165 results about "Slow virus" patented technology

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

The invention discloses a construction method and application of a gene modified mesenchymal stem cell. The construction method comprises the following steps: constructing a recombinant lentivirus carrying PD-L1 and ICAM1 genes; the recombinant lentivirus carrying the PD-L1 gene and the ICAM1 gene is transfected with mesenchymal stem cells, and the mesenchymal stem cells are screened by puromycin, so that an engineered mesenchymal stem cell strain modified by the PD-L1 gene and the ICAM1 gene is obtained; the mesenchymal stem cells provided by the invention are modified by engineering genes of PD-L1 and ICAM1, and are screened by puromycin to obtain a mesenchymal stem cell strain capable of stably and highly expressing the genes of PD-L1 and ICAM1; after the mesenchymal stem cells highly expressing the PD-L1 and ICAM1 genes are intravenously injected into an autoimmune hepatitis animal model, the autoimmune hepatitis symptom can be remarkably relieved, the liver injury degree can be reduced, the expression of inflammatory factors can be regulated, the inflammatory reaction can be inhibited, and the obvious curative effect can be achieved.

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.

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.

Provided herein are lipid particles, such as lenti viral particles, that incorporate or are pseudotyped with a variant Nipah Virus G (NiV-G) envelope glycoprotein, and in some aspects also a fusion (F) protein such as a NiV-F protein or a biologically active portion or variant thereof. Also provided are polynucleotides encoding the variant NiV-G and producer cells for preparation of the lipid particles, such as lentiviral particles, containing the variant NiV-G proteins, as well as methods for preparing and using the lipid particles, such as lentiviral particles.

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 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 discloses an anti-hepatoma drug screening cell model as well as a construction method and application thereof. The construction method of the model comprises the following steps: 1) fusing an EGFP (Enhanced Green Fluorescent Protein) gene to a C end of a Coilin gene, and constructing the fused gene to a CMV-MCS-EGFP-PGK-Puro lentiviral vector to obtain a virus vector for expressing Coilin-EGFP; 2) co-dyeing a virus vector and a virus packaging plasmid into a recipient cell for virus packaging, and centrifuging a collected cell culture solution containing the virus to obtain a virus supernatant for expressing the Coilinin-EGFP (Enhanced Green Fluorescent Protein); and (3) infecting the liver cancer cell line HCCLM3 by using the virus supernatant containing the expressed Coilinin-EGFP, and screening the cell strain capable of stably expressing the Coilinin-EGFP. According to the application, the obtained cell model is used for screening anti-hepatoma drugs. According to the method, the proper incision enzyme is selected, the enzyme digestion efficiency is high, the titer of the obtained virus vector expressing Coilinin-EGFP is high, the virus vector is applied to screening of anti-liver cancer drugs, positioning of Coilinin-EGFP agglomerates can be directly observed under a fluorescence microscope, the method is simple, visual and convenient to operate, and screening of compound small molecule drugs inhibiting cell nucleus positioning of Coilinin-EGFP is facilitated.

The invention provides a recombinant lentiviral vector and a preparation thereof. The recombinant lentiviral vector carries a coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or a coding sequence of shRNA targeting HIV genes. The recombinant lentiviral vector preparation comprises an effective dose of the recombinant lentiviral vector, a histidine hydrochloride buffer solution, saccharides and a surfactant. The recombinant lentivirus preparation disclosed by the invention has good stability under different environmental conditions.

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 belongs to the technical field of biology, and particularly relates to a 3D protein mutated O-type foot-and-mouth disease recombinant virus strain as well as construction and application thereof. The 403th methionine of the O-type foot and mouth disease virus 3D protein is mutated into alanine, the recombinant virus strain rFMDV-M403A with the mutated O-type foot and mouth disease virus 3D protein is successfully constructed and saved by using a reverse genetic technology, and amino acid of the recombinant virus strain can be stably mutated in a passage process; the growth speed of the mutant strain is slower than that of a wild strain, and the virus titer is remarkably reduced; in an individual experiment, neonatal mouse infection shows that the mutant strain can improve the survival rate of neonatal mice, and the virus load and tissue lesion in the liver are remarkably reduced. The attenuated foot-and-mouth disease recombinant virus strain provided by the invention provides important significance for deep understanding of a foot-and-mouth disease virus pathogenic mechanism and a prevention and control strategy, and also provides an important theoretical basis for further development of a foot-and-mouth disease genetic engineering vaccine.

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 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.

Disclosed in the present invention are an envelope glycoprotein for packaging a pseudotyped lentiviral vector for transduction of NK cells and a use of the envelope glycoprotein. The envelope glycoprotein comprises an extracellular region, a transmembrane region and a cytoplasmic region, wherein the amino acid sequence or the mutation sequence of the extracellular region is as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the amino acid sequence or the mutation sequence of the transmembrane region is as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and the amino acid sequence or the mutation sequence of the cytoplasmic region is shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. A BaEV envelope glycoprotein mutant replaces VSV-G to package a lentiviral pseudovirus, so that a high virus titer of 7.33E+07 TU / mL is maintained, NK cells can be effectively recognized, and entry of viruses into the cells can be mediated, thereby achieving the objective of efficiently transfecting the NK 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 discloses a lentiviral vector and lentiviral vector particles for treating hepatitis B infection. The lentiviral vector and particles of the invention contain a nucleotide sequence coding an hepatitis B virus antigen. The large S antigen of hepatitis B virus was in particular selected as candidate and can be applied in pharmaceutical compositions or vaccines for treating and / or preventing hepatitis B virus infection or treating and / or preventing diseases caused by hepatitis B virus infection, which has excellent therapeutic and preventive effects in subjects in need thereof.

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 invention relates to the technical field of virus packaging. The invention provides a packaging method of a CAR (chimeric antigen receptor) virus. The packaging method comprises the following steps: mixing a plasmid pCMX-dR8.91, a plasmid pCMV-BaEV, a plasmid pAdVAnti and an opti-MEM culture medium, standing, mixing with a PEI transfection reagent, standing, mixing with a 293T cell, culturing, replacing the culture medium, continuously culturing, and harvesting the virus. A second-generation lentivirus packaging system is adopted, transfer plasmids are second-generation transfer plasmids needing to be activated by a Tat element, packaging plasmids are second-generation packaging plasmids pCMV-dR8.91 containing ag, pol, rev and tat, enveloping plasmids are second-generation enveloping plasmids pCMV-BaEV, plasmids pAdVAnti beneficial to improvement of transfection efficiency are added, and the packaging efficiency of the CAR virus is improved through the system.