481 results about "Slow virus" patented technology

The invention belongs to the field of pharmaceutical and biological engineering, and relates to a CRISPR / Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and a lentivirus of the CRISPR / Cas9 recombinant lentiviral vector. The recombinant lentiviral vector is prepared by carrying out enzyme digestion on a lentiviral vectorlentiCRISPR by BsmBI and connecting into a BsmBI cohesive end-containing CXCR4 specific target sequence to recombine; the obtained CRISPR / Cas9 recombinant lentiviral vector is capable of mutating gene sequences at four different loci of ahuman immunodeficiency virusco-receptor CXCR4 and themutatuin rate is high and up to 25-75%. The cells transformed by the recombinant lentiviral vector cannot be infected by the human immunodeficiency virus. Compared with theRNAi-Knockdown, ZFN and TALEN technologies, the method has higher efficiency of suppressing the human immunodeficiency virus replication; the system is rapid to construct, simple and low in cost, is capable of preventing the invasion of the human immunodeficiency virus and is suitable for human immunodeficiency virus gene therapy.

Lentiviral vector particles comprising a Sindbis virus E2 glycoprotein variant and a lentiviral vector genome comprising a sequence of interest are provided. A lentiviral vector particle comprising: (a) an envelope comprising a Sindbis virus E2 glycoprotein variant; and (b) a lentiviral vector genome comprising a sequence of interest; wherein the E2 glycoprotein variant facilitates infection of dendritic cells by the lentiviral vector particle, and wherein the E2 glycoprotein variant has reduced binding to heparan sulfate compared to a reference sequence (HR strain).

In one aspect, the invention provides methods and compositions for the expression of small RNA molecules within a cell using a retroviral vector. The methods can be used to express double stranded RNA complexes. Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell, that interfere with a viral life cycle by down regulating either the viral genome, a viral genome transcript, or a host cell that. In another aspect the invention provides methods for treating patients having suffering from infection, particularly infection with HIV. In a further aspect, the invention provides methods for producing siRNA encoding lentivirus where the siRNA activity may interfere with the lentiviral life cycle.

The invention relates to a method for successfully inducing cell chemokine receptor CCR5 genes to be mutated into CCR5-delta32 deletion-type genes with a new genome editing technology CRISPR-Cas9. CCR5 is an important receptor for human immunodeficiency viruses (HIV) to invade personal host cells. CCR5-delta32 deletion means deletion of 32 basic groups occurs in a CCR5 coding region, so that the sequence after the 185th amino acid is changed, and early termination occurs. CCR5-delta32 biallelic-gene homozygous deletion has natural resistance to HIV infection, and can not be infected by HIV. By means of the method, a slow virus packaging system and the CRISPR technology are used at the same time; as the slow virus infecting host range is wide, the method can be applied to cells such as bone marrow stem cells and CD4T cells, and the CCR5-delta32 deletion-type genes hopefully become medicine for treating acquired immune deficiency syndrome or other diseases.

The invention discloses an application of virus-mediated Cpf1 protein in a CRISPR / Cpf1 gene editing system. The CRISPR / Cpf1 gene editing system provided by the invention consists of a recombinant virus or a recombinant vector. The recombinant virus is capable of expressing a Cpf1 gene; and the Cpf1 gene is capable of encoding the Cpf1 protein. The recombinant vector comprises a virus genome and the Cpf1 gene. The virus is either lentivirus or adenovirus. Experiments prove that virus-mediated FnCpf1 protein or AsCpf1 protein causes mutation of an hRb1 gene, an hP53 gene, an hDicer1 gene, an mRb1 gene, an mP53 gene, an mDicer1 gene, an rRb1 gene, an rP53 gene and an rDicer1 gene in the CRISPR / Cpf1 gene editing system. Therefore, the virus-mediated Cpf1 protein is applicable to gene editing in the CRISPR / Cpf1 gene editing system; and the Cpf1 protein has an important application value.

The invention belongs to the field of allogenic mesenchymal stem cells, and particularly relates to a preparation method of allogenic mesenchymal stem cells by CRISPR (clustered regularly interspaced short palindromic repeats) technique editing and IGF (insulin-like growth factor) optimization and application of the allogenic mesenchymal stem cells in treating myocardial infarction. The preparation method comprises the following steps: carrying out separation by density gradient centrifugation to obtain allogenic single karyocytes, and carrying out adherent culture to obtain mesenchymal stem cells; designing a mesenchymal stem cell surface antigen B2M-gRNA and an inflammatory factor TNF-alpha-gRNA; establishing recombinant slow virus particles, and transfecting the mesenchymal stem cells; optimizing the mesenchymal stem cells by using IGF-1; and preparing drugs for treating myocardial infarctions by using the modified and optimized mesenchymal stem cells. The CRISPR / Cas9 technique is utilized to remove the antigens capable of causing immunological rejection and the inflammatory factors capable of causing inflammatory reaction on the mesenchymal stem cell surface, and the IGF-1 is utilized to enhance the apoptosis resistance of the mesenchymal stem cells and promote the homing of the mesenchymal stem cells, thereby providing a new technical scheme for preparing drugs for treating cardiovascular diseases in clinic. The prepared allogenic mesenchymal stem cells can not cause immunological rejection after cell transplantation.

A modified polypeptide corresponding to an envelope glycoprotein of a primate lentivirus is described. The polypeptide has been modified from the wild-type structure so that it has at least two of the glycosylation sites proximal to the CD4 binding site or chemokine receptor site altered so that the alteration prevents glycosylation at that site or where glycosylation sites distal to these sites have been derivatized with a molecular adjuvant, while retaining the overall 3-dimensional structure of a discontinuous conserved epitope of the wild-type protein. Preferably, the polypeptide has both changes. Preferably, the primate lentivirus is HIV, and the protein is HIV-1 gp 120.

The invention discloses a targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats) / Cas9 slow virus system and application thereof. The sgRNA is selected from FTOsgRNAsp2 or FTOsgRNAsp3 with the following DNA sequence. The sgRNA has high cutting efficiency for the FTO gene. When the sgRNA-containing CRISPR / Cas9 slow virus system is used for transfecting SV-HUC-1, the FTO protein expression level of the obtained cell strain is obviously lowered. Therefore, the sgRNA disclosed by the invention can effectively implement targeted FTO gene knockout; and after the sgRNA is established into the CRISPR / Cas9 slow virus system, the system can knock out the FTO gene to obtain the FTO-gene-knockout cell strain, thereby being beneficial to researching the action mechanism of FTO in the cell strain.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeat) / Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof. A lentivirus of the CRISPR / Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 gene Delta 32 region is constructed that the lentivirus can introduce cells into a CRISPR / Cas9 system specific to CCR5, double-chain breakage occurs to a specific site of CCR5 gene, a random mutation is introduced to a breakage site after repairing by means of nonhomogeneous recombinant terminal binding, and the mutation rate reaches 90% and above. As gRNA is a nonhomogeneous region of CCR5 and CCR2, detection shows that the missing efficiency of the two gRNAs is lower than 0.2%. Cells modified via the recombinant lentivirus have significantly decreased efficiency of HIV (human immunodeficiency virus) infection. The system is quick to construct, simple and low in price, and is applicable to gene therapy of acquired immune deficiency syndrome.

The invention discloses gRNA target sequences for endogenous overexpression of 1ncRNA-XIST, a CRISPR / dCas9 lentivirus system and application thereof. The gRNA target sequences are respectively as shown in SED ID No. 1, SED ID No. 2 and SED ID No. 3. The CRISPR / dCas9 lentivirus system comprises the gRNA target sequences for endogenous overexpression of 1ncRNA-XIST. A method for screening stable strains according to the characteristics that lentivirus must be integrated into a host genome is cooperated with CRISPR / dCas9 to realize the endogenous overexpression of the large fragment gene 1ncRNA-XIST, so that the defect of incapability of stably expressing the large fragment gene 1ncRNA-XIST in a traditional method is overcome, the efficient overexpression stable cell strain of the large fragment gene 1ncRNA-XIST can be obtained in a short time, or cells obtained by screening can stably express target genes, thereby obtaining stably silenced 1ncRNA-XIST downstream specific gene cell strain. The gRNA target sequences and the CRISPR / dCas9 lentivirus system have important guiding significance on the research of the function of 1ncRNA-XIST in the trophocyte migration and the effect of 1ncRNA-XIST in the proliferation disorder and fetal growth restriction process.

The present invention provides lentiviral vectors that are useful in human gene therapy for inherited or acquired proliferative ocular disease. It furnishes methods to exploit the ability of lentiviral vectors to transduce both mitotically active and inactive cells so that eye diseases may be treated.

The invention provides a method for efficiently inducing multipotential stem cells, six inducible factors such as Oct4 (POU5f1), Sox2, c-Myc, Klf4, UFT1, Rex1 (ZFP42), and the like and p53 gene inhibitor are induced into the cells by utilizing a slow virus carrier, thereby the method can efficiently induce and produce the multipotential stem cells.

The invention discloses a low-expression CYP7A1 hepatic cell and a constructing method thereof. A method for inhibiting the CYP7A1 expression level of the hepatic cell comprises the step of introducing an encoding gene of small-interference RNA which is used for inhibiting the CYP7A1 gene expression into the hepatic cell so as to inhibit the CYP7A1 expression level of the hepatic cell. A slow virus vector system can be used for introducing the encoding gene of small-interference RNA which is used for inhibiting the CYP7A1 gene expression into the hepatic cell. An experiment proves that the method of the invention can obviously regulate the expression level of the CYP7A1 gene of the hepatic cell in an mRNA level and a protein level downwards and can further effectively reduce the secretoryvolume of total bile acid. The Low-expression CYP7A1 hepatic cell and the constructing method thereof can be used for the fundamental research and the clinical application of biologic artificial liver supporting treatment and have a wide application prospect.

Virus vectors, virus particles, and methods and uses of screening for, detecting, analyzing and determining amounts of virus antibody, or neutralizing antibody activity of samples are provided. Such virus vectors, virus particles, and methods and uses are applicable to a broad range of virus types, such as lentiviruses, adenovirus, and adeno-associated virus (AAV) serotypes. Methods and uses include virus antibody screening, such as anti-virus immunoglobulins screened for, detected, analyzed and amounts determined

The invention relates to the field of biomedical research, in particular to a gastric cancer cell line for expressing green fluorescent protein and luciferase, which is established by introducing the green fluorescent protein and the luciferase into a cell, and an application thereof in a cancer model. The gastric cancer cell line is characterized in that the gastric cancer cell line for stably expressing green fluorescent protein and luciferase is obtained by using a human gastric cancer cell line as mother cell and bringing the green fluorescent protein (GFP) and firefly luciferase (Fireflyluciferase, LUC) genes into the cell by using a slow virus infection method; and different gastric cancer models are established for the cell line by using hypodermic injection, tail vein injection and in situ injection methods. The invention provides establishment and application of the human gastric cancer cell line; and the gastric cancer cell line for expressing green fluorescent protein and luciferase is established by using the slow virus infection method, applied to the cancer model and used for gastric cancer preclinical studies.

A pseudo-virus packaging system of coronaviruses comprises a vesicular stomatitis virus (VSV) vector with a Fluc and EGFP double reporter gene replacing a GP gene and an assembly cell expressing coronavirus spike protein S. The double reporter gene is selected from luciferase and fluorescent protein, and the luciferase reporter gene is preferably a Fluc gene. The fluorescent protein reporter geneis preferably an EGFP gene, and the assembly cell is preferably 293T. The invention further relates to a one-step packaging method of the packaging system; a pseudo-virus with single-cycle infection,low background value and high titer can be quickly packaged through simple, convenient and rapid operation, and the packaging system has the characteristic of being rapid in detection compared with aslow virus mediated pseudo-virus system; the one-step packaging method can be used for researching COVID-19, SARS, MERS and other coronaviruses and other viruses, provides a powerful screening tool for evaluation of antiviral preparations and vaccine, and has wide application value.

The invention belongs to the fields of genetic engineering and cell engineering, pertaining to a human T-lymphocytic series model for screening and reactivating latent infection HIV-1 compound and a preparation method thereof. The invention discloses a T lymphocyte used for screening and activating latent infection HIV-1 drug, and the T lymphocyte is a Jurkat stable strain that is infected by but not expresses HIV slow virus carrying reporter gene. The preservation number of the T lymphocyte is CCTCC NO.C200821. The invention also provides a preparation method for the T lymphocyte, including infecting human T-lymphocytic series Jurkat with the HIV-1 slow virus carrying EGFO reporter gene, and carrying out cell sorting and HIV integration test so as to obtain the clone with HIV integration while not expressing EGFP. The invention is the cell model for the virus preservation library to control drug screening.

The invention discloses a double-reporter-gene skeleton vector. Double reporter genes of the double-reporter-gene skeleton vector are selected from luciferase and fluorescein, the fluorescein reportergene is preferably an mGFP gene, and the luciferase reporter gene is preferably a Fluc gene. A double-reporter-gene four-plasmid pseudovirus packaging system comprises the double-reporter-gene skeleton vector and three eukaryotic expression packaging plasmids which are respectively integrated with a slow virus gag-pol gene, a slow virus rev gene and an envelope protein gene containing a target virus. A packaged COVID-19 pseudovirus comprises the double-reporter-gene four-plasmid pseudovirus packaging system and a host cell, wherein the host cell is preferably selected from 293T-hACE2 cells. According to the invention, SARS-CoV-2 is specifically implemented, the pseudovirus with single-cycle infection and higher safety can be rapidly packaged, and the vector and the system can be used forresearching coronaviruses such as SARS-Cov2 and the like, provide a powerful screening tool for evaluation of antiviral preparations and vaccines, and have wide application value.

The invention discloses a triple minRNA for resisting virus infection of aids and the construction method thereof. The sequences of the triple minRNA are respectively designed according to gene sequences of ccr5, pol and vif and combined to be miRNA oligomeric and single strand DNA, then double-strand DNA is formed through annealing, then double-strand DNA is respectively inserted into the expression vector of pcDNA<TM> 6.2-GW / EmGFPmiR of miRNA to built the mi RNA expression plasmid, and the jamming effect of mi RNA expression plasmid to target gene can be detected through a Qpcr method. A miRNA string with the best gene interference effect to ccr5, vif and pol is inserted into a carrier of pcDNA 6.2 <TM>-GW / EmGFP, so as to obtain a series-wound interference vector of pcDNA6.2<TM>-GW / EmGFP-ccr5-pol-vif, and then the miR-ccr5-pol-vif in the series-wound interference vector is transferred to a lentiviral vector pLenti6.3 / V5-DEST so as to construct pLenti6.3 / V5-DEST miR-ccr5-pol-vif. The recombinant lentiviral vector can product three miRNA at the same time to cause interference effects to gene ccr5, gene pol and gene vif, so as to prevent the gene ccr5, the gene pol and the gene vif from expressing corresponding protein. Therefore, the effect of controlling Hiv infection and replication can be achieved.

The invention discloses an RNA with little interference for inhibiting the expression of an iron regulating element regulatory protein (HJV). The RNA with little interference is in reverse complementation with the iron regulating element regulatory protein gene mRNA, and the length of the RNA is 19-27bp. The invention further discloses a DNA for coding the corresponding shRNA of the RNA with little interference and a recombined slow virus capable of expressing the shRNA. The RNA with little interference, the DNA capable of coding the shRNA and the recombined slow virus can effectively inhibit the expression of the iron regulating element regulatory protein in vivo, improve the level of the iron regulating element and reduce the level of serum iron in vivo, thereby reaching the aim of curing diseases related to iron metabolism.

The invention discloses an induced slow virus expression system which comprises a target gene expression cassette and an rtTA expression cassette. The target gene expression cassette comprises an induced promoter containing a tetracycline cis-acting response element and a target gene, and the rtTA expression cassette comprises a promoter, an rtTA, a linker peptide and a screening gene, wherein a P2A linker peptide is adopted as the linker peptide, the screening gene is Puro, and the induced promoter is TetO6 or TRE3G. The invention further discloses a construction method of the induced slow virus expression system. The construction method comprises the steps of pLVX-Puro carrier reconstructing; TetON3G gene synthesizing and cloning; pLVX-rtTA3 constructing; EF1a promoter subcloning; red fluorescence protein (RFP) subcloning; subcloning of the induced promoter TRE3G or TETO6 containing the tetracycline cis-acting response element. The induced slow virus expression system can carry out inducible expression of the target gene to the greatest level while keeping low background expression after cells are introduced into the system, is sensitive in response for an inducer, high in efficiency and low in molecular weight and can be widely applied to gene function research.

The invention belongs to the field of micro-organism animal cell line and relates to a human hepatoma cell line which can emit high-intensity red or green fluorescence and has high transferring ability of lung and lymph node metastasis, and a method for establishing the same. The method comprises the following steps: using the human hepatoma cell line HCCLM3 and HCCLM6 which have high transferring ability of the lung and lymph node metastasis as mother cells, performing cotransfection on plasmid DNA of 239 cells through slow virus packaging plasmids to obtain false slow virus particles by expressing red or green fluorescent protein genes through eucaryon, and infecting liver cancer cell strains of the mother cells to obtain the chromosome integrated hepatoma cell line which has high transferring ability of the lung and lymph node metastasis and can stably expressing the red or the green fluorescence. The human hepatoma cell line which has high transferring ability of the lung and lymph node metastasis in vitro can be applied to the tracer studies on tumor cells, the molecular mechanism studies on the recurrence and transferring of liver cancer, as well as the pre-clinical drug efficacy studies on new anti-tumor drugs, thus the human hepatoma cell line has wide application prospect.

The invention relates to a method of inducing a human fibroblast to reprogram the human fibroblast into an osteoblast by utilization of Runx2 and a low-molecular-weight compound, particularly relates to a method of inducing a human fibroblast to transdifferentiate the human fibroblast into an osteoblast under the existence of dexamethasone by utilization of Runx2 gene expression, a low-molecular-weight compound CHIR99021 and / or a low-molecular-weight compound forskolin, and belongs to the fields of cytobiology and tissue engineering. The method includes: constructing a human skin fibroblast infected by a slow virus carrying Runx2-GFP, performing transdifferentiation of the human fibroblast under the existence of the dexamethasone by utilization of the low-molecular-weight compound CHIR99021 having a concentration of 5-10 muM and the low-molecular-weight compound forskolin having a concentration of 10 muM, changing into an osteoblast inducing liquid after 7-15 days, and inducing, and the fibroblast can be induced into the osteoblast after 5-20 days. The method can obtain osteoblasts simply and rapidly and can be used for cell therapy for bone metabolic disease, fracture, and the like. A pharmaceutical composition provided by the invention can be used for research, development and production of tissue-engineered bone, brings convenience to fundamental research and brings a prospect for clinical application.

The invention discloses an immortalized human liver cell line, a preparation method and an application thereof. The immortalized human liver cell line has the main function of primarily cultured humanliver cell. The preparation method comprises the following steps of: transferring SV40T antigen and human telomerase catalyzed subunit gene into primarily cultured separated human normal liver cell by slow virus carrier; and sieving in a cloning way to obtain the immortalized human liver cell line. The immortalized human liver cell line also can be extrinsically amplified in large scales in a microcarrier way. The immortalized human liver cell line can be used for preparing a biological artificial liver support system and preparing the medicament for curing the liver failure.

The invention belongs to the biomedicine field, and aims at providing an expression vector for stably indicating cell autophagy activities, an establishing method and an application method thereof. By constructing a slow-virus expression plasmid containing an EGFP-LC3 autophagy reporter gene and adopting a four-plasmid system, the invention establishes a slow-virus expression vector capable of enabling cells to stably express an EGFP-LC3 gene. By using the expression vector to infect a host cell, the EGFP-LC3 gene can be stably integrated in a genome of the host cell, and the host cell can stably and efficiently express the EGFP-LC3 and indicate cell autophagy changes. A stable autophagy indicating cell can be established by using the expression vector, and the cell autophagy changes are quickly and quantitatively analyzed by an image analysis method based on Top-hat operators and morphological filtration. The invention has the characteristics of stable and reliable autophagy indication, real-time sensitivity, quick and accurate autophagy quantification, simply and convenient operation and the like, greatly facilitates autophagy observation and analysis, and can be widely used for autophagy-correlated researches.

The invention discloses a tetracycline slow virus inducible expression vector as well as a building method and application thereof. The tetracycline slow virus inducible expression vector comprises a target gene expression frame and an antisense tetracycline transcription activating factor expression frame; an inducible CRISPR / Cas9 system places a Cas9 protein downstream of an inducible promoter, and places an sgRNA sequence of U6 prompting expression upstream of the whole tetracycline inducing system. The combination effect of TRE3Gp and TetON3G is best; after cells are introduced into the induction system, the expression of a target gene can be induced at the highest level while keeping low background expression. The induction system is sensitive to an induction agent, has high efficiency and a small molecular weight, and can be widely applied to gene function researches.

The invention discloses a CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an amicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treatingacute myelogenous leukemia (AML) clinically is outstanding.

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) primer, a kit and a method for detecting the virus titer of slow viruses. The primer falls on an envelope of the slow viruses, and the titer can be detected without a fluorescent label. In addition, the successfully integrated viruses infecting cells are detected, the real infection vitality of the virusescan be embodied, and the primer cannot be affected by cell genome DNA (deoxyribonucleic acid) extraction efficiency and has the advantages of simplicity and convenience in operation and high sensitivity.

The invention discloses a CD 30-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 30-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an ampicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of the eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; and an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treating hodgkin lymphoma or non-hodgkin lymphoma clinically is outstanding.