombinant feline herpesvirus. The recombinant feline herpesvirus can be used as a candidate vaccine to prepare a strain so as to resist feline herpesvirus and feline parvovirus infection.

The present invention includes composition and methods for making multivalent vaccines for immunization against Flavivirus and / or arboviruses including a multivalent Virus Like Particles (VLP) and mixtures thereof, the method comprising: method of making a Flavivirus and / or arboviruses Virus Like Particles (VLP) comprising: inserting two or more nucleic acids that encode at least one Flavivirus protein into a lentiviral backbone vector; generating a lentivirus by transfecting a first cell line with the lentiviral backbone vector and isolating the lentivirus therefrom; transducing a second cell line with the lentivirus; culturing the transduced cell line under conditions in which the multivalent Flavivirus Virus Like Particles (VLP) are released from the cell line; and isolating the Flavivirus Virus Like Particles (VLP) from a culture supernatant, wherein a cell line makes a virus-specific VLP, and the VLPs are purified and then mixed in different combinations to make the multivalent vaccine.

The invention relates to the technical field of biological medicine, and discloses a lentiviral vector for specifically targeting a target cell and a construction method and application thereof, the lentiviral vector comprises: (1) an antigen targeting artificial protein receptor, the receptor comprising a target cell surface antigen binding domain and a transmembrane domain; (2) a mutated Moreton vesicular disease virus envelope protein; and (3) an expression cassette, wherein the expression cassette comprises a heterologous transgene. The novel lentiviral vector based on the mutated Moreton vesicular disease virus envelope, provided by the invention, can realize specific infection on target cells in vitro and in vivo, and compared with a vesicular stomatitis virus envelope VSV-G, the lentiviral vector constructed based on mutated Moreton vesicular disease virus envelope protein is better in stability in serum, and has a good application prospect. The efficiency of specifically infecting target cells is higher, and the drug effect in a mouse tumor model is more prominent.

Provided herein are RNAi molecules for treating Huntington's disease. Further provided herein are expression cassettes, vectors (e.g., rAAV, recombinant adenoviral, recombinant lentiviral, and recombinant HSV vectors), cells, viral particles, and pharmaceutical compositions containing the RNAi. Yet further provided herein are methods and kits related to the use of the RNAi, for example, to treat Huntington's disease.

The invention provides a recombinant LLC-PK1 cell line capable of stably expressing a TGEV-N gene and application of the recombinant LLC-PK1 cell line in promotion of virus rescue, and belongs to the technical field of gene engineering. The invention provides a recombinant LLC-PK1 cell line capable of stably expressing TGEV-N. The recombinant LLC-PK1 cell line capable of stably expressing TGEV-N. The recombinant LLC-PK1 cell line capable of stably expressing TGEV-Nis obtained by cloning a TGEV-N fragment into a lentiviral vector to construct a recombinant lentiviral plasmid and infecting LLC-PK1 cells through lentivirus packaged by virus rescue. The recombinant LLC-PK1 cell capable of stably expressing the TGEV-N is beneficial to rescue of TGEV infectious cloned viruses, so that a tool is provided for researching a virus pathogenic mechanism and a novel genetic engineering vaccine.

The invention provides a human diploid stably transfected cell line suitable for coxsackie virus A6 culture and application of the human diploid stably transfected cell line. The construction of the human diploid stably transfected cell line comprises the steps of construction of an hKREMEN1 lentivirus overexpression vector, virus packaging and stably transfected cell screening. The problem that proliferation of the CVA6 virus in human diploid cells is not ideal is solved, the human KREMEN1 gene is over-expressed in the human diploid cells (KBM17) by utilizing a lentivirus technology, and the proliferation capacity of the CVA6 virus on the KMB17 cells is improved by constructing the KMB17 cells capable of stably expressing hKREMEN1 receptors, so that a good foundation is laid for research and development of hand-foot-and-mouth disease vaccines; vaccine preparation and effectiveness evaluation are facilitated.

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.

This invention relates to a replication-restricted recombinant feline herpesvirus, its construction method, and its applications, encompassing the fields of genetic engineering and preventive veterinary medicine. The recombinant feline herpesvirus expresses the exogenous feline parvovirus VP2 protein while simultaneously lacking the gL gene from feline herpesvirus. The construction method is as follows: 1. A QD-1 gL gene stably expressed F81 cell line is constructed using a lentiviral packaging three-plasmid system; 2. A PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid is constructed; 3. The gL gene sgRNA expression vector lentiCRISPRv2-gL-sgRNA is constructed; 4. The recombinant vectors from steps 2 and 3 are transfected into the F81-gL cell line to obtain the QD-1 ΔgL-FPV VP2 recombinant feline herpesvirus. The recombinant feline herpesvirus of this invention can be used as a candidate vaccine strain to resist feline herpesvirus and feline parvovirus infection.

The invention discloses a virus aerosol experiment system based on pseudovirus. The virus aerosol experiment system comprises the following steps: preparing a lentiviral vector for expressing green fluorescent protein; adding the lentiviral vector expressing the green fluorescent protein into a PBS solution to obtain a virus aerosol generation solution, adding the virus aerosol generation solution into an aerosol generator, taking a DMEM culture medium containing 1% of fetal calf serum as an aerosol collection solution, putting the aerosol collection solution into a liquid impact type aerosol collector for treatment, culturing HEK293T cells, inoculating the aerosol collection solution with the HEK293T cells, and culturing the HEK293T cells to obtain the lentiviral vector expressing the green fluorescent protein. Culturing the inoculated cells for 72 hours; and after culture is completed, measuring the proportion of cells expressing fluorescence, and calculating the number of active virus particles in the virus aerosol. According to the invention, a set of virus aerosol experiment system is established, the system adopts a pseudovirus expressing fluorescent protein as a model virus to carry out a virus aerosol active particle detection experiment, and the system can be applied to a method and a technology for evaluating detection and disinfection of virus aerosol active particles in air.

The invention relates to the technical field of medicine and biology, and particularly discloses a stress-inducible virus and application thereof.The stress-inducible virus comprises a stress-inducible adenovirus or a stress-inducible lentivirus, the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 1, and the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 2. The preparation method comprises the following steps: constructing a virus vector, wherein the virus vector has a nucleotide sequence as shown in SEQ ID NO: 1; and virus packaging: transfecting HEK293T cells by adopting shuttle plasmids containing SEQ ID NO: 1 and skeleton plasmids containing viral genome sequences, and collecting a virus stock solution to obtain the stress induced virus. The invention also discloses application of the stress-induced virus in preparation of medicines for preventing and treating oral tumors. Controllable release of the foreign protein under a specific signal is realized. The therapeutic protein produced by the invention can maximize the curative effect of drugs such as CD47 targeted protein and the like, and meanwhile, the toxic and side effects of the drugs on other tissues of the whole body are reduced.

Provided herein are a vector for eradicating human immunodeficiency virus (HIV) from a human comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV. Methods of treating of HIV and kits are also provided.

Described herein are lentiviral vectors and lentiviruses (comprising one or more lentivirus particles) with a payload capacity that is roughly double that of existing lentiviral vectors and lentiviruses. Kits and methods for making and using such vectors and viruses are also provided according to the present disclosure.

The invention belongs to the technical field of cell culture, and particularly relates to a construction method of an immortalized goat umbilical cord mesenchymal stem cell line. The method comprises the following steps: by taking newborn goat lamb umbilical cord tissues as a research object, cleaning goat lamb umbilical cords, stripping blood vessels, separating Wharton's jelly, cutting the Wharton's jelly into 1mm < 3 > tissues, culturing by using a DMEM / F12 complete culture medium containing fetal calf serum, carrying out passage, cryopreservation, repeated resuspension and centrifugation, and inoculating into a culture dish for culture; lentiviruses containing SV40 large T antigens are packaged in vitro and are transduced into goat umbilical cord mesenchymal stem cells, and immortalization is realized after long-term subculture. The result of the embodiment shows that the construction method of the immortalized goat umbilical cord mesenchymal stem cell line provided by the invention is strong in repeatability, simple and efficient in operation steps, good in stem cell growth state and stable in physiological state.

The embodiment of the invention discloses a method and a kit for rapidly and accurately quantifying an active lentiviral vector. The method comprises the following steps: treating a lentiviral vector sample by using a treating fluid to obtain a pretreated sample; carrying out first fluorescence labeling on the sample by using specific dye labeled lentiviral vector nucleic acid; performing second fluorescence labeling on the sample by using a specific protein antibody or aptamer; detecting fluorescence signals of the particles in the sample by using a flow cytometer; and counting the number of the particles jointly marked by the first fluorescence and the second fluorescence in the sample so as to calculate the number of the active lentiviral vectors. According to the quantitative method for the activity of the lentiviral vector, accurate qualitative and quantitative detection is carried out on the active lentiviral vector; the method can distinguish the complete lentiviral vector from other empty-shell viruses, free nucleic acids and the like, has the characteristics of accurate virus activity characterization and short measurement time, and can complete all detection within 1 hour.

The invention discloses a mouse M2 type macrophage induction method and application, and aims to solve the technical problem that mouse tumor cells cannot grow into tumors in a mouse body due to the fact that the polarization degree of current mouse M2 type macrophages is very low in the induction process. S2, after the cells are recovered and cultured, performing planking and virus infection on the recovered and cultured cells; s3, after the cells are subjected to planking and virus infection, protein and medicine configuration is carried out; s4, after the protein and the medicine are prepared, RAW264.7 M2 polarization condition setting is carried out; and S5, after the polarization condition of the RAW264.7 M2 is set. Protein drugs such as IL4, IL13, IL10, M-CSF, AZM, SOD and DEX are exogenously added, and lentivirus is used for mediating macrophages to stably express IL-4R receptors, so that M2 polarization of the macrophages is finally greatly improved, and a foundation is laid for subsequent research on growth and tumor formation of human / mouse tumor cells in mice.