40 results about "HEK 293 cells" patented technology

The invention discloses an HEK (human embryonic kidney) 293 cell line applicable to serum-free culture and an application thereof. According to the invention, the HEK 293 cell (293SF) applicable to serum-free culture is obtained by adopting a culture solution progressive substitution method, wherein the preservation number of the HEK 293 cell is CGMCC (China General Microbiological Culture Collection Center) No.5824. Detection shows that the 293SF cell has stable adenovirus proliferation capacity and foreign protein expressing ability; average lesion time is 97 hours and is reduced by 12.9% compared with the HEK 293 cell; virus virulence reaches up to 107.48TCID50 / mL and is improved by 43.3% compared with the HEK293 cell; and stability is good while generation number is improved, and the state and susceptibility of the 293SF cell are not changed after the 293SF cell is continuous passage is carried out for sixteen times. The cell line disclosed by the invention can be used for serum-free production of an adenovirus vector vaccine.

The present invention relates to recombinant blood coagulation factor IX (rFIX) mutants having improved FIX clotting activity. Three full length FIX proteins with combinations of mutations of amino acids important for functional activity of FIX and FIX wild type were cloned and expressed in HEK 293 cells. The proteins were tested by an activated partial thromboplastin time (aPTT) assays in FIX-depleted plasma. Two mutant proteins had increased specific FIX activity. Furthermore, a pre-activated FIX protein had an increased activity in FIX-depleted plasma. Therefore these FIX mutants can be used for the treatment of FIX associated bleeding disorders.

The present invention relates to recombinant blood coagulation factor IX (rFIX) mutants having factor VIII (FVIII) independent factor X (FX) activation potential. Five full length FIX proteins with combinations of mutations of amino acids important for functional activity of FIX and FIX wild type were cloned and expressed in HEK 293 cells. The proteins were tested by an activated partial thromboplastin time (aPTT) assay in FVIII-depleted plasma as well as in FVIII-inhibited patient plasma. In FVIII-depleted plasma functional activity of the FIX mutants was calculated as increased FVIII equivalent activity. The mutant proteins had increased FVIII equivalent activity. In FVIII-inhibited patient plasma the FEIBA equivalent activity was calculated for analysis of FVIII independent FX activation potential. The proteins had also increased FEIBA equivalent activity. Furthermore, the pre-activated FIX proteins had an increased activity in FIX-depleted plasma containing FVIII inhibitors. Therefore these FIX mutants are alternatives as bypassing agents for treatment of FVIII inhibitor patients.

The present invention relates to viral particles as immunogens against enterovirus infection and a method of producing the same. Specifically, the present invention features that human embryo kidney 293 (HEK 293) cells are used to produce viral particles of Enterovirus A, particularly Coxsackievirus A6 (CVA6) particles or Coxsackievirus A10 (CVA10) particles or both and optionally additional viral particles of other Enterovirus A e.g. Coxsackievirus A16 (CVA16) and / or Enterovirus A71 (EV71). The yield of the viral particles in HEK 293 cells is unexpectedly high and effective to induce an immune response against enterovirus infection, especially CVA6 and CVA10. The present invention also relates to an immunogenic composition against enterovirus infection for human use comprising the viral particles as described herein and a method of preventing enterovirus infection or a disease as caused, particularly Hand-Foot-Mouth diseases (HFMD), by administering the immunogenic composition to a subject in need thereof.

The invention discloses wild type EGFR stably and high expressing recombinant HEK 293-EGFR cells constructed on the basis of exogenous recombinant plasmids pcDNA3.1(+)-EGFR, which can improve the expression of EGFR by 4 to 5 times compared with the expression before transfection. EGF serving as an EGFR accelerator and gefitinib serving as a tyrosine kinase inhibitor are used to induce transfected HEK293 cells, and the examination of the change of the number of expressed phosphorylated EGFR and ERK in the cells shows that the transfected EGFR has activities; the medicinal gefitinib also has an inhibiting effect on the transfected EGFR, so EGFR-targeted medicament screening can improve the screening specificity and flexibility; and the wild type EGFR stably and high expressing recombinant HEK 293-EGFR cells can be used in comparative researches on the differences of EGFR pathway proteins.

The invention discloses a Coxsackie virus A10 domestication strain TA151R-1 with high titer and stable passage. The virus strain can infect various cell strains including RD cells, HEK 293 cells, Verocells, MRC-5 cells, Hep-2 cells, WI-38 cells and the like and can also be used for preparing a monovalent vaccine or a polyvalent vaccine; the prepared vaccine can protect an organism from damages from Coxsackie virus, can also completely prevent the attacks from other heterologous viruses, can effectively prevent and / or treat diseases caused by infection of the Coxsackie virus and has a wide application prospect.

The invention discloses a method for detecting an odor substance butanedione based on an olfactory receptor sensor. The method comprises the following steps of: at first, expressing olfactory receptor protein ODR-10 with a His6-tag on an HEK-293 cell surface, extracting and effectively immobilizing the olfactory receptor protein ODR-10 on a quartz crystal microbalance surface through an aptamer so as to form the olfactory receptor sensor; testing a series of odor material butanedione with different set concentrations, thus obtaining a butanedione-resonance frequency variation standard curve; and then, testing a gas which has unknown concentration and needs to be tested, thereby obtaining the concentration of the odor substance-butanedione in the gas to be tested according to the quartz crystal microbalance resonant frequency and the butanedione-resonance frequency variation standard curve. The method is simple in required instrument and convenient to operate, a problem that the olfactory receptor protein ODR-10a of a sensitive element and a quartz crystal microbalance are stably coupled with each other is solved, and the odor substance butanedione is quickly and specially detected.

The invention provides a strong loquat leaf extract refined with honey. The strong loquat leaf extract is prepared from 69g of loquat leaves, 15g of radix stemonae, 6g of root barks of white mulberry, 50g of poppy shells, 9g of cynanchum glaucescens, 6g of platycodon grandiflorum and 0.15g of menthol. The other medicinal materials except the menthol are subjected to extraction and alcohol sedimentation to obtain concentrated solution, supercritical extracts are obtained by supercritical extraction, concentration is continued to reach about 100ml, 2.5g of sodium benzoate is added and dissolved by stirring, about 100ml of processed honey and 300ml of maltose are added and heated to boil for 20 minutes, the solution is filtered and chilled, 0.5g of citric acid, the menthol and an appropriate amount of loquat essence dissolved by ethyl alcohol are added, stirred and uniformly mixed, and the processed honey is added to reach 100ml and uniformly mixed to obtain the strong loquat leaf extract refined with the honey. The platycodon D content of the prepared strong loquat leaf extract refined with the honey is high, and application of the strong loquat leaf extract refined with the honey to preparation of human embryo kidney HEK-293 cell proliferation inhibition medicines is discovered.

Disclosed are an in vitro method of producing a recombinant AAV virion in a mammalian host cell that comprises a functional adenoviral E1A gene (e.g., a HEK 293 cell) and rAAV virions made by the method.

The invention relates to a simple and easy dynamic detection method of potassium ion exchange inside and outside a human embryonic kidney epithelial cell (HEK 293) and erythrocyte. The method comprises the following steps of: preparing a nano capillary taking a boron silicon capillary as a material, and carrying out inner wall silanization, thus the nano capillary is changed to be hydrophobic; and filling a 1,2-dichloroethane (DCE) solution containing a K<+> vector in the nano capillary with a hydrophobic inner wall tube, wherein the solution belongs to an organic phase, the HEK293 cell and erythrocyte solution belongs to a water phase, Ag / AgCl belongs to the reference electrode in the water phase, Ag / AgTPBCI is a reference electrode in the organic phase. According to the invention, by utilizing a nano tube ion selective micro electrode, the dynamic detection of K<+> exchange inside and outside the HEK 293 cell and erythrocyte is carried out by cyclic voltammetry, thus the perfect combination of the ion selective electrode and an electrochemical detection technology is realized. The method combines the characteristics of a patch clamp technique and the ion selective electrode, and has the advantages of no destruction, low cost, rapid detection and high sensitivity.

The invention discloses a method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers. Through extraction and identification of micro vesicles, quantitative analysis of miRNA, protein extraction, Western blot, ELISA (Enzyme-linked Immuno Sorbent Assay) and EdU (5-Ethynyl-2'-deoxyuridine) proliferation assays, an in-vitro angiogenesis assay, a tumorigenesis assay and a tail intravenous injection micro vesicle assay, the situation that the quantitative analysis of the content of corresponding miRNA in artificially modified micro vesicles shows that the content of miR-29 is obviously increased by being compared with a negative control is discovered, the VEGF (Vascular Endothelial Growth Factor) protein secretion level of gastric cancer cells is obviously inhibited after the gastric cancer cells are hatched by the micro vesicles containing the miR-29, the proliferation of vascular endothelial cells co-cultured by the gastric cancer cells treated by the micro vesicles secreted by HEK (Human Embryonic Kidney)-293 cells treated by high-expression miR-29a and miR-29c is obviously inhibited, ring formation of the vascular endothelial cells co-cultured by the gastric cancer cells treated by the micro vesicles secreted by the HEK-293 cells treated by the high-expression miR-29a and the miR-29c is obviously inhibited, and the tumor volume in a mouse containing the micro vesicles which are subjected to the tail intravenous injection and high-expression miR-29a / c treatment is obviously smaller than the tumor volume in a mouse treated by the corresponding negative control.

The invention discloses a method for biologically synthesizing conjugated linoleic acid CLA. The invention provides a recombinant cell which is obtained in order to transfer a coding gene of SCD1 to a host cell, and the amino acid sequence of the SCD1 is a sequence 2 in a sequence table. The coding gene of the SCD1 is shown in a sequence 1 in the sequence table. The coding gene of the SCD1 is transferred to the host cell through a recombinant expression vector. In the method for biologically synthesizing the conjugated linoleic acid, the conjugated linoleic acid is obtained by culturing the recombinant cell. Experiments prove that: the coding gene of the Stearoyl-CoAdesaturase (SCD) gene sequence is transferred to an HEK 293 cell, a cell line stably expressing the SCD1 gene is obtained through G418 screening, heterologously expressed SCD enzyme has various bioactivities, and the content of conjugated linoleic acid and n-7 fatty acid components is obviously increased (p is less than 0.05).

The invention discloses a siRNA recombinant interference carrier based on an ALV-J gp85 gene conserved region as a target sequence, and a corresponding preparation method of the siRNA recombinant interference carrier. In the invention, a gp85 gene is used for designing and synthesizing siRNA to build a hairpin-shaped structure forming the siRNA; further an annealed double-chain DNA is obtained, and then is cloned into an adenovirus carrier pHBAd-U6-RFP to build a recombinant adenovirus carrier pAd-gp85-shRNA2; and after sequencing identification is built successfully, recombinant adenovirus carrier is subjected to homologous recombination with framework plasmids in HEK 293 cells. Experiments show that the carrier can effectively inhibit the replication of GX13HG03 virus in DF-1 cells, and can play a certain preventive and therapeutic effect on ALV-J in chickens. Therefore, the invention provides an effective adjunct means for the purification of J subgroup avian leukemia virus, thus providing a new way for AL prevention and control and antiviral research.

The invention relates to a construction method of recombinant HEK (human embryonic kidney) 293 cells highly expressing H3R (histamine receptor 3). The recombinant cells are constructed by using HEK 293 cells as host cells, and an exogenous expression vector transfecting the host cells is an expression vector comprising an H3R full length gene. The expression vector comprising the H3R full length gene is pEZ-M02 and is an enkaryotic expression vector. The recombinant HEK293-H3R cells highly expressing H3R are applied in screening antiallergic drugs. The construction method comprises extracting an enkaryotic expression vector, pEZ-M02 / H1R plasmid, transfecting the HEK293 cells with the pEZ-M02 / H1R plasmid, screening by utilizing G418 to obtain a genetic engineering cell line HEK293-H3R highly expressing the full length H1R, and confirming that the cell line can improve the expression of the full length H3R by 12 times.

The invention discloses a method for inducing de-differentiation of mature cells. The method for inducing de-differentiation of the mature cells comprises the following step of: culturing the mature cells in a solid medium to obtain globular cells, wherein the globular cells have the totipotency of embryonic stem cells, and the solid medium is obtained by mixing a 2*DMEM basal medium and a coagulant. Experiments prove that human embryonic kidney 293 (HEK 293) epithelial cells with a differentiating function grow in a three-dimensional globular shape through a low adhesion surface culture method; and experimental results show that the three-dimensional globular cultured HEK 293 cells have de-differentiation, and the properties of embryonic stem cells and renal progenitor cells are obtained.

The invention relates to a Galpha gene overexpression lentiviral vector and lentivirus and a construction method thereof, and belongs to the technical field of molecular biology. Construction is conducted by taking a pLVX-IRES-mCherry lentiviral expression vector as a basis through a gene recombination technology, a Galpha gene is integrated on the vector, and the Galpha gene overexpression lentiviral vector pLVX-IRES-mCherry-Galpha is obtained. The Galpha gene overexpression lentiviral vector pLVX-IRES-mCherry-Galpha is packaged through lentiviral packaging plasmids including pRSV-Rev, pMDLg-pRRE and pMD2.G, HEK 293 cell transfection is conducted, and the Galpha lentivirus is obtained. The Galpha gene overexpression lentiviral vector constructed through the method is high in host cell transfection efficiency and can specifically, continuously and efficiently promote stable expression of the Galpha gene in host cells.

The invention discloses a recombination HEK 293 cell highly expressed by KDR and an application thereof. The recombination cell takes an HEK 293 cell as a host cell, and an exogenous expression vector transfecting the host cell is an expression vector containing KDR full length genes. Compared with an extracellular region only containing KDR or the recombination cell constructed by partial gene sequence, the recombination HEK 293-KDR cell strain surface containing the KDR full length gene constructed by the invention can stably express KDR molecules, and has complete molecular activity. A cell membrane chromatography has a certain reservation to taspine when detecting the HEK 293-KDR cell and can be applied to drug screen taking VEGFR as a target spot.

The invention provides a method for differentiation of dopaminergic neuron by SHH / Lmx1a signal pathway regulation and control NTN modified rASCs, and belongs to the biomedical method. The coding sequence of Lmx1a gene and chimeric NTN gene are obtained by PCR according to the invention, recombinant adenovirus Ad-JLmx1a and Ad-NTN are respectively packed in the HEK-293 cell after homologous recombination. After rASCs is infected by Ad-Lmx1a, the SHH / FGF-8 and other cytokines induce the rASCs to a standby state, and ad-NTN is infected, and B27, N2supplement and bFGF and other derivants are added. The invention can substantially improve the cell differentiation potential of rASCs to Neuron, and related transcription factor can be applied to the future cell substitution treatment of Parkinsons disease.

The invention aims to provide a classical swine fever virus (CSFV) gene recombinant adenovirus and a production method thereof. An amino acid sequence of a gene segment used for preparing the CSFV gene recombinant adenovirus is SEQ ID NO:2 and a coded nucleotide sequence is SEQ ID NO:1. The CSFV gene recombinant adenovirus is prepared through homologous recombination of the gene segment to a multiple cloning site of a human type 5 replication-defective adenoviral genome. An optimal process for producing the CSFV E0-E2 gene recombinant adenovirus through adherent culture of HEK 293 cells is characterized by thawing the HEK 293 cells with 20% W-DMEM, after thawing for 48 hours, carrying out enlarged culture and subculture on the cells with 10% W-DMEM, after carrying out culture for 24 hours, inoculating the virus according to 3% until IFU is maximum, and along with repeated virus rejuvenation, infecting the cells for 48 hours according to the virus inoculation proportion of 1% and harvesting the virus, thus harvesting the virus with high virus titer. Therefore, the harvested virus solution is timely freeze-dried and different generations of freeze-dried virus seed batches are built, thus providing reliable virus seeds for preparing viruses with high virus titer levels in quantity for suspension culture.

The invention relates to a method for improving the thermal stability of interleukin IL-34, and belongs to the field of biotechnology. The method comprises the steps: carrying out site-specific mutagenesis of a codon agc encoding No.98 serine in a human-derived IL-34cDNA into a codon tgt encoding cysteine; co-transfecting an Sf9 insect cell with a transfer plasmid containing an S98C IL-34 mutant encoding sequence and BacVector-3000 baculovirus DNA to prepare a baculovirus for expressing S98C IL-34; and then adopting the baculovirus for transducing an HEK 293 cell to express the S98C IL-34. Compared with native IL-34, the S98C IL-34 obtained by the method has the thermal stability improved. Through M-NFS-60 cell proliferation experiments and CSF-1R phosphorylation experiments, the activity of the S98C IL-34 is proved to have no significant difference with that of the native IL-34.

The invention discloses a detection method for compound specificity through a G protein-coupled receptor (GPCR). The method comprises following steps: preparing recombination HEK-293 cell suspension; adding coelenterazine to the cell suspension to obtain recombination cell sap; dividing the recombination cell sap into a control group and a to-be-detested group, adding a to-be-detested compound solution to the to-be-detested group, and adding a Hank's balanced salt solution to the control group; if the strength of fluorescence signals emitted by the to-be-detested group is higher than the strength of the fluorescence signals emitted by the control group, significant differences are provided, then a to-be-detested compound acts activation on the GPCR of recombination HEK-293 cells, and otherwise the activation does not exist; continually adding agonist corresponding to the GPCR to the to-be-detested group to detect the fluorescence signals; and if the strength of the fluorescence signals of the to-be-detested group is significantly lower than the strength of the fluorescence signals of the control group, then the to-be-detested compound acts an inbibitional effect on the GPCR of the recombination HEK-293 cells, and otherwise the inbibitional effect does not exist. The method is a flexible and changeable detection mode, an unknown compound can be detected by means of the GPCR, and meanwhile reverse detection for the GPCR in specific binding with a known compound can be achieved by means of the known compound.

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a fructus cnidii extract and preparation and application thereof. The research is capable of using thefructus cnidii extract (aqueous extraction and alcohol extraction) as a research object, an electrophysiology patch clamp technology is combined, and in an HEK 293 cell for instantly transferring a human TRPV1, whether the fructus cnidii extract can activate or inhibit the TRPV1 is tested, so molecular targets of fructus cnidii for developing pharmacodynamic effects are studied. It is firstly discovered that both of an aqueous extract and an alcohol extract of the fructus cnidii activate a TRPV1 channel, and a transmembrane current is generated. So, the pungent-warm property of the fructus cnidii can be a result of TRPV1 activation, the partial efficacy of the fructus cnidii can be achieved through the TRPV1 channel, and the TRPV1 is one of the molecular targets for generating the effectsof warming kidney and tonifying Yang, killing parasites to relieve itching.

A cell test system is the CHO-K1 / d2EGFP and HEK-293 / d2EGFP lines, which contains 4xCPE and the DNA sequence of HSV-TK. A method for configuring said cell test system includes such steps as designing 4xCRE-HSV-TK sequence, artificially synthesizing relative oligonucleotide, designing primer, PCR amplification, cloning it to d2EGFP carrier, sequencing the linked resultant, choosing correct recombinant plasmid, tansferring it in CHO-K1 and HEK-293 cells, screening resistant clone and function test. Said cell test system can be used to screen the compound mutually acting on CAMP signal transmission path.

The invention provides a method for differentiation of dopaminergic neuron by SHH / Lmx1a signal pathway regulation and control NTN modified rASCs, and belongs to the biomedical method. The coding sequence of Lmx1a gene and chimeric NTN gene are obtained by PCR according to the invention, recombinant adenovirus Ad-JLmx1a and Ad-NTN are respectively packed in the HEK-293 cell after homologous recombination. After rASCs is infected by Ad-Lmx1a, the SHH / FGF-8 and other cytokines induce the rASCs to a standby state, and ad-NTN is infected, and B27, N2supplement and bFGF and other derivants are added. The invention can substantially improve the cell differentiation potential of rASCs to Neuron, and related transcription factor can be applied to the future cell substitution treatment of Parkinsons disease.

The invention relates to a cell line in which an expression construct is introduced into genomic DNA, the expression construct including: (a) a promoter operable in animal cells and heterologous to adenoviruses; and (b) a modified adenovirus El coding gene sequence of SEQ ID NO: 32 operatively linked to the promoter. The cell line of the invention is less likely to produce a replication competent adenovirus (RCA). The adenovirus producing cell line of the invention has a low possibility of producing RCA due to homologous recombination, when compared with conventional cell lines. Therefore, this makes it possible to regulate the required amount of virus during gene therapy using the adenovirus and prevent tissue damage and toxic effects caused by overproduction of the adenovirus. Also, the cell line of the invention shows superior adenovirus producing ability, as compared with an HEK 293 cell which is one of conventional adenovirus producing cell lines.

Somatic hypermutation promotes affinity maturation of antibodies by targeting the cytidine deaminase AID to antibody genes, followed by antigen-based selection of matured antibodies. Given the importance of antibodies in medicine and research, developing approaches to reproduce this natural phenomenon in cell culture is of some interest. The inventors use here the CRISPR-Cas 9 based CRISPR-X approach to target AID to antibody genes carried by expression vectors in HEK 293 cells. This directed mutagenesis approach, combined with a highly sensitive antigen-associated magnetic enrichment process, allowed rapid progressive evolution of a human antibody against the Human Leucocyte Antigen A*0201 allele. Starting from a low affinity monoclonal antibody expressed on Ag-specific naïve blood circulating B cells, they obtained in approximately 6 weeks antibodies with a two log increase in affinity and which retained their specificity. The strategy for in vitro affinity maturation of antibodies is applicable to virtually any antigen. It not only allows to tap into the vast naive B cell repertoire but could also be useful when dealing with antigens that only elicit low affinity antibodies after immunization. Accordingly as defined by the claims, the present invention relates to methods and kits for generating and selecting a variant of antibody binding protein with increased binding affinity and / or specificity.

The invention relates to a method for improving the thermal stability of interleukin IL-34, and belongs to the field of biotechnology. The method comprises the steps: carrying out site-specific mutagenesis of a codon agc encoding No.98 serine in a human-derived IL-34cDNA into a codon tgt encoding cysteine; co-transfecting an Sf9 insect cell with a transfer plasmid containing an S98C IL-34 mutant encoding sequence and BacVector-3000 baculovirus DNA to prepare a baculovirus for expressing S98C IL-34; and then adopting the baculovirus for transducing an HEK 293 cell to express the S98C IL-34. Compared with native IL-34, the S98C IL-34 obtained by the method has the thermal stability improved. Through M-NFS-60 cell proliferation experiments and CSF-1R phosphorylation experiments, the activity of the S98C IL-34 is proved to have no significant difference with that of the native IL-34.

The invention provides a preparation method for heart quieting and nerve soothing capsules. The preparation method comprises the following steps: weighing 100 grams of coptis chinensis, 50 grams of ambers, 250 grams of acorus gramineus, 417 grams of processed radix polygalae, 417 grams of wolfiporia extensa, 417 grams of salvia miltiorrhiza, 250 grams of licorice, 417 grams of red dates, 833 grams of wheat, 833 grams of calcined magnets and 833 grams of mother pearls, adding water in an amount which is 10 times the weight of the medicinal materials, performing impregnation for 0.5 h, performing decoction for 1 h, performing filtration, adding water in an amount which is 8 times of the weight of the medicinal materials into decoction dregs, performing decoction for 1 h, filtering medicinal liquor, mixing the decoction, concentrating the mixed decoction to relative density of 1.05 at 60 DEG C, adding ethanol until the volumetric ethanol content reaches 75 percent, performing stirring, standing and filtration, concentrating the filtrate to relative density of 1.25 at 65 DEG C, recycling the ethanol to obtain a concentrated solution, performing supercritical extraction to obtain supercritical extract, continuing concentration, drying the concentrated solution, and performing granulation and capsule filling to obtain the heart quieting and nerve soothing capsules. Special concentration and drying equipment is adopted, and the prepared heart quieting and nerve soothing capsules are high in extract content, and are found to be applied to preparation of a medicament for suppressing proliferation of HEK-293 cells.