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283 results about "Co transfection" patented technology
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Transient co-transfection of plasmids is a method that is commonly employed for cellular protein-protein interaction studies, transcription factor studies, and gene knockdown studies using shRNA encoding plasmids. Thus, co-transfection is useful for a broad scientific community.
Method for increasing non-homologous end joining efficiency of CRIPSR/Cas9 target knock-out genes
InactiveCN106906242AImprove accuracyVectorsOther foreign material introduction processesCancer researchBiomedical technology
The invention belongs to the technical field of molecular biology and biomedicine, and relates to a method for increasing the efficiency of CRIPSR / Cas9 target knock-out genes NHEJ. According to the method for increasing the efficiency of CRIPSR / Cas9 target knock-out gene NHEJ, small molecule interference RNA that efficiently interferes with expression of PTEN genes is screened out; by using CRISPR / Cas9 system to target knockout genes, the small molecule RNA is cotransfected into the target gene, thus the NHEJ efficiency of target genes is effectively improved and the NHEJ of target gene in cell multiple targets is significantly improved. The method has the advantages of low cost, simple operation and high efficiency, and has a good promoting effect on the efficiency and application of CRISPR / Cas9 technology.
Owner:重庆高圣生物医药有限责任公司
CRISPR/Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT
ActiveCN108690844AGood gene editing activityEasy to buildGenetically modified cellsNervous system cellsNerve cellsNeuron
The invention discloses a CRISPR / Cas9-gRNA targeting sequence pair, a plasmid and an HD model of HTT, and relates to the field of biotechnology. The targeting sequence pair of the invention comprisesan L sequence and an R sequence. The base sequence of the L sequence is shown as SEQ ID NO.1. The base sequence of the R sequence is shown as SEQ ID NO.2. The targeting plasmid of the invention comprises a first vector plasmid and the above-mentioned targeting sequence pair. The targeting sequence pair is constructed into the first vector plasmid. The HD cell model of the invention is obtained byco-transfecting cells with PolyQ Donor plasmids and the above-mentioned targeting plasmids. By using differentiated neuron cells as a carrier to construct the HD cell model, the invention provides a research platform for exploring the influences or changes of mHtt protein on the internal environment of differentiated nerve cells, and studying the changes of various signal pathways, metabolic pathways and intracellular homeostasis caused by mHtt protein in differentiated neuronal cells.
Owner:SOUTHWEST UNIV
Method for obtaining sheep with different hair colors on basis of CRISPR/Cas9 and sgRNA of targeted ASIP gene
ActiveCN105950626AStrong specificityPrecise targeted modificationHydrolasesMicroinjection basedAnimal scienceGenome editing
The invention discloses a method for obtaining sheep with different hair colors on the basis of CRISPR / Cas9 and sgRNA of a targeted ASIP gene. The invention provides sgRNA (ASIP-sgRNA) capable of achieving specific and targeted modification of a sheep ASIP gene, and the sgRNA (ASIP-sgRNA) is RNA as shown from the third nucleotide to the 22<nd> nucleotide of 5' tail end of a sequence 4 of a sequence list or RNA with the nucleotides from the third nucleotide to the 22<nd> nucleotide of the 5' tail end of the sequence 4 of the sequence list. The ASIP-sgRNA particularly can be the RNA as shown in the sequence 4 of the sequence list. The invention further provides a method for obtaining sheep with changed hair colors. The method comprises the following steps that co-transfection of sgRNA capable of achieving specific and targeted modification of the sheep ASIP gene and Cas9mRNA on sheep cells is conducted, and therefore the sheep ASIP gene is deleted, and the sheep with the changed hair colors are obtained. According to the method for obtaining the sheep with different hair colors on the basis of CRISPR / Cas9 and sgRNA of the targeted ASIP gene, a CRISPR / Cas9 genome-editing technology is combined with a microinjection technology, and an effective technological means is provided for artificially changing the hair colors of the sheep.
Owner:新疆畜牧科学院生物技术研究所
IGM production by transformed cells and methods of quantifying said IgM production
ActiveUS20070154469A1High IgM activityImprove safety and efficacyAnimal cellsSugar derivativesPentamerPolymeric IgM
IgM can be obtained in the form of a pentamer by placing the genes encoding the H, L, and J chains on the same vector to transform appropriate host cells. The gene encoding the J chain may be introduced by co-transfection. When no J chain is expressed, the IgM is produced as a hexamer. The transformants obtained according to the present invention achieve a high yield of IgM. The present invention also provides methods which enable separation and quantification of polymeric IgM.
Owner:CHUGAI PHARMA CO LTD
Method for detecting promoter activity by utilizing double luciferase reporter genes
InactiveCN103382505AConvenient researchAvoid influenceMicrobiological testing/measurementLipofectaminePromoter activity
The invention discloses a method for detecting promoter activity by utilizing double luciferase reporter genes. The method for detecting the promoter activity by utilizing the double luciferase reporter genes comprises step 1, building a pGL3-basic-MSTNpro recombinant which contains 7 sections of MSTN gene 5' control region fragments with different lengths; step 2, performing cultivation and planking on target cells, configuring a mixture of the pGL3-basic-MSTNpro of the step 1 and a lipidosome and enabling a renilla luciferase carrier pRL-TK to be served as an internal reference to perform cell co-transfection; step 3, performing detection on luciferase activity through the double luciferase reporter genes.
Owner:GUIZHOU UNIV
Recombinant adeno-associated virus expressing human antisense gene CyP2J2 and its preparation methods
ActiveUS8273344B2Inhibit proliferation and migrationPromote apoptosisBiocideGenetic material ingredientsNucleotideGenetics
Owner:XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
IgM Production by Transformed Cell and Method of Quantifying the Same
ActiveUS20100172899A1High IgM activityImprove safety and efficacyImmunoglobulins against cell receptors/antigens/surface-determinantsAntibody ingredientsPentamerPolymeric IgM
IgM can be obtained in the form of a pentamer by placing the genes encoding the H, L, and J chains on the same vector to transform appropriate host cells. The gene encoding the J chain may be introduced by co-transfection. When no J chain is expressed, the IgM is produced as a hexamer. The transformants obtained according to the present invention achieve a high yield of IgM. The present invention also provides methods which enable separation and quantification of polymeric IgM.
Owner:CHUGAI PHARMA CO LTD
Preparation method and application of CAR-T cell targeting B7H3
ActiveCN109880804AHas a lethal effectEnhance killing activityMammal material medical ingredientsAntineoplastic agentsBiologyLung cancer
The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.
Owner:XUZHOU MEDICAL UNIV
Method for constructing fixed-point integrated exogenous DNA transgenic pigs
ActiveCN108285906AEasy to use in productionEasy to breedVector-based foreign material introductionAnimal husbandryExogenous DNAHigh activity
The invention discloses a method for constructing fixed-point integrated exogenous DNA transgenic pigs. The method comprises the following steps: S1, performing safety target screening and target binding gRNA cleavage efficiency verification; S2, constructing a homologous arm donor plasmid and obtaining a fixed-point integrated transgenic cell line; S3, constructing the fixed-point integrated exogenous DNA transgenic pigs. The method disclosed by the invention has the benefits that a gRNA target sequence is introduced in the donor plasmid, the donor plasmid is linearized while a Cas9 nucleasecleavage target gene is induced by utilizing intracellular transcription gRNA, the test steps are significantly simplified, the labor is saved, and the co-transfection efficiency is favorably improved; carriers used for the fixed-point integration are less, a homologous arm is moderate in size, and the transgenic cell line is more favorably obtained; an efficient site-directed integration technology, a high-activity site-specific transgenic cell culture technology and a somatic cell cloning technology are combined, so that the efficient preparation of fixed-point integrated transgenic animalsis facilitated, and the breeding speed of new transgenic animal varieties is accelerated.
Owner:WENS FOOD GRP CO LTD +1
Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins
ActiveCN104962581AImprove protectionEffective protectionViral antigen ingredientsMicroorganism based processesDiseaseNewcastle disease virus NDV
The invention provides a preparation method of a recombined new castle disease virus vaccine strain which expresses African swine fever virus p72 proteins and a recombined new castle disease virus vaccine strain. The method provided by the invention comprises the following steps: constructing a recombinant transcriptional plasmid which is inserted with a p72 gene of African swine fever virus (ASFV); constructing a transcriptional helper plasmid system; carrying out a contransfection for the transcriptional plasmids and the transcriptional helper plasmids into host cells BHK-21 which can be duplicated in new castle disease attenuate strains; and saving and obtaining the recombined virus stain. The vaccine strain for expressing the African swine fever virus p72 proteins provided by the invention has important preservation and strategy meaning in the aspect of animal epidemic disease prevention and control, and can be applied to the treatment and prevention of African swine fever virus.
Owner:LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
Human liver cancer high-transfer cell strain with stable expression of fluorescent protein and construction method thereof
InactiveCN101381706AAvoid pollutionImprove efficiencyVector-based foreign material introductionForeign genetic material cellsCarcinoma cell lineCancer cell
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.
Owner:ZHONGSHAN HOSPITAL FUDAN UNIV
Allele double knockout targeting vector system and construction method thereof
InactiveCN102094036AHigh target shooting efficiencyShorten experiment timeVector-based foreign material introductionFluorescenceTransgene
The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo / GFP and Hyg / RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.
Owner:NORTHWEST A & F UNIV
Recombinant Adeno-Associated Virus Expressing human Antisense Gene Cyp2j2 and Its Preparation Methods
ActiveUS20080131403A1Inhibit migrationPromote apoptosisBiocideGenetic material ingredientsHuman tumorLymphatic Spread
A recombinant adeno-associated virus expressing antisense human CYP2J2 gene and preparation method thereof are provided, the recombinant adeno-associated virus is prepared through cloning human CYP2J2 cDNA (1509 bp, which encode the protein containing 503 amino acids) from human leucocyte DNA by PCR, cotransfecting three plasmids by the calcium phosphate precipitation technique to pack and prepare the recombinant adeno-associated virus containing the antisense target gene, and purifying. The recombinant adeno-associated virus obtained from the above method can be transfected into different kinds of human tumor cell lines, inhibiting the proliferation and migration of tumor cells, promoting the apoptosis of tumor cells and suppressing the growth and metastases of tumor. Therefore, it can prove that the selective inhibitor of CYP2J2 and the recombinant adeno-associated virus expressing antisense human CYP2J2 gene are the potential medicine for treating tumor.
Owner:XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
Polynucleotides encoding anti-ganglioside antibodies
ActiveUS7709615B2High IgM activityImprove safety and efficacySugar derivativesImmunoglobulins against cell receptors/antigens/surface-determinantsPentamerNucleotide
Owner:CHUGAI PHARMA CO LTD
Preparation method of bone mesenchymal stem cell carrying NK4 gene and application thereof
InactiveCN101912618AGrowth inhibitionImprove tumor inhibition rateGenetic material ingredientsDigestive systemBALB/cGastric carcinoma
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.
Owner:祝荫
Construction method of SOD (superoxide dismutase) gene modified mesenchymal stem cell and application thereof
InactiveCN102660580AHigh activityConducive to survivalPeptide/protein ingredientsGenetic material ingredientsCytopathic effectSuperoxide dismutases
The invention provides a construction method of a SOD (superoxide dismutase) gene modified mesenchymal stem cell. The construction method comprises the following steps: (A) connecting an SOD3 gene to an adenovirus vector to obtain a recombinant adenovirus vector; B) co-transfecting the recombinant adenovirus vector and a packaging plasmid to a 293 cell line by utilizing a transfecting agent, culturing until the 293 cell line completely generates a cytopathic effect, collecting an adenovirus strain, storing the strain and confirming that the SOD3 gene is connected to the adenovirus vector; C) amplifying the adenovirus strain, infecting the 293 cell line, collecting the adenovirus supernatant, and determining the titer after ultrafiltration, purification and concentration; and D) infecting the mesenchymal stem cell in different conditions respectively by the adenovirus, and determining the best infection condition to obtain the SOD gene modified mesenchymal stem cell. The EC-SOD (extracellular superoxide dismutase) gene over-expressed SOD gene modified mesenchymal stem cell can be prepared by the method, and can be applied to treatment of mouse with radiation so as to prolong the survival period of the radiated mouse.
Owner:SHENZHEN BEIKE BIOTECH
High-activity glutathion peroxidase GPX 1 mutant and its preparation method
ActiveCN103320406AIncrease vitalityImprove protectionOxidoreductasesVector-based foreign material introductionGenes mutationNatural source
A high-activity glutathion peroxidase GPX 1 mutant and its preparation method belong to the field of biotechnology. A mutant gene is obtained by a gene mutation or synthesis method, and then a catalytic group SeCys of GPX is introduced into a substrate binding part of the mutant through an auxotroph prokaryotic expression system or an auxotroph and SPP low-temperature combined expression system so as to endow the mutant with high GPX activity; or a target gene, along with a SeCys insertion sequence, is firstly assembled onto a secreting type mammalia cellular expression vector, and then a binding protein 2 of the SeCys insertion sequence is assembled onto an intracellular mammalia cellular expression vector, contransfection of the same lactation cell strain is carried out by the two vectors, and GPX is synthesized by the lactation cell in the presence of sodium selenite. The method provided by the invention is simple. The mutant provided by the invention has advantages of high activity, high yield and good stability. Yield decreasing and inactivation caused by renaturation of an inclusion body are avoided. Thus, natural GPX source limitation and unstable properties are solved.
Owner:JILIN UNIV
Integration plasmid and cell line expressing recombinant human FVIII, and construction method and application thereof
InactiveCN103215308APrevent buildupAvoid purification processFermentationVector-based foreign material introductionPromoterRecombinant coagulation factor VIIa
Owner:SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
Screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells
ActiveCN109880851AAchieve enrichmentImprove HDR efficiencyStable introduction of DNAVector-based foreign material introductionResistant genesMammal
The invention discloses a screening report vector and screening method for enriching CRISPR / Cas9-mediated homologous recombination repairing cells. The screening report vector includes a universal sgRNA expression cassette, a Cas9 expression cassette, a resistance gene expression cassette and a resistance gene homologous recombination repair template, and the screening reporter vector itself can be repaired by homologous recombination of the resistance gene sequence to make the cells have resistance. The screening report vector, a targeting vector for cell genome HDR editing and a donor vectorform a co-transfection system which can be applied to HDR positive cell cloning with efficient enrichment point editing, fragment insertion and fragment deletion, and can be widely applied to a variety of cells of mammals. The screening efficiency of HDR-edited cells is significantly improved, the reintroduction of double-strand breaking and DNA integration in the unrelated positions of the cellgenome is not required, and an effective way is provided to promote research and application of accurate gene editing.
Owner:NORTHWEST A & F UNIV
Leukemia mouse model based on gene co-transfection technology and preparation method thereof
ActiveCN102864172AIncrease success rateImprove featuresFermentationGenetic engineeringBone marrow cellViral vector
The invention relates to the technical field of biology, in particular to a leukemia mouse model based on a gene co-transfection technology and a preparation method thereof. The preparation method of the leukemia mouse model comprises the following steps: performing construction and packaging of K-ras mutants and AML1-ETO fusion gene lentivirus vectors; performing bone marrow cell separation and virus infection condition monitoring; implanting infection cells in a mouse to build a leukemia animal model; and performing model identification. The method of building the leukemia mouse model in a mode of utilizing caudal vein injection to lead in the manual site-directed mutagenesis K-ras mutants and AML1-ETO fusion gene lentivirus vectors is adopted initiatively. The leukemia mouse model is high in success rate, pathological characteristics are high in similarity to morbidity conditions of clinical leukemia, and the novel animal model can be provided for leukemia extramedullary infiltration mechanism research, leukemia medicine screening and gene and molecule target treatment.
Owner:SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof
ActiveCN104195116AViral antigen ingredientsMicroorganism based processesF proteinNewcastle disease virus NDV
The invention discloses a recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and a construction method thereof, belonging to the field of recombinant virus vaccines. The recombinant Newcastle disease virus is a goose isolate, the cleavage site amino acid sequence of F protein of the recombinant Newcastle disease virus is GRQGRL, and the P3 gene is positioned in a noncoding region between the P gene and M gene of Newcastle disease virus. The transcription plasmid pCI-NA-VP3(SEQ ID NO.1) and the transcription helper plasmid pcDNA-N, pcDNA-P and pcDNA-L(SEQ ID NO. 2-4) cotransfect a host cell licensed by application of the Newcastle disease virus to culture a transfected host cell, and the recombinant Newcastle disease virus can be saved from a cell suspension of the transfected host cell. The recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes can be used as a bivalent living-vector vaccine for preventing Newcastle disease virus and goose parvovirus.
Owner:JILIN UNIV
Cell line for stable expression of peste des petits ruminants virus receptor Nectin-4 and construction method of cell line
InactiveCN107805628AEasy to separate workGood genetic stabilityImmunoglobulin superfamilyFermentationVector systemCell culture supernatant
The invention discloses a cell line for stable expression of a peste des petits ruminants virus receptor Nectin-4 and a construction method of the cell line. According to the invention, by virtue of asynthetic Nectin-4 gene and on the basis of a lentiviral vector system, a recombinant plasmid pLOV-eGFP-Nectin-4 is constructed. The recombinant plasmid, together with pSPAX2 and pMD2.G plasmid, is applied to co-transfection of a 293T cell, so that retrovirus-like particles are obtained. A supernatant-infected MDBK cell is cultivated by virtue of cells containing the retrovirus-like particles, and puromycin screening and purifying are conducted, so that the MDBK cell for stable expression of Nectin-4 protein is obtained, and the MDBK cell is named as MDBK-Nectin-4. Through the construction ofthe cell line, an experimental material is provided for deep research of an interaction between PPRV and the receptor Nectin-4 and for the clinical rapid isolation of the PPRV is provided.
Owner:ANHUI AGRICULTURAL UNIVERSITY
Detection method of human myelin oligodendroglia glycoprotein self immune antibody
ActiveCN107271680AImprove detection efficiencyGood specificityDisease diagnosisBiological testingGlycoproteinAntigenic protein
The invention discloses a detection method of a self immune antibody. The method comprises the following steps of performing cotransfection cells by expression vectors of allergen protein of an antibody to be detected and a chaperone protein expression vector; after the transfected cells are fixed and after washing, adding serum to be tested for incubation; adding second antibodies for incubation; then, performing analysis, wherein the antigen protein of the antibody to be detected is MOG protein. The detection method has the characteristics of high detection efficiency, high specificity and high sensitivity. The common problem of false positive or false negative detection result in the immunoblotting and enzyme linked immunosorbent assay are successfully solved; very important study and practical values are realized.
Owner:广州敏特生物技术有限公司
Cre fusion protein functional test cell and modified Cre fusion protein
InactiveCN102827873AHigh membrane penetration efficiencyImprove biological activityMicrobiological testing/measurementTransferasesHEK 293 Cell LineDrug biological activity
The invention discloses a Cre fusion protein functional test cell and a modified Cre fusion protein. The Cre fusion protein functional test cell takes an HEK (human embryonic kidney) 293 cell as a host cell, a pAcmy (African cassava mosaic virus)-stop-EGFP (enhanced green fluorescent protein) carrier and phi C31 integrase are used for co-transfection of the host cell. Through the action of the phi C31 integrase, the carrier which carries an LoxP (locus of X-over P1) element having a Cre recombinase detection function is integrated to a pseudo attP (attachment P) site of an HEK 293 cell line in a single copying manner, so a stable and reliable cell model is provided for researching the Cre fusion protein membrane penetrating efficiency and biological activity. The Cre fusion protein HNTC (hemagglutinin-neuraminidase cytotoxic T) which is modified by TAT (transactivator) and NLS (nuclear localization signal) polypeptides has high membrane penetrating efficiency and good biological activity, can be effectively used as a tool for genetic manipulation at a mammalian cell chromosome level.
Owner:NORTHWEST A & F UNIV +1
Recombined rhabdovirus containing double valence insect resisting gene
InactiveCN1597938AHas insecticidal effectImprove insecticidal efficiencyBiocideViruses/bacteriophagesCotton bollwormChitinase
The invention clones insect specific neurotoxin gene BmkIT of East Asian scorpion and insect chitinase gene Chi to an insect cell-converting medium carrier pAcPDIEI to obtain a recombinant plasmid containing divalent anti-insect genes pAcPDIEI-Bmkit-Chi and transfecting the recombinant plasmid and DNA of AcNPV genome together in sfg cells, and obtaining a recombinant rhabdovirus containing divalent anti-insect genes AcNPV-BmKIT-Chi. The cooperation of the insect specific neurotoxin with the chitinase improves the poisoning effect of virus on cotton bollworms and other lepidopteral pests.
Owner:SHANXI UNIV
COVID-19 vaccine and preparation method and application thereof
PendingCN112386684AImprove securityWide variety of sourcesSsRNA viruses positive-senseVirus peptidesImmunocompetenceT cell
The invention provides a COVID-19 vaccine and a preparation method and application thereof. The COVID-19 vaccine comprises T cells expressing SARS-CoV-2 S protein. The preparation method of the COVID-19 vaccine comprises the following steps that (1), coding genes of the SARS-CoV-2 S protein are inserted into an adenovirus vector to construct a recombinant adenovirus vector; (2), the recombinant adenovirus vector and packaging helper plasmids are used for co-transfecting mammalian cells to prepare recombinant adenovirus; and (3), the recombinant adenovirus is adopted for infecting the T cells to obtain the COVID-19 vaccine. According to the COVID-19 vaccine and the preparation method and application thereof, a virus system is utilized for constructing the recombinant T cells expressing theSARS-CoV-2 S protein, the recombinant T cells are input into a body to continuously express the S protein, the body is induced to generate a specific humoral immune response or a cellular immune response, and the body obtains the protective immunity capacity on SARS-CoV-2.
Owner:广东昭泰细胞生物科技有限公司
Preparation method of novel coronavirus pneumonia bivalent vaccine
PendingCN111317816AGood effectQuick resultsSsRNA viruses positive-senseViral antigen ingredientsEnzyme digestionShuttle vector
The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.
Owner:翁炳焕
Method for preparing and screening cell line expressing bispecific antibody
InactiveCN104818295AFermentationVector-based foreign material introductionBispecific antibodyMaterial resources
The present invention provides a method for preparing and screening a cell line expressing bispecific antibody. The method comprises: constructing two plasmids expressing bispecific antibody, wherein the two plasmids contain double promoters and respectively express different fluorescent proteins; and carrying out transformation, culture and extraction on the two recombinant plasmids, co-transfecting the two recombinant plasmids into host cells, screening the positive monoclonal cell line expressing the double fluorescence, and evaluating the yield and the stability according to the fluorescence intensity. With the method of the present invention, the high yield cell line can be conveniently and rapidly screened in advance, the test period can be shortened, the manpower and material resource investment can be reduced, and the accuracy is high.
Owner:WUHAN YZY BIOPHARMA CO LTD
Recoverable immortalized rat bone marrow mesenchyme stem cell as well as preparation method and application thereof
InactiveCN102250841AHave biological propertiesEnhance in vitro proliferative abilityFermentationGenetic engineeringHypoxic ischemic brain injuryBrain damage
The invention discloses a recoverable immortalized rat bone marrow mesenchyme stem cell. The cell carries an SV40T (Simian Virus 40T) gene and a hygromycin resistance gene and two ends of the SV40T gene also have orthokinetic Loxp sites. The preparation method of the recoverable immortalized rat bone marrow mesenchyme stem cell comprises the following steps of: co-transfecting an HEK293 (Human Embryo Kidney 293) cell by SSR69 (Simple Sequence Repeat 69) plasmids and pAmhpo plasmids and screening by the hygromycin to obtain the recoverable immortalized rat bone marrow mesenchyme stem cell with hygromycin resistance; the cell has the same biological property with a primary rat and also has the advantages of strong external multiplication capacity, stable biological property, capability of being recovered to a previous state, good biological safety and the like; and the cell provided by the invention can be used as a seed cell induced and differentiated by nerves and a seed cell for treating hypoxicischemic brain damage of a newly-born rat.
Owner:CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
Specific targeted hDGK theta gene luciferase reporting system
The invention discloses a specific targeted hDGK theta gene luciferase reporting system. The specific targeted hDGK theta gene luciferase reporting system is composed of a targeting carrier and a targeting donor, wherein an exogenous DNA fragment required to be introduced is carried by the targeting donor, the exogenous DNA fragment comprises T2A small peptide, a luciferase gene cDNA sequence, aneGFP expression frame, a Neomycin gene sequence, and an upstream and downstream homologous arm sequence; and the targeting carrier contains a nuclease Cas9 expression frame and a guiding chain sgRNA expression frame of the targeted hDGK theta. An establishing method of the reporting system comprises the following steps: screening sgRNA of a targeted hDGK theta gene 3' non-coding region, constructing an eukaryotic expression carrier carried by Cas9 and a sgRNA expression element as the targeting carrier, constructing the upstream and downstream homologous arms carried with a luciferase gene cDNA sequence and used for targeted integration as well as the targeting donor used for gene screening, performing co-transfection of target cells by two carriers, screening by using a resistant gene, and establishing the luciferase reporting system of targeted hDGK theta in a target cell.
Owner:SHAANXI NORMAL UNIV
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