73 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, 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 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.

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.

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.

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.

Formulas for use in enhancing the transduction efficiency of lentiviral vector (LV) particles into various target cells are disclosed herein. For example, a formulation for use in enhancing the transduction of paramyxovirus pseudotype lentiviral vector particles into target cells may comprise a cell-permeable peptide (CPP) comprising at least one of Vectofusion-1 or LAH4-C; and poloxamer 407, wherein the concentration of poloxamer 407 is greater than or equal to the concentration of CPP. Additionally, a method of using such formulations to enhance the transduction of LV particles, including particularly in vivo transduction, is disclosed herein.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

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).

The present invention relates to an integration-defective lentiviral vector (IDLV) comprising a nucleic acid, the said nucleic acid comprising, between a 5′ LTR sequence and a 3′ LTR sequence, at least one nucleus export signaling sequence; at least one nucleic acid sequence of interest; and at least one nuclease site. The invention further relates to an isolated cell comprising said IDLV, a pharmaceutical composition comprising said IDLV or said isolated cell, and their pharmaceutical use in the treatment of a disease selected from the group consisting of immune diseases, viral infections, tumors and blood diseases; and / or a disease caused by the lack of a protein or by the presence of an aberrant non-functional one in an individual in need thereof.

The invention provides a lentiviral vector purification method and application thereof. The lentiviral vector obtained by the method is high in purity and low in impurity content, and very high transfection efficiency can be obtained. Besides, the volatility among all batches of lentiviral vectors produced by the method is extremely small, the lentiviral vectors are generally stable in the aspects of yield and recovery rate, and all batches of lentiviral vectors can meet the requirement of virus injection level in the aspect of impurity level.

The invention discloses a tongue squamous cell carcinoma in-situ PDX model construction method based on SERINC3 high expression and application of SERINC3, and belongs to the technical field of tumor biology and animal model construction. The method comprises the following steps: segmenting small tissues from cancer tissues of a patient suffering from tongue squamous cell carcinoma, implanting the small tissues into the armpit of an NCG mouse to culture tumor tissues, and then subculturing the tumor tissues; the subcultured tumor tissue is taken and implanted into the lingual margin of an NCG mouse, and after the tumor is cultured, lentivirus carrying sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids is injected into the tumor at multiple points to obtain the tongue squamous cell carcinoma in-situ PDX model. The model constructed by the invention can better simulate the natural occurrence and development process of TSCC, reconstructs a tumor microenvironment closer to clinical practice, and particularly has remarkable advantages in the aspect of simulating head and neck lymph node metastasis of tongue squamous cell carcinoma.

The application belongs to the technical field of biology, and particularly relates to a 3D protein mutant O foot-and-mouth disease recombinant virus strain and construction and application thereof. The application mutates methionine at the 403th position of the 3D protein of the O foot-and-mouth disease virus into alanine, and successfully constructs and rescues a 3D protein mutant O foot-and-mouth disease recombinant virus strain rFMDV-M403A by using reverse genetic technology. The amino acid of the recombinant virus strain can be stably mutated in the process of subculture, and the growth speed of the mutant strain is slower than that of the wild strain, and the virus titer is significantly reduced. In individual experiments, the infection of the mutant strain to suckling mice shows that the mutant strain can improve the survival rate of the suckling mice, and significantly reduce the viral load in the liver and the tissue lesions. The application provides an attenuated foot-and-mouth disease recombinant virus strain, which has important significance for in-depth understanding of the pathogenic mechanism and prevention and control strategy of the foot-and-mouth disease virus, and provides an important theoretical basis for further development of the foot-and-mouth disease genetic engineering vaccine.

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.