137 results about "Stable transfection" patented technology

Production of clinical grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. Disclosed herein are systems based on a stably trans formed insect cell lines harboring helper genes required for vector production. Specifically exemplified are system embodiments that take advantage of DNA regulatory elements from two unrelated viruses—AcMNPV and AA V2. System embodiments utilize rep and/or cap genes either stably transfected in cell lines or which are introduced into cells as an expression cassette in a vector. Rep and cap genes that are designed to remain silent until the cell is infected with a viral vector. Infection with viral initiates rescue/amplification of integrated AAV helper genes resulting in dramatic induction of the expression and assembly of rAAV. The arrangement of this specific embodiment provides high levels of Rep and Cap proteins in every cell thus improving rAAV yields by 10-fold. The described vectors are modular in design and may be utilized for the production of other multiprotein complexes.

The present invention is directed to isolated nucleic acid molecules that encode LIM mineralization protein, or LMP. The invention further provides vectors comprising nucleotide sequences that encode LMP, as well as host cells comprising those vectors. Moreover, the present invention relates to methods of inducing bone formation by transfecting osteogenic precursor cells with an isolated nucleic acid molecule comprising a nucleotide sequence encoding LIM mineralization protein. The transfection may occur ex vivo or in vivo by direct injection of virus or naked plasmid DNA. In a particular embodiment, the invention provides a method of fusing a spine by transfecting osteogenic precursor cells with an isolated nucleic acid molecule having a nucleotide sequence encoding LIM mineralization protein, admixing the transfected osteogenic precursor cells with a matrix and contacting the matrix with the spine. Finally, the invention relates to methods for inducing systemic bone formation by stable transfection of host cells with the vectors of the invention.

The invention pertains to the field of biomedical technology, in particular to an orthotopic transplanted mouse liver cancer model which takes the research of the occurrence and development mechanisms of liver cancer and the development of anti-tumor drugs as the purposes, and the preparation method and the application thereof. The H22 cell strain of stable transfection EGFP is injected into the BALB/c mouse peritoneal cavity for amplification; the ascites is extracted from the H22 ascites cancer mouse peritoneal cavity, then cell separation is carried out and cell suspension is prepared; the mouse peritoneal cavity is opened, and the cell suspension is extracted by a micro-syringe to be injected into the mouse liver; a cotton bud with 75 percent alcohol is pressed on the needle hole till the surface of the liver does not bleed any more, and the needle hole is sealed by bonding agent; then the liver surface is washed by physiological saline, and layer separation is carried out and the peritoneal cavity is closed. The results show that the occurrence rate of mouse liver cancer is 100 percent and the natural remission rate is zero. The model is an ideal orthotopic liver cancer model which can be used in the research of the development and metastasis mechanisms of orthotopic liver cancer and can also be applicable to the development of anti-tumor drugs, the experimental treatment and diagnosis research of liver cancer.

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 snake venom metal protease inhibitor BJ46a capable of resisting attack and transference of tumor and application thereof, and belongs to the field of medical organism. In the invention, according to the BJ46a gene sequence (AF294836) in GenBank, the BJ46a full gene is designed and synthesized. An expression vector cloned to baculovirus can generate the recombined BJ46a protein capable of inhibiting the activity of the substrate metal protease in Sf9 insect cells, and in vivo and in vitro experiments show that the BJ46a protein has the functions of resisting the attack and transference of melanoma cells B16. The inhibitor adopts the genetic transfection technique, and can establish B16/pcDNA3.1HisC-BJ46a cell strains with stable transfection, and the in vivo and in vitro experiments prove that the BJ46a can inhibit the attack and transference of B16 cells at the gene level. The inhibitor is applied to preventing and treating the attack and transference of tumor, and has great application prospect.

The invention discloses a lentiviral plasmid expression vector as well as a construction method and an application of the lentiviral plasmid expression vector. The lentiviral plasmid expression vector is pCDH-MCS-MuSK-GFP and the nucleotide sequence of the lentiviral plasmid expression vector is shown in SEQ ID NO. 2 in a sequence table. The invention further discloses an application of the lentiviral plasmid expression vector in construction of an anti-muscle-specific receptor tyrosine kinase antibody detection. According to the invention, a stable transfected cell line of MuSK is established by constructing a viral vector, a MuSK antibody is qualitatively and quantitatively detected clinically by an immunofluorescence staining method with relatively high sensitivity and specificity in combination with a flow cytometry, the detection results are more stable and manpower and time costs are greatly saved. The diagnosis and treatment levels of myasthenia gravis are greatly improved by the establishment of the project, the labor capacity and life quality of patients are improved and the lentiviral plasmid expression vector has significant social and economic benefits.

The invention provides a method for suspension culture of sensitive cells, which comprises the following steps that: (1) expression vectors containing gene sequences, capable of weakening the cell adhesion performance, are used for stably transfecting Marc145 cells or MA104 cells; (2) a stable transfection cell clone is screened and separated; (3) the gene expression in the stable transfection cell clone is detected; and (4) the stable transfection cell clone is subjected to suspension culture. The method further relates to a method for producing blue ear disease vaccine vaccine by using the Marc145 cells or MA104 cells obtained through the suspension culture.

The invention relates to a recombinant human IL (Interleukin)-23 receptor extracellular region/Fc fusion protein capable of combining with IL-23 and antagonizing a function of IL-23. The fusion protein is characterized in that a fusion protein encoding gene is rich in mammalian cell preferred codons, and is highly expressed by a CHO (Chinese Hamster Ovary) stable transfection cell strain. The protein is formed by fusion of a gene optimized IL-23R-CHR active fragment and a human immune globulin molecule Fc fragment; the defects that the half-life period of the prokaryotic expression IL-23R-CHR fragment is short, the stability is poor and endotoxin is difficult to remove are overcome; the biological activity of IL-23-CHR is reserved; in addition, the fusion protein does not have ADCC (Antibody Dependent Cellular Cytotoxicity) and CDC (Complement Dependent Cytotoxicity) effects, so that the fusion protein is more suitable for treatment of chronic diseases such as an autoimmune disease and a chronic infection; and the contents of molecular design, expression, purification, construction of a stable expression cell strain and disease treatment and the like of the fusion protein are included.

The invention provides a method for suspension culture of subculture cells, which comprises the following steps that: (1) expression vectors containing gene sequences, capable of weakening the cell adhesion property, are used for stably transfecting pig testis cells or pig kidney cells; (2) a stable transfection cell clone is screened and separated; (3) the gene expression in the stable transfection cell clone is detected; and (4) the stable transfection cell clone is subjected to suspension culture. The method further relates to a method for producing hog cholera vaccine by using the pig testicle cells or the pig kidney cells obtained through the suspension culture.

The present invention relates to a method for constructing a stable cell line carrying an orthogonal tRNA/aminoacyl tRNA synthetase, and the method stably integrates an orthogonal tRNA/aminoacyl tRNAsynthetase gene into cell genomes by double lentiviral stable transduction and plasmid stable transfection. The invention further relates to the stable cell line obtained by the method and the use ofthe stable cell line in the expression of a protein containing non-natural amino acids, and by utilizing unique reactive groups on the non-natural amino acids, the purpose of high-efficiency and specific protein modification can be achieved.

The invention constructs prokaryotic recombinant plasmid and eukaryotic recombinant plasmid of mouse Beta-defensin-1-polypeptide (mBD-1) by the gene engineering technology, leads the prokaryotic recombinant plasmid to transfer into engineering bacteria to carry out induction to effective expression mouse Beta-defensin-1-polypeptide (mBD-1) and carry out the extraction and purification of expression products and enzyme cutting of enterokinase so as to release mBD-1 which is mature and has activity; by experiment, the invention proves that the mBD-1 polypeptide and MDCK cell strain which can transfect the eukaryotic recombinant plasmid stably has anti-influenza virus function so as to develop a novel drug used for curing or preventing influenza.

The invention relates to a polysaccharide LBP1a1-1 extracted from fructus lycii, application of the polysaccharide in preparation of medicines or health care products for preventing and/or treating neurodegenerative diseases, and application of the polysaccharide in preparation of medicines or health care products for inhibiting Abeta42 and in preparation of medicines or health care products for preventing and treating Alzheimer's disease. Specifically, the invention relates to arabinogalactan extracted from the fructus lycii. The preparation method comprises the following steps: extracting crude polysaccharides in the fructus lycii by combining cellulase, amylase and papain with water of 55 DEG C, and performing alcohol precipitation; and identifying by combining multiple column chromatography purification methods with spectrum analysis, thereby obtaining the arabinogalactan. In vitro experiments prove that the polysaccharide is capable of inhibiting production of the Abeta42 in CHO/APPBACE1 cells for stable transfection of APP and BACE1 in a dosage dependent manner. Therefore, the polysaccharide has a potential effect of treating the Alzheimer's disease, and is expected to be developed into a carbohydrate drug for treating the Alzheimer's disease.

The invention provides a tetracycline strictly controlled eukaryotic expression vector. Following DNA elements including hCMV, rtTA2-M2, IRES, EGFP-Puro, element (Luc) of coding luciferase and Ptetbi-RFC are respectively introduced and are used for the expression and screening of exogenous interest genes in eukaryotic cells. The vector is strictly controlled by Tet or Dox after being transfected. And the expression level of the genes is quantitatively regulated through the amount of the added Tet or Dox. The expression level of the interest genes can be indirectly detected through the detection of the activity of Luc luciferase. The transfection efficiency of the vector can be completed through the detection of the proportion of green fluorescyte. The stably transfected cells are sorted by a flow cytometry. Thus, the expression of the interest genes is ensured to be simple and convenient. And in a stable cell line obtained through screening, the interest genes are integrated with a host genome. The tetracycline strictly controlled eukaryotic expression vector has long stable expression characteristics and advantages.

The present invention relates to a a cytochrome c-reporter fusion protein construct comprising a modified cytochrome c protein which targets the mitochondria and has a reduced ability to induce apoptosis in a living cell. The invention also relates to nucleic acid constructs encoding such protein fusions and cells stably transfected with such constructs. The stably transfected cells of the invention can be used in assays to detect apoptosis.

The invention relates to polysaccharide extracted from lycium barbarum, an extraction method of the polysaccharide and the application of the polysaccharide in preparation of a drug for treating an Alzheimer's disease and specifically relates to a method for extracting polysaccharide LBP1C-2 (lycium barbarum polysaccharide 1C-2) from the lycium barbarum. The method comprises the following steps: extracting enzyme-water combined extraction lycium barbarum crude polysaccharide from the lycium barbarum and purifying the crude polysaccharide, so as to obtain the polysaccharide LBP1C-2; determiningthe polysaccharide LBP1C-2 as pectin through acid hydrolysis, methylation and nuclear magnetic resonance analysis. An in-vitro experiment shows that the polysaccharide LBP1C-2 can inhibit the generation of CHO/APPBACE1 cells of stable transfection APP and BACE1 and A beta 42 in HEK293-APPsw cells formed by mutation of stable transfection APP Switish in a dose-dependent manner, and the pectin caninhibit the aggregation of A beta 42.

The invention discloses a method for detecting the biological activity of chicken interferon alpha by a luciferase reporter gene method and an application thereof. The method comprises the following steps: using PCR amplification to obtain the pMx1 gene fragment of the chicken Mx1 protein; inserting the pMx1 gene fragment into the 5' end of the pGL3-basic vector luc gene, and then using PCR amplification to obtain the pMx1-luc fusion gene fragment; Replace the pCMV and EGFP gene fragments in the pEGFP-N1 vector with pMx1-luc fusion gene fragments, construct pMx1-luc plasmids, transfect cells, and select stable transfected cell lines; Cloning culture; prepare a standard curve with the chicken interferon-α standard diluted in gradient, add the chicken interferon-α sample to be tested into the cells after cloning culture and co-incubate, then detect the fluorescence intensity, and evaluate the chicken interferon to be tested in combination with the standard curve Titer of alpha sample.