56results about How to "Efficient Proliferation" patented technology

The invention provides a coxsackievirus A16-type virus strain and a use thereof. The coxsackievirus A16-type virus strain has the preservation number of CGMCC No. 5371. The full-length sequence analysis and the mass spectrometry analysis on a VP1 protein produced by the coxsackievirus A16-type virus strain prove that the oxsackievirus A16-type virus strain is a good CA16 virus strain which is not polluted by allothigenes and has good immunogenicity. The CA16 virus strain can efficiently proliferate in Vero cells and has virus titer of 7.41g CCID 50 / ml. The CA16 virus strain or a vaccine prepared from the CA16 virus strain can be used for preventing diseases caused by CA16 viruses, and has the characteristics of stable titer, good immunogenicity and less immunizing dose.

The invention discloses a porcine epidemic diarrhea virus attenuated vaccine strain. The porcine epidemic diarrhea virus attenuated vaccine strain is an attenuated vaccine obtained by PEDV / CH / NB / 2014 passage attenuation and has an accession number of CGMCC No. 13299. The invention also discloses a use of the attenuated vaccine strain NB120 in prevention of porcine epidemic diarrhea diseases and the use belongs to the field of biotechnology. The attenuated vaccine strain NB120 can be processed to form a single vaccine or a multi-vaccine and the vaccine can effectively prevent porcine epidemic diarrhea diseases. The attenuated vaccine strain NB120 has the advantages of good safety and good immunogenicity. After applying to a sow or piglet, the vaccine strain can produce protective immune response to a prevalent strain of porcine epidemic diarrhea virus and thus the vaccine strain can effectively prevent infection caused by the prevalent strain of porcine epidemic diarrhea virus, can reduce a disease loss and has a good application prospect.

The invention belongs to the field of biomedical detection, and in particular relates to a culture method of tumor specific TIL cells. According to the culture method of the tumor specific TIL cells disclosed by the invention, the tumor specific TIL cells can be acquired by screening specific magnetic beads twice. The method disclosed by the invention is simple in operation, and is capable of efficiently obtaining the tumor specific TIL cells through multiplication culture and greatly enhancing the tumor-cytotoxicity effect of the TIL cells; and meanwhile, through serum-free culture, the method can solve the potential risks of transmission of viruses and other dangers in the prior art, so as to provide a foundation for extensive application in the future.

The invention provides a culturing method for ammonia oxidizing bacteria. The culturing method comprises the following steps: inoculating ammonia oxidizing bacteria liquid into a fermentation tank; carrying out fermentation culture for 5-7 days; collecting fermentation liquid, so as to obtain the ammonia oxidizing bacteria, wherein the inoculation amount is 5%; the culturing conditions refer to that a pH (Potential of Hydrogen) value is controlled to be 7.5-8.0 by adding a base liquid, a temperature is 25-30 DEG C, a ventilation amount is 1.5-2.0M<3> / h, a tank pressure is 0.05-0.1MPa, and a rotation speed is 180-200r / min; a culture medium is a nitrobacteria culture medium. The culturing method uses a fermentation tank yeast propagation method, so that infectious microbes are difficult to pollute; various parameters, such as the temperature, the pH value, dissolved oxygen and a stirring rotation speed and the like, can be stably controlled; compared with the traditional opened type yeast propagation method, the culturing method for the ammonia oxidizing bacteria can be used for more economically and effectively preparing an ammonia oxidizing bacteria agent, so that the agent can be rapidly and effectively propagated; the culturing method for the ammonia oxidizing bacteria is suitable for large-scale industrial production and has a wide application prospect in a sewage treatment field.

The invention provides a serum substitution combination for immune killer cell amplification in vitro. Insulin with the final concentration being 0.5-20 mg / L, transferrin with the final concentration being 0.69-22 mg / L, bovine serum albumin with the final concentration being 3000-6000 mg / L, ethanolamine with the final concentration being 0.61-2.44 mg / L, alpha-thioglycerol with the final concentration being 5.41-10.82 mg / L, linolenic acid with the final concentration being 0.5-5.0 mg / L and cholesterol with the final concentration being 0.8-20 mg / L are added into a conventional culture medium. In the serum substitution combination, ferric ammonium citrate with the concentration being 0.46-2.3 mg / L, beta-disodium glycerophosphate with the concentration being 300-1500 mg / L and putrescine with the concentration being 0.25-1.25 mg / L can be added. The components are definite, can be added into different culture media such as an RPMI1640 culture medium, a DMEM / F12 culture medium and an IMDM culture medium, can support immune killer cell amplification in vitro, and are efficient and universal. In vitro serum-free amplification of immune killer cells can be achieved, the biological therapy of tumors is promoted, and conduction of the somatic cell therapy is promoted particularly.

The invention provides an adapting method of a rabies virus (RV) CTN-1 strain to a primary chicken embryo fibroblast. The adapting method comprises the following steps of carrying out 10 continuous passages on RV CTN-1V5 in a vero cell to obtain a CTN-1V15 strain; carrying out one passage on a virus seed of the CTN-1V15 strain in a chicken embryo to obtain a first-generation virus of the RV CTN chicken embryo; carrying out passage on the first-generation virus of the RV CTN chicken embryo in the chicken embryo fibroblast to enable the first-generation virus of the RV CTN chicken embryo to gradually adapt to the chicken embryo fibroblast. The invention provides the adapting method of the RV CTN-1 strain to CEC (chicken embryo cardiomyocytes); by using the method, the CTN strain can be rapidly and efficiently multiplied in the CEC; moreover, the obtained RV CTN chicken embryo cell adapting strain can be stably multiplied on the CEC, and has favorable stability and immune protection. The invention also provides an inactivated vaccine prepared by using the RV CTN chicken embryo cell adapting strain, and the inactivated vaccine has favorable immune protection and can be used for producing refined and purified RV for people.

The present invention belongs to the technical field of health-care products and particularly relates to a probiotic nutritional preparation suitable for astronauts. The probiotic nutritional preparation comprises 15%-20% of bifidobacterium lactis, 8%-12% of lactobacillus plantarum, 10%-15% of lactobacillus casei, 9%-16% of lactobacillus rhamnosus, 25%-30% of fructooligosaccharide and 20%-25% of xylooligosaccharide; the bifidobacterium lactis, lactobacillus plantarum, lactobacillus casei and lactobacillus rhamnosus are bacterium strain freeze-dried powder. The product aims at special needs ofthe astronauts and properly matches bacterium flora and prebiotics, during the preparation process of the freeze-dried powder, a self-made protective agent is added to improve activity of the variousmicroorganisms, so that the final product has high activity, can directly reach intestinal tracts and is good in effects, and the probiotics match the prebiotics and are efficient in reproduction.

The invention provides a microorganism expanding culture device for water environment treatment. The microorganism expanding culture device comprises an equipment shell, an automatic quantitative microbial agent adding system, a raw water quality filtering and disinfecting system, a clear water supply system, a microbial agent constant-temperature culture system and an automatic control system, the microbial agent automatic quantitative adding system, the raw water quality filtering and disinfecting system and the clear water supply system are all connected with the microbial agent constant-temperature culture system through pipelines; the automatic control system is provided with an operation control program and operation requirements in advance. According to the invention, the effective flora to be supplemented and added into the water body is subjected to enlarged culture by controlling key environmental factors such as temperature, dissolved oxygen and the like which influence microbial proliferation, so that under the condition of the same dosage of the microbial agent, the number and the biological activity of the effective flora added into the water body are increased, and the treatment effect is improved.

The invention discloses a lamb testis support cell immortalized cell line and an establishment method and application thereof. The lamb testis support cell immortalized cell line is named a lamb testis support cell immortalized cell line hTERT-LSC with a collection number CCTCC NO: C2018202. Tests find that the hTERT-LSC cell line can realize continuous passage for more than 60 generations, and the growth and proliferation characteristics are not changed. The cell line is relatively sensitive to inoculation of the sheep pox virus, can efficiently proliferate the sheep pox virus, and can ensurethe uniformity and stability of the virus. The titer of the sheep pox virus proliferated by the cell line is equivalent to that of the lamb testis support cell LSC, and the cell division rate is higher than that of the LSC and can reach 1:3. Furthermore, by utilizing the immortalized cell line for preparing a vaccine, the production process is simplified, the production cycle is shortened, the production cost is reduced, and simultaneously, the quality stability of a prepared sheep pox virus vaccine is also ensured. Therefore, the invention provides a new technical means for large-scale production of the sheep pox virus vaccine.

Disclosed are: a culture method for pancreatic precursor cells derived from pluripotent stem cells, said culture method comprising step (A) for three-dimensionally culturing pancreatic precursor cellsderived from pluripotent stem cells in a medium that contains a factor belonging to the epidermal growth factor (EGF) family and / or a factor belonging to the fibroblast growth factor (FGF) family, and (2) a Wnt agonist; a production method for islet cells from pancreatic precursor cells derived from pluripotent stem cells, said production method comprising step (E) for inducing the differentiation of the pancreatic precursor cells cultured by the aforesaid method into islet cells; and a cryopreservation method for pancreatic precursor cells derived from pluripotent stem cells, said cryopreservation method comprising step (F) for freezing the pancreatic precursor cells cultured by the aforesaid method.

The invention discloses a porcine epidemic diarrhea virus and its cultivation method and application. The virus strain is a porcine epidemic diarrhea virus of the family Coronaviridae, named as PEDV / CH / NB / 2014, and the preservation number is CGMCC No.12041. The virus was cultured using DMEM without trypsin and supplemented with 2% fetal calf serum as a maintenance solution. And stably proliferate efficiently in Vero cells, the virus titer can reach 108.0TCID50 / mL, no exogenous virus pollution, has strong pathogenicity to piglets, when the virus titer reaches 103TCID50, it can cause piglets to become ill, which is the current domestic The popular variant virulent strain is used for preparing PEDV diagnostic reagents and vaccines, has good immunogenicity, and can make up for the shortage of existing vaccine types.

The invention relates to a method for proliferating avian influenza virus by adopting an MDCK cell line. The method comprises the following steps: domesticating adherent MDCK cells into a serum-free full-suspension culture type MDCK cell line which is stably proliferated, inoculating a bioreactor with the MDCK cell line, completing primary cell culture, preparing avian influenza H5N1 subtype virusseed viruses for suspension MDCK cell culture, inoculating suspension MDCK cells with the avian influenza H5N1 subtype virus seed viruses, performing culturing, and separating and purifying virus particles. The cells obtained by domestication are stable in passage, good in cell morphology, high in quality, capable of keeping sensitivity and biological characteristics to viruses, high in virus multiplication capacity and capable of effectively multiplying avian influenza virus strains.

A Scophthalmus maximus rahbdovirus (SMRV0207, CCTCC No.202006) is prepared through sampling the liver, kidney, heart and spleen tissues from the scophthalmus maximus in ill, shearing, adding PBS, homogenizing, adding double antibody, freezing, thawing, centrifugal extracting, filtering, inoculating it to CLC 0207 cell, and constant-temp. continuous culture. Its advantages are high reproduction speed, and high reproduction valency up to 10 to the power 8 TCID 50 / ml. It can be used to prepare vaccine.

The invention discloses a marine-derived vibrio bacteriophage, a microecological preparation and a preparation method and an application of the marine-derived vibrio bacteriophage, the marine-derived vibrio bacteriophage is preserved in China Center for Type Culture Collection, the preservation time is August 6, 2020, and the preservation number is CCTCC NO: M2020397. The vibrio bacteriophage has the potential of infecting and efficiently cracking pathogenic vibrios, the efficiency of cracking vibrio alginolyticus can reach 80-90%, and the bacteriophage can be used for preparing a biological microecological preparation to be applied to the related fields of prevention and control of pathogenic vibrios in aquatic water and the like.

The invention provides a cell culture medium, a preparation method and application, and belongs to the technical field of cell culture. The cell culture medium is prepared from a basic culture medium,cytokines, hormones, proteins, biogenic amines, an inhibitor and solution declining elements, wherein the cytokines, the hormones, the proteins, the biogenic amines, the inhibitor and the solution declining elements are added into the basic culture medium. The basic culture medium can effectively maintain the stability of the cell environment while maintaining the activity of the cells. By addingthe cytokines, the hormones, the proteins, the biogenic amines, the inhibitor and the solution declining elements into the basic culture medium, on the one hand, through the synergy between the components, animal-derived components in a traditional culture medium can be replaced, the stability of the cell culture medium can be ensured, and the risks of disease transmission and immune response arereduced; and on the other hand, the components cooperate with one another other, the cell activity is maintained, cells can proliferate steadily and rapidly, the primary separation efficiency of thecells is greatly improved, and the self-renewal and tissue repair capabilities of the cells are improved.

The invention relates to an efficient proliferation method and application of an H5 subtype avian influenza virus, and mainly relates to improvement of a virus seed diluent. According to the efficientproliferation method and application of the H5 subtype avian influenza virus, a phosphate buffer solution is used as a basis, and aspartic acid, ZnCl2 and an epidermal growth factor are added, so that efficient proliferation of the H5 subtype avian influenza virus in SPF chicken embryos and non-immune chicken embryos is realized; in a vaccine preparation process, an antigen meeting a production standard can be obtained only by centrifuging and filtering treatment, and the virus content and HA titer which are the same as those of an antigen solution concentrated by a traditional production process can also be achieved; and the level of an immunized HI antibody has no significant difference from that of an HI antibody obtained by the traditional process. Compared with the traditional process, the efficient proliferation method has the advantages that concentration treatment is not needed, so that the production cost, particularly the cost of concentration equipment and a concentration process, is reduced, the production efficiency is improved, and the pollution risk in product production can be reduced.

The purpose of the present invention is to provide a cell culture medium, particularly a serum-free cell culture medium, that makes it possible to raise cell growth efficiency without using feeder cells. The present invention provides a serum-free cell culture medium containing growth arrest-specific 6 (GAS6).

By improving the seed virus diluent, adding aspartic acid, ZnCl2 and epidermal growth factors in a specific proportion on the basis of a phosphate buffer, so that the H7 subtype avian influenza virusin the low-immunity chick embryo can be efficiently proliferated. The level of virus proliferation and the immune effect of the prepared vaccine are basically close to those of SPF chick embryos and non-immune chick embryos, however, the production cost is effectively reduced, the production threshold is lowered, which has a positive significance in production of H7 subtype avian influenza vaccines.

The problem of the present invention is to provide a method for industrially and efficiently cultivating seaweed having alginic acid-producing capability and an industrially advantageous method for producing an alginic acid-containing composition. The method for cultivating seaweed relating to the present invention is characterized in comprising a step for heterotrophically cultivating seaweed having alginic acid-producing capability belonging to the genus Parachlorella. Moreover, the method for producing an alginic cid-containing composition relating to the present invention comprises a step for obtaining an alginic acid-containing composition from a seaweed cultivation product obtained by the cultivation method.

The Scophthalmus maximus rhabdovirus, SMV0207, with preservation number of CCTCC No. V202006, is prepared through taking liver, kidney, heart and spleen tissues of suffering Scophthalmus maximus, crushing, adding PBS for homogenation, adding amphitolerant, freezing, thawing, centrifugating to obtain coarse virus extracting liquid, filtering, inoculation to CLC0207 cell for further culture at constant temperature. The said process is simple and practical, and the virus strain may be amplified in CLC0207 cell fast in the amplification potency up to 108 TCID50 / ml. The virus strain may be used in preparing sea water fish rhabdovirus cell engineering vaccine.

The present invention pertains to: cloned MDCK cells which exhibit an expansion factor of 4.5-fold or more when being cultured using microcarriers; a method for culturing the MDCK cells; and a methodfor multiplying a virus by using the MDCK cell culturing method.

The invention provides a culture medium for flounder paralichthys lymphocyte proliferation. The culture medium is formed by adding concanavalin A, lipopolysaccharide, phytohemagglutinin, America pokeweed lectins, flounder paralichthys serum, fetal calf serum, mixed deoxygenation mononucleotide, L-glutamine and sodium bicarbonate into a basic culture medium. The culture medium contains the flounder paralichthys serum and various types of mitogen for promoting proliferation of lymphocyte, through effective matching of all ingredients, efficient and rapid proliferation of the lymphocyte can be achieved, the activity index of the lymphocyte is high, the average proliferation activity is as high as 85.33%, and meanwhile the culture medium has good stability and can completely meet the requirement of culturing lymphocyte of the fish molecular immunology and the cytogenetics.

The invention relates to a method for effectively preparing mesenchymal-like stem cells having anti-inflammatory efficacy and immunosuppression, the method including: inducing differentiation of the human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines to select cystic embryoid bodies; loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells; isolating only monolayer-shaped cell clusters from the cells passing through the cell-permeable 3D culture unit, and uniformizing the same. Further, the invention relates to mesenchymal-like stem cells prepared by the preparation method, a transporter or a therapeutic composition including the mesenchymal-like stem cells, and a therapeutic composition for prevention or treatment of diseases that includes an active ingredient or exosomes secreted from the mesenchymal-like stem cells.

The invention provides a culturing method for ammonia oxidizing bacteria. The culturing method comprises the following steps: inoculating ammonia oxidizing bacteria liquid into a fermentation tank; carrying out fermentation culture for 5-7 days; collecting fermentation liquid, so as to obtain the ammonia oxidizing bacteria, wherein the inoculation amount is 5%; the culturing conditions refer to that a pH (Potential of Hydrogen) value is controlled to be 7.5-8.0 by adding a base liquid, a temperature is 25-30 DEG C, a ventilation amount is 1.5-2.0M<3> / h, a tank pressure is 0.05-0.1MPa, and a rotation speed is 180-200r / min; a culture medium is a nitrobacteria culture medium. The culturing method uses a fermentation tank yeast propagation method, so that infectious microbes are difficult to pollute; various parameters, such as the temperature, the pH value, dissolved oxygen and a stirring rotation speed and the like, can be stably controlled; compared with the traditional opened type yeast propagation method, the culturing method for the ammonia oxidizing bacteria can be used for more economically and effectively preparing an ammonia oxidizing bacteria agent, so that the agent can be rapidly and effectively propagated; the culturing method for the ammonia oxidizing bacteria is suitable for large-scale industrial production and has a wide application prospect in a sewage treatment field.

The invention discloses an outdoor large-scale spat intermediate culture method. The method comprises the following steps that 1) an outdoor shellfish bed is constructed in a pond; 2) a water level height difference is gradually formed among a higher-place pond, an algae pond and the outdoor shellfish bed in the vertical direction, high-density shrimp culture is carried out in the higher-place pond, fish and shrimp mixed culture is carried out in the algae pond, and intermediate culture on spats is carried out in the outdoor shellfish bed; and 3) culture wastewater is purified: the culture wastewater in the higher-place pond is discharged into the algae pond for ectopic dilution and conversion firstly, the wastewater is changed into fertilizer water to promote the growth of beneficial unicellular algae, then the fertilizer water in the algae pond is introduced into the outdoor shellfish bed, the fertilizer water is changed into purified water through the filter feeding activity of bivalve shellfish, and meanwhile, the unicellular algae are used as bait organisms to promote the growth of the spats, so that the culture wastewater is purified in an ex-situ manner. According to the method, the breeding cost is greatly reduced, the productivity of intermediate culture of the spats is greatly improved, high economic benefits are achieved, the problem of treatment of culture wastewater can be effectively solved, and the method is suitable for popularization and application in areas suitable for breeding of bivalve spats such as clams and razor clams.

The present invention has successfully established a mixed culture system capable of actively proliferating a Kupffer cell in a primary culture of a cell population derived from a liver. Additionally, the present invention has successfully established a novel production method for efficiently producing a large amount of highly purified Kupffer cells using this mixed culture system.