41 results about "Slow virus" patented technology

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 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 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 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.

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.

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 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.

The application provides an application of a reagent overexpressing a HS3ST5 gene in promoting virus infection and a recombinant CHO-K1 cell line constructed by the reagent, and belongs to the technical field of genetic engineering. The application provides that a HS3ST5 fragment is cloned into a lentivirus vector to construct a recombinant lentivirus plasmid, and then the lentivirus is packaged by virus rescue, and then the lentivirus is used for infection of CHO-K1 cells to obtain a recombinant CHO-K1 cell line expressing the HS3ST5. The recombinant CHO-K1 cell stably expressing the HS3ST5 can promote adsorption, internalization, gene replication and progeny virus generation of FMDV, which provides a tool for studying a virus pathogenic mechanism and provides a basis for preparing a virus infection cell model subsequently.

The application provides application of a reagent for overexpressing an RNMT gene in promoting virus infection and a recombinant LLC-PK1 cell line constructed by the reagent, and belongs to the technical field of genetic engineering. The application provides that an RNMT fragment is cloned into a lentivirus vector to construct a recombinant lentivirus plasmid, and then the lentivirus is packaged by virus rescue, LLC-PK1 cells are infected, and a recombinant LLC-PK1 cell line for expressing RNMT is obtained. The recombinant LLC-PK1 cell line for stably expressing RNMT can promote generation of PEDV offspring viruses, which provides a tool for studying a virus pathogenic mechanism and provides a basis for preparing a virus infection cell model subsequently.

The invention discloses a nanoparticle antigen based on a ferritin vector, a recombinant expression vector, a recombinant lentiviral vector and a preparation method and application of the nanoparticle antigen. The nanoparticle antigen comprises recombinant protein formed by fusion of a swine transmissible gastroenteritis virus spike protein receptor binding domain and a ferritin subunit through a flexible connecting peptide, the recombinant protein is self-assembled to form the spherical nanoparticle antigen. According to the invention, eukaryotic expression plasmids and lentivirus packaging plasmids are successfully constructed, and efficient expression and purification of the fusion protein in HEK293T cells and ExpiCHO cells are realized. The purified recombinant protein can be spontaneously assembled in vitro to form spherical nanoparticles with uniform structure and regular form. Transmission electron microscope detection proves that the compound has good structural integrity and high-density multivalent antigen display capability. The vaccine has the advantages of high safety, strong immunogenicity, easiness in industrialization and the like, and a new technical path is provided for development of TGEV and related coronavirus vaccines.

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 invention discloses a method for improving H1N1 influenza pseudovirus infection titer based on a lentivirus system and application of the method, and belongs to the technical field of pseudoviruses. The technical problem to be solved is that the infection activity of the H1N1 influenza pseudovirus is low, and the key point of the technical scheme is to establish a method for improving the infection activity of the H1N1 influenza pseudovirus by treating the H1N1 influenza pseudovirus by using TPCK-pancreatin before infection, so that the virus infection activity is remarkably improved, and the infection efficiency is about 50% instead of being almost infected with cells originally.

This invention discloses a nucleic acid molecule encoding a modified retroviral Gag protein and its applications. The Gag protein contains an engineered acylated tag sequence at its N-terminus, comprising: a glycine residue at the second position after the initiating methionine for myristylation modification; and / or a cysteine ​​residue in the N-terminal region for palmitoylation modification. The tag sequence is selected from SEQ ID NO:1, 3-9, or derivative amino acid sequences having at least 80% identity and retaining function. Preferably, the modification involves replacing the first 9 amino acids at the N-terminus of the wild-type HIV-1 Gag protein with the aforementioned tag to enhance membrane anchoring and improve viral packaging efficiency. This invention also provides packaging plasmids containing this molecule, host cells, packaging systems, and preparation methods. This invention can significantly improve viral yield and titer, and is particularly suitable for overcoming the efficiency bottleneck in packaging large-load lentiviruses of 7-10 kb.

Provided herein are lipid particles containing at least two attachment proteins derived from a paramyxovirus envelope attachment protein and at least one paramyxovirus fusion (F) protein. Also provided are lipid particles, such as lentiviral vectors or lentivirus-like particles, as viral vectors. Also provided are producer cells and compositions containing such lipid particles as well as methods of making and using the lipid particles.

The invention discloses a BHK-21 cell line capable of stably expressing a human ENT1 gene and a construction method and application thereof, the cell line is hamster kidney tissue fibroblast BHK-ENT1, the cell line carries the human ENT1 gene, and the ENT1 provides substances and energy basis necessary for replication for viruses by maintaining the steady state of nucleotide and energy in the cell, so that the human ENT1 gene can stably express the human ENT1 gene. The virus titer generated after the Newcastle disease virus infects host cells is improved. The construction method of the cell line comprises the following steps: amplifying a human ENT1 gene and constructing a stable expression vector, co-transfecting the stable expression vector and packaging plasmids psPAX2 and pMD2. G to an HEK-293T cell, culturing, and collecting a supernatant rich in lentiviral particles; and infecting the BHK-21 cell with the collected lentivirus, screening through a puromycin drug, and carrying out enlarged culture, so as to obtain the BHK-ENT1 cell.

The application discloses application of MARCH2 gene as a target point in inhibition of replication of avian leukosis virus (ALV), and further discloses a MARCH2 gene overexpression cell line, a construction method thereof and application of the MARCH2 gene overexpression cell line in inhibition of infection of different subgroups of ALV. The MARCH2 gene overexpression cell line is successfully constructed by a lentivirus packaging infection technology, and experimental results show that the cell line can significantly inhibit replication of different subgroups of ALV. The application proposes that overexpression of the MARCH2 gene or expression of a coded protein of the MARCH2 gene can effectively inhibit replication of different subgroups of ALV. The finding provides a technical means for future anti-disease breeding work against ALV infection by taking the MARCH2 gene as a target point, and has a wide application prospect.

The application belongs to the technical field of biological medicine, and discloses a spinal cord injury treatment drug based on a LINC00311 / miR-4318 / MYLK regulation axis and an application thereof, and aims to solve the technical problems of poor targeting and large side effects of the existing anti-inflammatory treatment of spinal cord injury. The effective component of the drug is an inhibitor of LINC00311, the inhibitor is selected from one or more of shRNA, siRNA, antisense oligonucleotide and CRISPRi system, can target the specific nucleotide sequence of LINC00311, and can be used in combination with miR-4318 mimic, and is administered by intrathecal injection, slow virus carrier delivery and the like. The in-vitro experiment proves that the inhibitor can significantly inhibit the release of TNF-alpha, IL-1beta, IL-6 and other pro-inflammatory factors by microglia cells; the in-vivo experiment shows that it can significantly improve the BBB motor function score of spinal cord injury rats, and reduce spinal cord tissue edema and necrosis. The application first discloses the mechanism of the LINC00311 / miR-4318 / MYLK ceRNA regulation axis in spinal cord injury, has the advantages of novel target, significant effect and high transformation potential, and provides a new scheme for the clinical treatment of spinal cord injury.

Provided for herein are pharmaceutical compositions comprising a virus, such as lentiviruses, including pseudotyped lentiviruses and uses thereof. Also provided for herein are methods of purifying and / or concentrating viral vectors and methods of producing sterile compositions, such as, sterile pharmaceutical compositions, wherein the methods can perform steps after passing the viral vector through a protein coated sterile filter. Also provided for herein are methods of using the pharmaceutical compositions.

The invention relates to the technical field of virus utilization, and particularly provides a method for improving lentivirus titer, which is used for screening compounds, compound concentration and harvesting time used in a production process. On one hand, the mechanism provided by the invention is helpful for reducing reinfection of produced lentivirus BaEVless on production cells and improving virus titer; on the other hand, in the production process, the heparin sodium can remarkably reduce apoptosis after production cell fusion, prolong the cell survival time and increase the virus yield.

The invention discloses a preparation method and application of a conditionally replicated lentiviral vector, and the preparation method comprises the following steps: based on an attenuated HIV-1 (R8.71) without an auxiliary protein coding regulatory gene, inserting a tetracycline regulated transcriptional activator rtTA and a tetO-containing cis-response element TRE, and carrying out function inactivation on a transcriptional regulation system Tat-TAR of the attenuated HIV-1 (R8.71), so as to obtain the conditionally replicated lentiviral vector. The conditional replicating type lentivirus vector plasmid is obtained; the auxiliary protein coding regulatory gene comprises a Vif gene, a Vpr gene, a Vpu gene and a Nef gene; the nucleotide sequence of the transcription activator rtTA is as shown in SEQ ID NO. 1; 1, and the nucleotide sequence of the cis-response element TRE is as shown in SEQ ID NO. 2. The conditionally replicated lentiviral vector provided by the invention meets the requirements of'catalog of pathogenic microorganisms infected by humans', can be tested in a P2-level biosafety laboratory with the qualification easier to obtain than P3, is used as a positive control virus for RCL detection, and greatly improves the flux of RCL release detection while meeting the requirements of a quality system for data integrity.

This invention provides a method for characterizing recombinant viral particle preparations using analytical ultracentrifugation. The recombinant viral particles include recombinant adeno-associated viral particles, recombinant adenovirus particles, recombinant lentivirus particles, and recombinant herpes simplex virus particles. Different variants of recombinant viral particles, including empty capsids and recombinant viral particles with different genomes (e.g., truncated genomes, aggregates, recombinants), can be identified and quantified. This method can be used to characterize recombinant viral particle preparations regardless of the genomic sequence of the recombinant virus or the serotype of the recombinant viral capsid.

The invention relates to a lentiviral vector or retroviral vector and application thereof in infection of B cells, establishment of acquired immunity in subjects and preparation of broad-spectrum neutralizing antibodies. A virus envelope of the lentiviral vector or the retroviral vector contains a targeting molecule targeting B cells and carries a broad-spectrum neutralizing antibody gene; the viral glycoprotein of the lentiviral vector or retroviral vector may further comprise a first mutation that attenuates its ability to bind to a receptor and a second mutation that enhances its ability to antagonize complement inactivation, the capacity of the lentiviral vector or the retroviral vector for targeted transduction of B cells in a subject and preparation of broad-spectrum neutralizing antibodies is further enhanced.