246 results about "Slow Virus Diseases" patented technology

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 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.

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 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.

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 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 non-integration slow virus vector system and the preparation and the application thereof. The non-integration slow virus vector system comprises a pLVTH vector, a pHelper1.0 vector, a pCMV-VSV-G vector and host cells. The pLVTH vector, the pHelper1.0 vector and the pCMV-VSV-G vector in the vector system are commonly transfected with the host cells to obtain the non-integration slow virus vector in the host cells by packing. The non-integration slow virus vector system of the invention can provide highly efficient gene transfer, longtime gene expression and wide host cells, cannot randomly integrate carried genes into host gene groups and provides a new method for the gene therapy.

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 preparation method of bone mesenchymal stem cells carrying NK4 genes and application thereof in medicine for treating gastric cancer. The preparation method comprises the following steps: extracting the NK4 genes from an HGF plasmid, using an in-Fusion technology to directionally clone the NK4 genes to a slow virus expression carrier plasmid, constructing an NK4-EGFP fusion gene recombinant slow virus carrier plasmid (pGC-FU-NK4) and then co-transfecting a 293T cell with the pGC-FU-NK4, a pHelper1.0 carrier and a pHelper2.0 carrier, packaging to produce NK4 overexpression slow virus particles (Lenti-NK4), using slow virus (Lenti-NK4) carrying NK4-EGFP fusion gene to transfect the bone mesenchymal stem cells to establish in vitro bone mesenchymal stem cells to stably express the NK4 genes. Balb / C nude mice animal experiments prove that the NK4 gene therapy taking BMSCs as a carrier can obviously inhibit the growth of subcutaneous transplant tumor of gastric cancer with high tumor inhibition efficiency, and the NK4 gene therapy is a good gene therapeutic method for gastric cancer.

The invention discloses a preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, the prepared CAR-T cells and application thereof. The preparation method comprises the following steps: S1, separating, activating and amplifying T lymphocytes: after activating separated CD3+CD8+T lymphocytes, culturing and amplifying; S2, infecting the T lymphocytes treated in step S1 bya recombinant slow virus carrying a CAR, so as to prepare the CAR-T cells, wherein a CAR gene carried in the recombinant slow virus carrying the CAR comprises a CD28 intracellular domain and a 4-1BBintracellular domain; nucleotide sequences of the CD28 intracellular domain and the 4-1BB intracellular domain are shown as SEQ ID7 and SEQ ID9 respectively. The CAR-T cells prepared by the method canbe widely applied to treatment of various cancers. Compared with the prior art, the proliferation rate of the prepared CAR-T cells is remarkably improved when compared with CAR-T cells prepared by aconventional method.

The invention belongs to the field of biotechnology, particularly relates to a titer detection method for a slow virus carrier, and discloses a quality index detecting method conducting real-time fluorescence quantification nucleic acid amplification detection (qPCR and RT-qPCR) by using a sequence specific primer and a fluorescent dye, a used primer and a standard product. The detecting method isefficient and accurate, the operation is easy, the use amount of a sample is low, the repeatability is good, and the limit problem of inaccurate carrier quantitation in the fields of eucaryon geneticengineering and gene therapy is solved. The method has the advantages of detection time and simple and convenient operation step, the analysis process after the amplification is not needed, comparedwith the Taqman probe method, the SYBR Green I does not need the design of a synthesis fluorescent probe, the design is simplified, the cost is lowered, and a dissolution curve is combined in the analysis to judge the specificity of the reaction.

The invention discloses ribonucleic acid interference (RNAi) for inhibiting porcine reproductiion and respiratory syndrome virus (PRRSV) replication and a preparation method of RNAi, The RNAi comprises a small interfering RNA (siRNA) sequence. The preparation method comprises the steps of constructing a short hairpin RNA (shRNA) slow virus expression vector, preparing replication-defective slow virus, infecting slow virus Marc-145 cells (green monkey kidney cells) and the like. The invention also discloses a method for verifying the effect of inhibiting PRRSV from replication. The RNAi sequence has the obvious effect of inhibiting the PRRSV replication on sensitive cells. According to the invention, the exploration of RNA interference on in vitro and vivo replication of hog cholera virus is carried out, a slow-virtue-mediated stably-integrated RNA interfering technology for special conserved gene segments of a targeted hog cholera virus genome is constructed, and transgenic animals with the siRNA of targeted hog PRRSV are hopeful to construct. The necessary experimental data is accumulated for gene function research of RNAi applied to PRRSV and prevention and treatment of hog cholera, and early-stage preparation is provided for disease resistance breeding of animals.

The invention discloses a preparation method of a pseudotype virus for nucleic acid detection of 2019-nCoV, and relates to the technical field of biology. The preparation method comprises a method forpreparing standard quality control products which contain but are not limited to a 2019-nCoV nucleotide sequence and are used for nucleic acid detection through a non-integrated slow virus vector system, a method which is used for preparing the standard quality control products for nucleic acid detection, and a method for constructing a non-integrated slow virus through mutating 64-locus amino acid Asp of integrase of slow virus auxiliary plasmid into Asn, and amino acids Arg, Arg and Lys at 262-264 loci into Ala, Ala and His. In the manner of removing elements of promoters and the like of slow viruses, the load quantity of virus vectors can be promoted, a pseudotype virus which can be used for nucleic acid detection and complete flow quality control is constructed, and the preparation method can be used for constructing a pseudotype virus for corona viruses and can also be used for constructing a pseudotype virus for SARS-COV, MERS-COV, influenza viruses and the like.

The invention discloses novel application of a human YTHDF1 (Youth-Tech-Health Domain Family Member 1) gene, that is, a human YTHDF1 gene expression amount detection reagent is applied to preparationof lung cancer clinical diagnosis reagents, a human YTHDF1 gene is applied to preparation of lung cancer medicines, compared with normal pulmonary epithelial cells, the YTHDF1 gene is highly expressed in lung cancer cells, RNA (Ribonucleic Acid) designed according to the YTHDF1 gene is capable of interfering slow viruses, inhibiting expression of YTHDF1, remarkably inhibiting proliferation of thelung cancer cells, retarding tumor cell division at a G0 / G1 phase and promoting cell apoptosis, inhibiting formation of nude mouse transplanted tumour, and furthermore the purpose of treating lung cancer can be achieved; YTHDF1 gene knockout mouse tumor formation capability detection shows that cell proliferation is remarkably inhibited, and cell apoptosis is remarkably increased; the results show that the YTHDF1 gene can be used as a target for lung cancer treatment, and wide prospects are provided for development of lung cancer treatment medicines based on YTHDF1 genes in future.

The invention relates to bioengineering technologies, and particularly relates a method of inducing differentiation of neural stem cells into dopaminergic neurons by using recombinant slow virus. The method comprises the following steps: constructing a recombinant slow virus vector; separating, culturing and identifying the neural stem cells; transfecting the recombinant slow virus into NSCs; identifying the NSCs transfected by the recombinant slow virus, wherein the process of transfecting the recombinant slow virus into the NSCs comprises the following steps: digesting NSCs after being subjected to three times of passages to prepare a single-cell suspension, and inoculating into a culture plate; transfecting the TH+Brn4 gene recombinant slow virus; adding the slow virus and ploybrene, uniformly shaking, putting the culture plate into an incubator to culture for 1-2 hours, and replacing fresh differential culture medium; meanwhile, screening by using puromycin to obtain stable transfected cells. The method has the advantages as follows: by constructing the slow virus vectors of target genes TH and Brn4, exogenous genes after transfecting the NSCs can be stably expressed in cells; high proportion of TH positive dopaminergic neuron is generated after differentiation; the Brn4 can promote high expression of neurotrophic factors and has the function of inhibiting apoptosis.

The invention discloses a cell strain MSCs for overexpression of an Nrf2 gene as well as a preparation method and application of the cell strain MSCs. The preparation method comprises the following steps: performing PCR amplification on a human-derived Nrf2 gene ORF; performing purification and double enzyme digestion on the PCR product obtained in the step (1), then connecting the PCR product with the framework plasmid pLV-CMV-XbaI-BamHI-GFP to construct an Nrf2 slow virus recombinant vector, and after conversion, selecting clone and determining positive clone; sequencing the positive clone, and after no mutation is determined, performing amplification massively; leading the recombinant vector and an auxiliary plasmid in a 293 FT cell to obtain a virus; using the virus to infect the 3-5 th generation hUC-MSCs cells so as to obtain the cell strain MSCs for overexpression of the Nrf2 gene. According to the invention, the overexpression of Nrf2 in MSCs is stably performed to increase the cell activity, and a higher anti-apoptosis feature is also realized under the anoxic and oxidative stress conditions; Nrf2-MSCs with the treatment level amount is obtained in vitro and can serve as an excellent tool cell for preclinical study of MSCs and can be used for preparing a medicine for enhancing preclinical study of MSCs transplanting.

The invention discloses a modified enhanced type DC-CIK cell targeting immune cell mass, and a preparation method and application thereof. A method for preparing a DC cell comprises the following steps: performing isolated culture on monocyte, thereby acquiring an immature DC cell; and performing tumor antigen load and induced differentiation culture on the immature DC cell, thereby acquiring a mature DC cell, wherein the monocyte is acquired by separating from a peripheral blood mononuclear cell of a sample. The method for preparing CIK comprises the following steps: performing in vitro differentiation culture on a CIK cell, thereby acquiring an immature CIK cell; preparing a modifying recombined slow virus for performing in vitro modification on the CIK cell; and co-culturing the modified CIK cell and the DC cell, thereby acquiring the mature modified CIK cell (Triumph-DC-CIK), so as to be used for achieving a better tumor immunotherapy effect.

The invention discloses an inducing method for danio-rerio inductivity multi-potential stem cells. The induction method includes the following steps that danio-rerio tissues or danio-rerio embryos are gotten, and danio-rerio fibroblasts are obtained through in-vitro isolated culturing; the danio-rerio fibroblasts are infected through viruses, the infected danio-rerio fibroblasts are induced, and the danio-rerio inductivity multi-potential stem cells are obtained through manual selecting cloning and subculturing. Based on the same technical scheme, the invention further discloses an inductive culture medium and an iPS culture medium used for the inducing method. RT-PCR analysis indicates that iPS cells induced through the embryo fibroblasts have the potency of being differentiated into various triploblastic-source cells. By means of the method, the inductive culture medium and the iPS culture medium, the technical difficulty that the infection efficiency of slow viruses in fish cells is low is overcome, and new tool cell sources and new concepts are provided for researching a danio-rerio somatic-cell reprogramming mechanism to select cell materials.

The invention discloses a method for establishing a tree-shrew breast cancer model for slow virus by nipple injection. Slow virus is introduced to a tree-shrew breast duct by adopting a nipple injection technology, so that a cancer gene is over-expressed or an anti-cancer gene is knocked down after epithelial cells in a mediated duct are infected, and therefore, tree-shrew breast tumors are induced. According to the method for establishing a tree-shrew breast cancer model for slow virus by nipple injection disclosed by the invention, the breast tumors can be touched within two weeks after expressing the cancer gene PyMT; and moreover, the induction success rate is almost 100%, and the method is short in incubation period, strong in specificity, extremely high in induction rate, stable and convenient and simple to operate. The method disclosed by the invention can be used for quickly and efficiently establishing the tree-shrew breast cancer model.

The invention discloses a natural killer cell modified by specific chimeric antigen receptor gene and a preparation method and use thereof. The natural killer cell modified by specific chimeric antigen receptor gene comprises a NY-ESCO-1 specific chimeric antigen receptor gene and a truncated spleen tyrosine kinase Syk gene. The preparation method simultaneously infects the natural killer cell with a slow virus containing the NY-ESCO-1 specific chimeric antigen receptor gene and a slow virus containing the truncated spleen tyrosine kinase Syk gene. By the combined action of the NY-ESCO-1 specific chimeric antigen receptor gene and the truncated spleen tyrosine kinase Syk gene, the natural killer cell modified by the specific chimeric antigen receptor gene improves the targeted tumor cell specific lethality of the natural killer cell, improves the ability of activating downstream signals of the natural killer cell, avoids the escape of tumor immunity, and accordingly enhances the killing activity on the tumor cell.

Production of secretory insulin dry cell system by encoded various exogenous gene slow virus carrier is carried out by copying pWPST, cloning various insulin regulating genes to the carrier, transfecting 293T cell, packing, collecting virus grains, concentrating and transfecting human embryo pancreas dry cell. It's simple, efficient and practical, it can obtain one, two or three kinds of exogenous gene cells, which are in different expression and meet different needs.