19results about How to "Stable passage" patented technology

The invention discloses a method for directly inducing generation of organoids from IPS and application, and belongs to the field of cell engineering. The invention provides a method for directly and rapidly generating a target organ based on human IPS, and the lung organ, the placenta organ and the blood vessel organ are obtained. According to the method, the organoid represented by three germ layers is induced, so that the method is suitable for constructing various organoid. Compared with the prior art, the method has the advantages that step-by-step induction is not needed, one-step construction is successful, the operation is simple, the construction time is greatly shortened, the process is repeatable, long-term stable culture and passage are realized under in-vitro conditions, and the freezing survival rate is high; the method has the advantages that the method is simple in operation, the cultured organoid is good in homogeneity, RNA-seq results show that high expression of the blood vessel related genes appears in the organoid, and the maximum length of the organoid can reach 400 micrometers, so that the culture method can spontaneously induce expression of part of the blood vessel related genes to support growth of the organoid. The invention provides an important tool for researching pathogenesis of diseases and subsequent drug development.

The invention discloses a yak intramuscular precursor fat immortalized cell line and a preparation method thereof. The method comprises the following steps: (1) obtaining yak primary intramuscular precursor fat cells; (2) performing SV40T lentivirus infection on the precursor fat cells in the yak muscle and screening cell strains: performing SV40T lentivirus infection by utilizing a 1 / 2 small-volume infection method to obtain immortalized cells after the precursor fat cells in the yak primary muscle are infected with SV40T, namely immortalized cells for short; puromycin is adopted to screen immortalized cells; and diluting the screened cells by using a limited dilution method, and carrying out enlarged culture to obtain the immortalized cell line. The yak intramuscular precursor fat immortalized cell line constructed by the invention has genetic homogeneity, the heterogeneity influence of cell populations is eliminated, and an in-vitro cell model is provided for subsequent research related to yak intramuscular fat cells.

The application belongs to the technical field of biology and particularly relates to an African swine fever virus pNP868R protein monoclonal antibody and a preparation method and application thereof. The application takes the pNP868R protein prepared by eukaryotic expression as an antigen, immunizes BALB / c mice, uses hybridoma technology, and obtains a hybridoma cell line capable of stably secreting the anti-African swine fever virus pNP868R protein monoclonal antibody through cell fusion, indirect ELISA method screening, limited dilution method subcloning and hybridoma continuous passage culture. The application also obtains an African swine fever virus pNP868R protein monoclonal antibody. The monoclonal antibody has high affinity with the pNP868R protein and can be applied in Western-Blot experiments, indirect immunofluorescence experiments, immunoprecipitation experiments and the capture of proteins interacting with pNP868R, and can be used for the diagnosis, prevention or treatment of African swine fever.

The invention discloses a gene VII type Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E, the preservation number of the gene VII type Newcastle disease attenuated marker vaccine strain is CGMCC (China General Microbiological Culture Collection Center) No.46292, and the whole-genome nucleotide sequence of the vaccine strain is as shown in SEQ ID No: 1; wherein the 256th amino acid glycine (G) of the HN protein is replaced by glutamic acid (E). A Newcastle disease virus reverse genetic manipulation platform is utilized, different sites are mutated on the basis of a Newcastle disease virus MG7 strain, a gene VII type Newcastle disease virus marker vaccine attenuated strain rMG7-HN-G256E which is low in pathogenicity, good in growth characteristic, high in virus titer, stable in passage and excellent in immunogenicity is rescued, and the marker vaccine strain is good in immunogenicity, high in immunogenicity, high in immunogenicity, high in specificity, high in specificity and high in specificity. The vaccine can induce a high-level protective antibody, completely protects immunized chickens, can be used for preventing and controlling the current epidemic gene VII type Newcastle disease virus, and lays a foundation for identifying vaccine immunity and wild virus infection.

The present invention relates to the technical field of recombinant enzymes. The present invention provides a phospholipase A1 and a method and application of its integrated secretory expression in Bacillus subtilis. The amino acid sequence of the phospholipase A1 is shown in SEQ ID NO.1. The present invention constructs various signal peptides and expression vectors combined from the Bacillus subtilis homologous protein signal peptide library, and combines a high-throughput screening method to screen out signal peptides that can improve PLA1 secretion, thereby optimizing the secretion of PLA1. Compared with the original signal peptide, the secretion amount of PLA1 is increased by 10 times, and the enzyme activity is increased by 10 times, which is of great significance for the reduction of PLA1 production cost and further large-scale application. In addition, the phospholipase A1 enzyme preparation prepared by the present invention can reduce the phosphorus content of rice oil to 70-75ppm at a temperature of 55°C and acidic conditions.

The present invention belongs to biotechnology, and specifically relates to monoclonal antibodies against p30 protein of African swine fever virus and its preparation method and application. The main technology includes using recombinant p30 protein prepared by prokaryotic expression as antigen, immunizing BALB / c mice, and then using hybridoma technology, through cell fusion, indirect ELISA method screening, limited dilution method subcloning, and hybridoma continuous passage culture, to obtain a hybridoma cell line that can stably secrete monoclonal antibodies against p30 protein of African swine fever virus. The present invention also discloses the sequence of the prepared monoclonal antibody immunogen, the subtype of the monoclonal antibody, the CDR sequence, the accession number of the hybridoma cell line, and the application of the monoclonal antibody in Western Blot experiment, indirect immunofluorescence experiment, neutralization experiment and blocking ELISA method for detecting African swine fever virus antibodies in pig serum.

This disclosure belongs to the field of cell culture technology, and specifically relates to a serum-free culture medium for MSCs and its preparation method. The culture medium contains a fixed ratio of basic nutrients and six key regulatory factors, namely transferrin, ascorbic acid, human serum albumin, human fibroblast growth factor rhFGF, platelet-derived growth factor PDGF-BB, and transforming growth factor TGF-β1. By optimizing the combination and concentration of these six key regulatory factors, the proliferation rate and activity of MSCs are significantly improved. It also provides four suitable formulations: high proliferation, high activity, balanced, and economical. The culture effect is stable and reproducible, with high safety and a simple process, making it suitable for laboratory research and large-scale clinical-grade MSC culture.

The invention relates to an in-vitro culture medium, an in-vitro culture method and application of a human amniotic fluid source organoid. The in-vitro culture medium of the human amniotic fluid source organoid comprises a DMEM-F12 basal culture medium, a glial cell source neurotrophic factor, 8-bromoadenosine-3 ', 5'-cyclic monophosphate and aldosterone. The culture medium provided by the invention is clear in component and high in culture success rate. The human amniotic fluid organ cultured by using the culture medium and the method disclosed by the invention can be differentiated into multiple types of functional cells including neuron-like cells, myocardial cells and the like through in-vitro induction, and the differentiation capacity is obviously superior to that of an existing 2D cell line model.

The invention discloses a gene VII type Newcastle disease attenuated marker vaccine strain rMG7-F-N471A, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.46293. The whole-genome nucleotide sequence of the vaccine strain is as shown in SEQ ID No: 1; wherein the 471st amino acid asparagine (N) of the F protein is replaced with alanine (A). A Newcastle disease virus reverse genetic manipulation platform is utilized, different sites are mutated on the basis of a Newcastle disease virus MG7 strain, a gene VII type Newcastle disease virus marker vaccine attenuated strain rMG7-F-N471A which is low in pathogenicity, good in growth characteristic, high in virus titer, stable in passage and excellent in immunogenicity is rescued, and the marker vaccine strain is good in immunogenicity, high in immunogenicity, high in immunogenicity, high in specificity, high in specificity, high in specificity and high in specificity. The vaccine can induce a high-level protective antibody, completely protects immunized chickens, can be used for preventing and controlling the current epidemic gene VII type Newcastle disease virus, and lays a foundation for identifying vaccine immunity and wild virus infection.

The invention discloses a gene VII type Newcastle disease attenuated marker vaccine strain rMG7-L-V1291I, the preservation number of the gene VII type Newcastle disease attenuated marker vaccine strain rMG7-L-V1291I is CGMCC (China General Microbiological Culture Collection Center) No.46291, and the whole-genome nucleotide sequence of the vaccine strain is as shown in SEQ ID No: 1; wherein the 1291th amino acid valine (V) of the L protein is replaced by isoleucine (I). A Newcastle disease virus reverse genetic manipulation platform is utilized, different sites are mutated on the basis of a Newcastle disease virus MG7 strain, a gene VII type Newcastle disease virus marker vaccine attenuated strain rMG7-L-V1291I which is low in pathogenicity, good in growth characteristic, high in virus titer, stable in passage and excellent in immunogenicity is rescued, and the marker vaccine strain is good in immunogenicity, high in immunogenicity, high in specificity, high in immunogenicity and capable of being used as a marker vaccine for the Newcastle disease virus. The vaccine can induce a high-level protective antibody, completely protects immunized chickens, can be used for preventing and controlling the current epidemic gene VII type Newcastle disease virus, and lays a foundation for identifying vaccine immunity and wild virus infection.

The application provides a proliferation culture medium for long-term stable passage of bovine cells, a method for promoting long-term stable passage of bovine cells and application. The components of the proliferation culture medium include complete culture medium, cycloastragenol and metformin hydrochloride. Research shows that the proliferation culture medium can make bovine fetal fibroblasts (BFFs) break through the proliferation limit in the process of in-vitro passage culture, form a continuous cell line, and be beneficial to the production of cell culture meat products of excellent quality. The culture medium provided by the application can support the continuous passage of BFFs to 145 generations, and the cell type of the bovine fetal fibroblast cell line passed to 145 generations is unchanged, having the advantage of long-term stable passage. The induction by the proliferation culture medium can make BFFs break through the aging limit, realize long-term stable passage (≥100 generations) and maintain a positive rate of >90% of vimentin, meet the demand of cell culture meat mass production for the number of cells, and have very broad application prospects.

This invention provides a *Chitozobacterium mangroveense* FCG-7 strain and its application in the preparation of N-acetylglucosamine, belonging to the field of microbial technology. The *Chitozobacterium mangroveense* FCG-7 strain provided by this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 63488. This strain exhibits good low-temperature resistance and the ability to produce extracellular chitinase, enabling it to efficiently degrade chitin under low-temperature conditions to produce N-acetylglucosamine with excellent biological effects. Furthermore, this strain remains stable after subculturing and holds promise for application in the production of high-value-added N-acetylglucosamine from chitin-based biomass under low-temperature conditions.

The application discloses screening of aspergillus oryzae with high yield of mycelial protein and application thereof, and belongs to the technical field of fermentation engineering. The aspergillus oryzae strain WL03 with high spore production capacity, good adaptability, stable subculture, and high mycelial concentration and mycelial protein content is screened, the culture medium for aspergillus oryzae fermentation is optimized, and the scale-up culture is further carried out in a 5L fermenter and a 20L fermenter. The strain of the application achieves the effects of 21g / L mycelial concentration and 46% mycelial protein content in the 5L fermenter, and achieves the effects of 20g / L mycelial concentration and 46% mycelial protein content in the 20L fermenter, and has the industrial application potential.

The invention relates to the technical field of metabolic engineering, and discloses a by-product eliminated gibberellin high-yielding strain as well as a construction method and application thereof. In gibberella zeylanica, a bicarpin gene cluster and a fusarium gene cluster are relatively large, the size of the bicarpin gene cluster is about 17 kb, and the size of the fusarium gene cluster is about 16.8 kb, so that the difficulty of knocking out the bicarpin gene cluster and the fusarium gene cluster at the same time is relatively large. According to the present invention, with the CRISPR-Cas9 gene editing technology, the simultaneous knockout of the bicarpin gene cluster and the fusarium gene cluster is achieved, such that the influence of the fermentation by-product bicarpin and fusarium is eliminated, the gibberellin synthesis is increased, and the high-yield gibberellin gene engineering strain is obtained.

The invention provides a Guangxi Bama miniature pig fetus kidney fibroblast subculture method, and belongs to the technical field of cell subculture, and the method comprises the following steps: step 1, obtaining a cell material, step 2, carrying out cell subculture, step 3, carrying out cryopreservation and recovery, step 4, drawing a cell growth curve, and step 5, identifying cell types. According to the method, a mechanical method and an enzyme digestion method are combined, primary kidney fibroblasts of Guangxi Bama miniature pig fetuses are separated and cultured, and a primary cell line capable of being subjected to conventional subculture, freezing and resuscitation is obtained; the proliferation potential is detected through a CCK8 method, cell identification is carried out through immunofluorescence, and the result shows that newborn Guangxi Bama miniature pig kidney fibroblasts are successfully separated and can be subjected to mass passage and freezing in vitro, and the newborn Guangxi Bama miniature pig kidney fibroblasts are already subjected to passage to 20 generations or above at present and grow well; after freezing recovery, normal culture and passage can still be realized.

The invention belongs to the technical field of organoid culture, and particularly relates to a passage method of a human induced pluripotent stem cell-derived small intestine organoid and application of the passage method. According to the passage method disclosed by the invention, after matrigel is mildly removed by a dissociation reagent, long-range stable passage of the small intestine organoid can be maintained by a method for slightly breaking the small intestine organoid through a suction head, and the structure and cell type diversity of the small intestine organoid can be maintained. Meanwhile, enteroviruses are infected into the small intestine organoid obtained by adopting the passage method, and the enteroviruses can be effectively copied in the small intestine organoid and infect one or more cells including intestinal epithelial cells, proliferative cells, panning cells and intestinal endocrine cells; therefore, the method can be used for related research of enterovirus infection and host interaction.

The invention provides a percocypris pingi kidney cell line and a construction method thereof, and belongs to the technical field of aquaculture. Wild percocypris pingi is subjected to artificial temporary rearing, kidney tissue is collected, disinfected and crushed into tissue blocks, primary culture and subculture are carried out in an L-15 or DMEM / F12 culture medium containing penicillin, streptomycin, amphotericin B and gentamicin and having the osmotic pressure of 260-300 mOsm / L, and the kidney cell line of the percocypris pingi is obtained. The percocypris pingi kidney cell line obtained by the construction method disclosed by the invention is in a fibroblast shape, can be continuously passed and directly applied to biological characteristic research, and can meet the requirements of germplasm resource preservation and theoretical research of a rare species of the percocypris pingi; and a foundation is laid for research on environmental adaptability of the cell level of the percocypris pingi.

Provided are: a method for producing retinal pigment epithelial cells, which improves the efficiency of differentiation induction in which pluripotent stem cells are differentiated into retinal pigment epithelial cells, and which can provide high-purity retinal pigment epithelial cells in a short period of time by means of a simple and convenient operation; the present invention relates to a method for culturing retinal pigment epithelial cells which can stably grow and culture cells, a method for evaluating toxicity / drug efficacy using retinal pigment epithelial cells useful in transplantation therapy, and a therapeutic drug for retinal diseases. The present invention relates to a method for producing retinal pigment epithelial cells comprising a step for adherent culture of human pluripotent stem cells by using a coated laminin-E8 fragment, a method for culturing retinal pigment epithelial cells comprising a step for adherent culture of retinal pigment epithelial cells by using a coated laminin-E8 fragment, and a method for culturing retinal pigment epithelial cells comprising a step for adherent culture of human pluripotent stem cells by using a coated laminin-E8 fragment. A method for evaluating the toxicity or efficacy of the retinal pigment epithelial cells produced or cultured by the method; and a therapeutic agent for retinal diseases comprising retinal pigment epithelial cells.

The application provides a new goat endemic intranasal adenocarcinoma cell ENA-1. The cell strain is preserved in China Center for Type Culture Collection (Wuhan), and the preservation number is CCTCC NO: C2022232. It is identified that the ENA-1 cell strain can grow rapidly in vitro, can be stably and continuously passed, retains the immunohistochemical characteristics of adenocarcinoma, can form a tumor after being inoculated subcutaneously in an immunodeficient nude mouse, and can form a tumor after being inoculated in a healthy goat for 8 months. The ENA-1 can be used as a cell material for the in-vitro treatment research of the goat endemic intranasal adenocarcinoma, can be used for constructing an animal model of the goat endemic intranasal adenocarcinoma, and has important significance for the research of the goat endemic intranasal adenocarcinoma.