102 results about "Culture viruses" patented technology

The invention discloses a porcine pseudorabies virus strain as well as an inactivated vaccine and applications thereof, belonging to the field of separation and application of the porcine pseudorabies virus strain. The invention firstly provides a porcine pseudorabies virus BJ strain separated from diseased pig tissues, and the microbial preservation number of the porcine pseudorabies virus BJ strain is CGMCC (China General Microbiological Culture Collection Center) No.7351. The invention discloses a method for preparing the inactivated vaccine by applying the porcine pseudorabies virus BJ strain. The method comprises the steps of culturing a virus strain to obtain a virus solution; adding an inactivator, and inactivating and concentrating the virus solution; and evenly mixing an adjuvant and the virus solution, and emulsifying to obtain the inactivated vaccine. The technological parameters of the inactivated vaccine preparation method are further optimized, and the immune protection efficacy and safety of the inactivated vaccine can be improved. Shown by the immune protection efficacy and safety tests, the porcine pseudorabies inactivated vaccine prepared has good immune protection efficacy and safety, and can be clinically used for preventing or treating porcine pseudorabies.

Influenza viruses for use in preparing human vaccines have traditionally been grown on embryonated hen eggs, although more modern techniques grow the virus in mammalian cell culture e.g. on Vero, MDCK or PER.C6 cell lines. The inventor has realised that the conditions used for influenza virus 5 culture can increase the risk that pathogens other than influenza virus may grow in the cell lines and have identified specific contamination risks. Suitable tests can thus be performed during manufacture in order to ensure safety and avoid iatrogenic infections.

A preparation method of porcine reproductive and respiratory syndrome vaccines by utilizing a bioreactor utilizes a stream perfusion type bioreactor as a cultivation tool. The preparation method comprises the steps of a cultivating cells for preparing the vaccines; b vaccinating and cultivating viruses; c harvesting virus liquor; d preparing the vaccines and the like. The preparation method has the advantages that the preparation method is simple and convenient in operation, low in contamination probability and capable of remarkably reducing preparation cost, the vaccines are large in yield, uniform and stable in quality and good in immune effect, and the like. The whole preparation process does not involve other problems of biological safety and public hygiene so that the preparation method is suitable for large-scale preparation of the porcine reproductive and respiratory syndrome vaccines.

The invention relates to a method for preparing a vaccine by breeding infectious bursal disease virus (IBDV) by a chicken embryo source cell line. The method mainly comprises the following steps of: 1) subculturing cells DF-1 for preparing vaccines; 2) breeding IBDV HQ cell seeds; 3) breeding virus liquid for preparing the vaccines; 4) concentrating and inactivating the virus liquid for preparingthe vaccine; 5) preparing other virus liquid of newcastle disease method, newcastle disease-infectious bronchitis virus method, and newcastle disease-infectious bronchitis virus-egg drop syndrome method combined vaccine and concentrating; and 6) proportioning inactivated combined vaccine, emulsifying and sub-packaging. The production process is simple, and stable and is easy to operate, eliminates biological potential safety hazard existing in the conventional vaccine production, and overcomes the defects that large-scale production of the vaccines is limited by supply of chicken embryos; cost and batch-to-batch variation are reduced; the virus titer and the quality of vaccine are improved; basis is laid for culturing virus liquid on large scale by a suspension culture technology in vaccine industry; and the produced IBDV inactivated vaccine and combined vaccine have high safety and immune efficacy, and have the complete immune protection effect on IBDV attack.

The safe high-efficiency continual closed cell culture virus generation/deactivation system comprises: a cell amplification unit connected to a gas supplying unit, a steam generator, a supplying device for cell growth medium, a primary fill and collection device, a heat source supply device, and a acid/alkali liquid supply device; a virus amplification unit connected to a gas supplying unit, a basic medium supply device, a cell/carrier separator, a heat source supply device, and a acid/alkali liquid supply device; and a old carrier collector. This invention integrates cell and virus amplification processes for automation control and standard production with little pollution.

The invention discloses a production method of a transmissible gastroenteritis virus vaccine. The production method comprises the steps of carrying out subculture on cells for production so as to form monolayer cells; multiplying virus seeds for production, namely inoculating the basic virus seeds to the monolayer cells for culturing; dissociating the monolayer cells into a monolayer cell suspension liquid, and inoculating the cell suspension liquid into a bioreactor for culturing; multiplying vaccine culturing virus liquid, namely inoculating the virus seeds for production to the cells to be cultured after the quantity of the cells reaches 5*106-5*107 unit/ml; and harvesting the virus liquid. The production method has the advantages that the production cost can be greatly lowered, the production cycles are short, each production cycle only lasts for 5-7 days, and compared with that of transmissible gastroenteritis virus generated by an existing spinner bottle culture method, the titer of the transmissible gastroenteritis virus produced by the method is higher; the automation degree is high, few workers are required, the production process is simple and stable, the operation is easy, the yield is high, the occupied area is small, the production scale can be easily and rapidly expanded, and the quality is balanced and stable basically; the environmental pollution is slight and is easy to avoid.

The invention belongs to the technical field of jonquil tissue culture, and especially relates to a tissue culture rapid propagation method for jonquil. The method comprises the following steps: preparing mediums, wherein the mediums comprise a basic medium and mediums in all tissue culture stages, the compositions of the basic medium comprise MS, cane sugar with a concentration of 8% and 0.7% of agar, the pH value of the basic medium is 5.6-5.8, the compositions of an induction callus medium comprise MS + 1.0 mg/L of 6-BA + 0.2 mg/L of NAA or MS + 1.0 mg/L of 6-BA + 0.1 mg/L of NAA, the compositions of a propagation medium comprise MS + 1.0 mg/L of 6-BA + 0.1 mg/L of NAA, and the compositions of a rooting medium comprise MS + 0.2 mg/L of NAA; culturing virus-free tissue culture seedlings; selecting explants and disinfecting, specifically choosing jonquil leaves as explants, washing with alcohol with a concentration of 75% for half a minute, and then performing disinfection processing for 10 min by using HgCl with a concentration of 0.1%; and transplanting the tissue culture seedlings. The method is high in propagation coefficient, short in period and low in cost.

The invention discloses a method for replicating and proliferating avian influenza viruses in the passage cells which are absorbed and grow in the microcarrier of a bioreactor. The invention is based on the method which uses cells as carrier to proliferate viruses in the bioreactor. The obtained avian influenza viruses and the avian influenza virus vaccine product have uniform quality and stable toxicity. The automatic control of vaccine production can be realized, the problem of large-scale production and application can be solved, the production matrix of the influenza vaccine can be completely changed, the technology that chick embryo is adopted to culture viruses can be changed, the problems caused by chick embryo such as adventitious agent pollution and viral pollution and the interferences caused by heterologous proteins such as chick embryo can be reduced, the purity, safety and immune effect of vaccine can be increased and the method can play an important role in the prevention of avian influenza.

The invention belongs to the field of biological products, in particular to a process for preparing rabies vaccine by culturing human diploid cells WI-38 and MRC-5 (called as human diploid cells as follows) as toxigenic cells through a Celligen310 bioreactor and taking a rabies virus PM strain as a virus seed, wherein the finished vaccine is prepared by comprising the steps of resuscitating human diploid cells, culturing and proliferating the human diploid cells, inoculating the virus seed of the rabies virus PM strain onto the human diploid cells, domesticating, inoculating and culturing virus seeds, collecting virus filtrate, inactivating, purifying, concentrating, adding a protective solution and the like. Verification indexes of the vaccine meet standards of Chinese Pharmacopoeia of 2010 version. The process is characterized in that the human diploid cells are cultured and prepared as matrix cells by using the Celligen310 bioreactor; therefore, exogenous pollution factors and tumorigenicity of animal passage cells can be avoided. As the inoculated virus strain is the rabies virus PM strain in the preparation process of the vaccine, the immune effect is better than that of the traditional vaccine. The vaccine prepared by using the process has the advantages of being high in purity, good in immune effect and high in safety.

The invention discloses an H9N2 subtype avian influenza virus strain as well as an inactivated vaccine and application thereof, and belongs to the field of separation and application of avian influenza virus strains. The H9N2 subtype avian influenza virus strain J/ZY strain is separated from a suspected H9N2 avian influenza pathogenetic chicken flock, and the microbial collection number of the strain is CGMCC No. 8853. The invention further discloses a preparation method for the H9N2 subtype avian influenza inactivated vaccine, and the preparation method comprises the following steps: culturing viruses to obtain a virus supernate; adding an inactivating agent to inactivate the virus supernate; mixing adjuvants and the virus supernate uniformly, and emulsifying to obtain the inactivated vaccine. Immune protection experiments prove that the separated H9N2 subtype avian influenza virus strain J/ZY strain serving as a vaccine strain has good immunogenicity and a relatively high antibody level can be produced after an animal is immunized by the strain.

The invention relates to a novel process for preparing influenza virus split vaccine, comprising the following steps of: (1) inoculating and culturing viruses: inoculating working seed lot viruses diluted to virus amount of 2.0-0.6LgEID50/ml in chick embryo allantois, and culturing at 33-35 DEG C for 48-72h; (2), harvesting and inactivating viruses and concentrating a virus harvest liquid; (3) purifying and splitting viruses: adding TritonX-100 with volume ratio of final concentration of 0.3-0.5% and deoxysodium cholate in a purified virus liquid, mixing evenly and placing at 20-30 DEG C for 90-120min; (4) adding a PB split agent; and (5) sterilizing, filtering and then preparing a semi-finished product: diluting each stock solution to hemagglutinin content of 30micrograms/strain/ml by 0.01mol/L of PBS (Phosphate Buffered Saline) with a pH value of 7.2, and filtering by a filter membrane with aperture of 0.22micron to obtain a semi-finished product. The novel process for preparing influenza virus split vaccine has the beneficial effects that: due to the increase of a novel purification method, high yield can be achieved, effective antigen content can be improved and the contents of residual ovalbumin and split agent causing inoculation reaction can be greatly reduced.

The invention relates to a PK15 cell acclimation suspension method and a two-stage virus production process. According to the PK15 cell acclimation suspension method, PK15 cells are acclimated to adapt to serum-free suspension culture and used as host cells for producing viruses which can have cytopathic effect or sensitivity on suspension PK15 cells by two-stage culture virus inoculation; when the cells grow to 2.0-20.0*10<6> cells/mL, diluting with a production culture medium by 1-5 times is performed to reach a density range of 1.0-10.0*10<6> cells/mL, and then virus inoculation is carriedout or virus inoculation is performed before dilution. The suspension PK15 cells are adaptive to suspension growth in the serum-free chemical-defined culture medium, and the two-stage virus productionprocess is simple and easy in amplification; liquid changing is avoided, and high culture medium utilization efficiency is achieved; production and growth culture media can be identical or not and can be mixed proportionally to realize cell secondary growth and viral expression promotion.

The invention relates to a method for preserving tissue culture bulbs of fritillary viruses, which belongs to the technical field of plant virus preservation and comprises following steps: identifying pathogeny species of the viruses, preparing culture medium, culturing virus-free tissue culture bulbs, testing virus ELISA, vaccinating virus by friction, and preserving virus bulbs. The invention has the advantages that the tissue culture bulbs of the fritillary viruses are used to infect the purified viruses, and then the viruses of fritillary tissue culture bulbs are preserved, which overcomes the defects in traditional preservation, can prevent viruses from confounding and losing, is easy to extract and prepare infectious titer, provides virus with high purity for preparing antiviral breeding and antiviral serum, lowers preserving cost, increases preserving effects, and is easy to carry and communicate.

The invention discloses a preparation method for an avian influenza virus split vaccine. The preparation method comprises the following steps of constructing H7N9 avian influenza working seed lot virus seeds, inoculating and cultivating viruses, inactivating the viruses, clarifying and concentrating virus harvest liquid, purifying the viruses, splitting the viruses, inactivating the viruses again, removing sugar and a splitting agent, filtering bacteria, performing filtration and preparing semi-finished products. The H7N9 influenza vaccine prepared by the method is high in immunogenicity, wide in antigen spectrum, stable in quality, high in purity, high in safety, low in cost and suitable for large-scale production and has a large application value.

The invention provides a slow virus purification method. The slow virus purification method comprises the following steps: S1, culturing viruses; S2, carrying out purification pretreatment on the viruses; carrying out centrifugal separation on the slow viruses obtained through amplification, and collecting supernatant as a virus harvesting liquid; firstly carrying out primary filtration treatment on the virus harvesting liquid, carrying out secondary filtration treatment on the virus liquid obtained through filtration, collecting a virus filtrate, carrying out ultrafiltration concentration, then centrifuging, collecting the supernatant, then carrying out tertiary filtration treatment, and collecting a filtrate; and S3, carrying out membrane chromatography treatment on the collected virus purification pretreatment sample, then carrying out ultrafiltration treatment, and purifying by adopting molecular sieve chromatography, wherein matrix used by a chromatography membrane is a regenerated cellulose skeleton during the membrane chromatography. By adopting the technical scheme of the invention, virus recycling efficiency is improved on the basis of guaranteeing slow virus activity; and operation is simple, amplification is easy, the virus harvest liquid can be treated in a large scale, and the slow virus purification method provided by the invention has good repeatability and stability.

The invention discloses a method for large scale cultivation in a bioreactor, comprising the following steps: (1) making a recombinant H5N1 avian influenza virus adapt to MDCK cells; (2) preparing a seed lot by using the adapted virus strain as a reactor large-scale culture virus, wherein the seed lot virus generation is no less than F6, the hemagglutination titer is no less than 28, and EID50 is no less than 107.5; (3) culturing the MDCK cells by using polyester sheets as a carrier and the reactor as a basket fixed bed, and pouring the cells on every gram of the carrier, wherein the culture medium is newborn calf serum DMEM; (4) inoculating the virus on the cultured MDCK cells, washing the cells by using a virus diluent, conducting virus inoculation, after absorption, and replacing a cell maintenance media; (5) maintaining the MDCK cells and conducting virus propagation, stirring, and replacing the cell maintenance media; and (6) monitoring the hemagglutination titer and harvesting the virus, and harvesting the reactor to culture a volume virus liquid. According to the invention, the method is easy to operate and the operation is simple, the HA titer of the virus after adaption is higher than 2<7>, and the problem that the culture virus titer cannot satisfy the demand of produce vaccine in the existing culture of the recombinant H5 subtype avian influenza virus on the MDCK cells is solved.

The invention belongs to the field of biological products, and in particular relates to an inactivated vaccine for porcine epidemic diarrhea virus which is produced by ST cells and a preparation method of the inactivated vaccine. The method comprises four steps, namely amplification culture of the ST cells, virus inoculation, multiplying of vaccine producing virus liquid, and product preparation. The vaccine, which is produced by the method, is high in immunization level and short in immunological window phase; an immunity period is obviously prolonged; and total secretory antibody (total SIgA) in intestinal mucosa is remarkably improved.

The invention provides a comprehensive detoxification method of lily bulbs. The method concretely comprises the following steps: 1, picking off explant growing points; 2, carrying out heat treatment detoxification; 3, cleaning and disinfecting explants; 4, and carrying out collection and induction culture on the growing pints; 5, carrying out detoxifying culture; and 6, detecting viruses. The method solves disadvantages in the prior art, solves the disadvantages of individual methods through comprehensively utilizing heat treatment detoxification, meristem culture detoxification and callus detoxification methods, and also has the advantages of effective reduction of the virus cross infection change, high detoxification rate, high survival rate, and effective shortening of the bulb culture period.

The invention discloses a method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation. The method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation comprises the following steps: 1) selecting an implant and carrying out pre-treatment; 2) peeling stem tips; 3) carrying out induction culture on the stem tips; 4) carrying out multiplication culture; 5) carrying out rooting culture; 6) culturing virus-free seedlings; and 7) detecting viruses and bacteria. The method for carrying out virus removal on prunus salicina during tissue culture and rapid propagation has the advantages that a stem tip virus removal method, a high-temperature treatment virus removal method and a chemical reagent virus removal method are combined, a good virus removal effect in tissue culture and rapid propagation of prunus salicina seedling culture is realized, the obtained virus-free seedlings are subjected to virus detection, and results show that viruses of all the seedlings are successfully removed, and no prune dwarf virus (PDV) or prunus necrotic ringspot virus (PNRSV) infection is found. By utilizing the method disclosed by the invention for carrying out virus removal on prunus salicina, new virus-free prunus salicina seedlings are cultured through tissue culture, and then domestication and transplantation are carried out, so that the aims of increasing the yield and improve the fruit flavour are realized.

The invention discloses a large-scale culture method of an H9N2 subtype avian influenza virus. According to the large-scale culture method, the H9N2 virus is rapidly adapted to MDCK, the HA titer of the virus is 28, MDCK cells are cultured as seed cells by taking a 7.5L reactor as a seed cell tank and cytodex1 as a carrier, the seed cells are amplified to large-scale culture in a 40L fixed bed basket type stirring system reactor, and 80L of H9N2 viruses of which the hemagglutination titer (HA) is up to 210 can be obtained. The virus produced by using the method is high in titer and large in yield, the method is easy in standardization and is applicable to all H9 subtype influenza viruses, and the difficulty that the culture virus titer cannot meet vaccine production when the H9N2 avian influenza virus is cultured on the MDCK cells is solved.