606results about "Recovery/purification" patented technology

The invention belongs to the technical field of nano antibodies, and particularly relates to a nano antibody specifically bound with AAV9 and application of the nano antibody. The nano antibody provided by the invention can be specifically combined with the AAV9 adeno-associated virus without being combined with other AAV serotypes such as AAV2, AAV5 and AAV8, so that high-sensitivity and high-specificity detection of the AAV9 adeno-associated virus is realized. In practical application, the nano-antibody coupled solid-phase carrier provided by the invention is adopted as an affinity filler, the AAV9 adeno-associated virus can be effectively captured and mildly eluted, the virus purification and recovery effects are effectively improved, and the nano-antibody coupled solid-phase carrier is suitable for high-purity recovery of the AAV9 adeno-associated virus.

A mammalian or avian cell line that expresses high levels of human influenza virus receptors is provided. In one embodiment, the cell line supports human influenza virus, e.g., human A / H3 influenza virus, isolation and growth much more effectively than corresponding conventional (unmodified) cells or in corresponding human virus receptor-overexpressing cells, and the propagated viruses may maintain higher genetic stability than in the corresponding cells.

The invention belongs to the field of virus detection and diagnosis, and relates to an anti-AAV2 monoclonal antibody as well as a preparation method and application thereof. The invention provides an anti-AAV2 rabbit monoclonal antibody, and amino acid sequences of a heavy chain variable region and a light chain variable region of the anti-AAV2 rabbit monoclonal antibody. The anti-AAV2 rabbit monoclonal antibody provided by the invention can be specifically combined with the VLP of AAV2, and can be used for detecting the AAV virus antigen. The anti-AAV2 monoclonal antibody provided by the invention provides an effective detection tool for the detection of AAV2 in the research and development process of a gene therapy vector and the detection of AAV2 in basic scientific research.

Polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods for expressing AAV Rep and Cap proteins are provided. In certain aspects, these polynucleotides, cells, and methods may produce higher levels of one or both of Rep and Cap transcripts. In some aspects, these polynucleotides, cells, and methods may produce higher levels of one or both of Rep and Cap proteins. In various aspects, these polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods use one or more heterologous polyA sequences with one or both of the Rep and Cap coding sequences. In numerous aspects, these polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods may use one or more enhancers to increase expression of Rep proteins, where such enhancers can be transcriptional enhancers, translational enhancers, or combinations thereof. In certain aspects, these polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods may be used to produce recombinant AAV (rAAV).

The invention provides an engineered nuclease with high salt tolerance. The polypeptide comprises one or more mutations, so that the three-dimensional structure of the polypeptide has more surface areas with positive charges. Compared with the nuclease with the SEQ ID NO: 1 sequence, the polypeptide still has at least 60% nuclease activity under the condition that the solution ion strength exceeds 200 mM.

The invention discloses a serum-free full-suspension MDCK (Madin-Darby Canine Kidney) cell and application thereof in preparation of influenza vaccines, and belongs to the field of virology and biological medicines. The serum-free full-suspension MDCK cell is preserved in the China Center for Type Culture Collection, is classified and named as a canine kidney cell MDCK-JHS, and has a preservation number of CCTCC NO: C2024373. The invention further provides a method for culturing the influenza virus by using the cell line, high-titer amplification of the influenza virus can be realized, and the method is suitable for industrial production.

The invention belongs to the technical field of nano antibodies, and particularly relates to a nano antibody specifically bound with AAV2 and application of the nano antibody. The nano antibody provided by the invention can be specifically combined with the AAV2 adeno-associated virus without being combined with other AAV serotypes such as AAV5, AAV8 and AAV9, so that high-sensitivity and high-specificity detection of the AAV2 adeno-associated virus is realized. In practical application, the nano antibody coupled solid-phase carrier provided by the invention is adopted as an affinity filler, the AAV2 adeno-associated virus can be effectively captured and mildly eluted, the virus purification and recovery effects are effectively improved, and the nano antibody coupled solid-phase carrier is suitable for high-purity recovery of the AAV2 adeno-associated virus.

The invention belongs to the technical field of antibody preparation, and particularly relates to a nano antibody for resisting different serotypes of adeno-associated viruses and application of the nano antibody. The nano antibody is selected from one of Nb1 to Nb24; the amino acid sequences of the heavy chain variable regions of the nano antibodies Nb1 to Nb24 are respectively shown as SEQ ID NO. 1 to 24. The nano antibody provided by the invention can sensitively and specifically bind to AAV, has excellent broad-spectrum recognition and binding capacity to various serotypes such as AAV2, AAV5, AAV8 and AAV9, and has a good application prospect in the fields of AAV immunodetection, affinity purification and the like.

Currently, the steps performed prior to release of influenza strains to vaccine manufacturers involve passaging influenza virus through eggs. The invention aims to provide procedures useful in manufacturing influenza vaccines, in which the use of eggs is reduced, and preferably is avoided altogether. For instance, rather than use chicken eggs for influenza vaccine isolation, MDCK cells (Madin Darby canine kidney cells) may be used e.g. growing in suspension, growing in a serum-free medium, growing in a protein-free medium, being non-tumorigenic, grown in the absence of an overlay medium, etc.

The invention relates to a culture medium for high-density serum-free suspension culture of BHK-21 cells and application of the culture medium. The culture medium contains inorganic salt, amino acid, vitamins, energy substances, a buffer substance, an antioxidant, sterol, polyamine, an additive and an indicator, and the antioxidant comprises dihydrolipoic acid and S-acetyl-L-glutathione. According to the invention, the culture medium for serum-free suspension culture of the BHK-21 cells, which is clear in components and remarkable in culture effect, can be provided. The added dihydrolipoic acid and S-acetyl-L-glutathione are combined as antioxidants of the culture medium, so that the survival rate of BHK-21 cells cultured in the culture medium is improved while the oxidation resistance of the serum-free suspension culture medium is improved.

A Janus particle, comprising a particle core, the particle core comprising at least a first surface functionalized with a functional group and a second surface functionalized with a metal particle.

The present disclosure provides materials and methods related to the purification of viral vectors. In particular, the present disclosure provides peptides, compositions, adsorbents, and related methods, capable of removing process-related impurities (e.g., host cell proteins, nucleic acids, and media components) and product-related impurities (e.g., product fragments, product aggregates, and inactive forms derived from product degradation by or association with other species in the cell culture harvest) from samples during the production and purification of adenovirus.

Provided is a method for large-scale preparation of a purified preparation of a recombinant lentiviral vector at the GMP grade. The method comprises: (a) providing raw material feed liquid to be purified that comprises recombinant viral vectors; (b) carrying out a microfiltration treatment on the feed liquid to obtain a microfiltered filtrate comprising the recombinant viral vectors; (c) optionally concentrating the filtrate to obtain a concentrated filtrate; (d) purifying the filtrate obtained in the previous step by means of chromatography to obtain a crude pure product comprising the recombinant viral vectors; and (e) subjecting the crude pure product obtained in the previous step to liquid exchange and elaborate purification to obtain the purified recombinant viral vectors.

The present disclosure is directed to methods of improving the transduction efficiency of non-transducible cells, such as cells with low hLDLR. The cells may be labeled with 5'FAM and transduced with a scFv-truncated VSV-G pseudotyped lentivirus which comprises an anti-fluorescein scFv. Further provided are methods of gene therapy, cell engineering, genetic screening, and disease models using the fluorescein-labeled cells transduced with the scFv-truncated VSV-G pseudotyped lentivirus.

The invention belongs to the technical field of rabies vaccines, and particularly relates to a purification method of a rabies virus inactivated vaccine, which comprises the following steps: step 1, carrying out passage by using a Walvax-2 cell strain through a cell factory method, and carrying out multiplication culture on the passage production cells in a microcarrier bioreactor; 2, cleaning the cells with a treating fluid, inoculating the cells with a rabies virus fixed strain, and preparing and culturing a rabies virus solution; step 3, carrying out ultrafiltration concentration on the rabies virus stock solution; step 4, purifying the rabies virus concentrated solution; step 5, inactivating the rabies virus; and 6, hydrolyzing to obtain the rabies vaccine stock solution. By adopting the composite Capto Core 700 column chromatography, impure proteins can be effectively removed, the recovery rate is high, the quality of the rabies vaccine obtained by adopting a mode of firstly purifying and then adopting beta-propiolactone inactivation is excellent, and the effect of the finally obtained human diploid cell rabies vaccine stock solution is good.

The invention relates to an efficient enrichment method for low-concentration bacteriophages in river and lake water bodies, which remarkably improves the recovery rate and the concentration multiple of the bacteriophages through a multi-stage synergistic technology in combination with physical interception, chemical precipitation and biological targeted adsorption. The method specifically comprises the following steps: pretreatment: removing large particles and suspended solids through gradient filtration and low-speed centrifugation; ultrafiltration concentration: adopting a hollow fiber ultrafiltration membrane with the molecular weight cut-off of 30-100 kDa, and realizing 10-50 times concentration under the pressure of 0.1-0.3 MPa; chemical precipitation strengthening: optimizing synergistic precipitation conditions of PEG and NaCl, and reducing phage inactivation in combination with low-temperature standing; specific adsorption of host bacteria: combining the host bacteria in the logarithmic phase with bacteriophage according to the volume ratio of 1: 100, and carrying out pH regulation and control elution to obtain a high-purity enriched solution. The method has the advantages that the total concentration multiple reaches 500-1000 times, and the recovery rate is gt; the activity retention rate is gt; and the method is suitable for a complex water body environment. Through a multi-technology combination strategy and targeted adsorption design, an efficient tool is provided for environmental monitoring, phage therapy and microbiological research.

The invention discloses a tetravalent influenza virus subunit vaccine and a preparation method thereof, and relates to the technical field of biology, each dose of the tetravalent influenza virus subunit vaccine comprises two influenza A viruses and two influenza B viruses, the tetravalent influenza virus subunit vaccine is prepared by virus inoculation, virus multiplication culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, splitting and ultracentrifugation purification, mixing, filtration sterilization, split charging and packaging. Inactivation comprises the following steps: adding a cross-linked carboxymethyl glucan solution into the monovalent virus harvesting liquid, adding formaldehyde, and inactivating at 5 DEG C for 45 hours; then genipin is mixed into the virus liquid, the stirring speed is 200 rpm, the stirring time is 8 h under the conditions that the pH is 7.2 and the temperature is 25 DEG C, residues of free methanol can be further reduced, migration of small molecules is inhibited, and the long-term storage stability is guaranteed.

The invention provides a system and a method for clarifying a single virus harvest solution of Vero cells in real time. Belongs to the technical field of biological product production. The method comprises the following steps: assembling and checking the system, and sterilizing the system; controlling the liquid level and starting clarification; filter element selection and parallel arrangement are carried out, and pressure monitoring and filter element switching are carried out; cooling and storing the clarified virus harvesting liquid; online cleaning is conducted, and the cleaning effect is monitored; the clarification process is monitored and recorded in real time; and recording data according to the monitoring system. Through multi-point temperature monitoring and real-time regulation and control, the temperature uniformity in the production process is ensured, adverse effects such as quality reduction of the virus harvesting liquid caused by temperature fluctuation are avoided, and the production safety is improved.

The present invention relates to the field of biological detection technology, and in particular to a method for isolating a solanacearum phage and its application. The method is based on the co-culture of host bacteria and phage, and the addition of a resin azure indicator to detect the turbidity OD of the bacteria. 600 The advantages are: 1. Adding resin azure can cause redox reaction and color change during bacterial growth, making the detection of bacterial activity more sensitive and accurate for bacterial solution OD 600 The detection is also more sensitive; 2. Liquid culture medium can be used to separate Ralstonia solanacearum phages by monitoring bacterial turbidity OD 600 The bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance can be determined from two aspects: the bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance, thereby screening out high-quality phage combinations that are highly effective in killing bacteria and not prone to developing resistance; 3. This method can also be used for the screening of highly effective bactericidal phage groups and for antibacterial experiments involving the combination of phages with other drugs such as antibiotics.

Mammalian or avian cell lines that express human influenza virus receptors at high levels are provided. In one embodiment, the cell lines support the isolation and propagation of human influenza viruses, e.g., human A / H3 influenza viruses, much more efficiently than in corresponding conventional (unmodified) cells or corresponding human virus receptor-overexpressing cells, and the propagated viruses are able to maintain greater genetic stability than in the corresponding cells.

An oncolytic poxvirus encoding a truncated human CD19 is used in conjunction with a chimeric antigen receptor to treat solid tumors.

The invention relates to a preparation method of a Vero cell influenza virus vaccine, and belongs to the technical field of virus culture. In the culture of the influenza virus, red algae protein and soybean protein hydrolysate are added into a culture medium at the same time, the red algae protein and soybean protein hydrolysate are subjected to low-temperature ammonium sulfate precipitation and PD-10 desalination column purification, natural conformation is kept, integrin-FAK-Src and IGF-1R signal channels can be activated, and cell adhesion and proliferation are promoted; the latter contains soybean protein isolate, polysaccharide and RGD motif peptide, and can form an adhesion film on a substrate and mediate adhesion through integrin. According to the invention, the Vero cells and the recombinant plasmid are combined to improve cell adherence, metabolic homeostasis and oxidation resistance, so that the Vero cells grow at high density and efficiently replicate influenza viruses under a serum-free condition, the virus titer (TCID50) reaches 8.5 log10 / mL, and the Vero cells and the recombinant plasmid are suitable for industrial vaccine production.

The invention relates to the technical field of virus antigens, and particularly discloses an inactivation method for improving the recovery rate of enterovirus antigens. The method disclosed by the invention specifically comprises the following steps: adding a protective agent with the final concentration of 50-250 [mu] g / ml into purified enterovirus liquid; after fully and uniformly mixing, stirring and incubating the mixed solution; the protective agent is prepared by mixing TritionX-100, arginine and lysine; the incubation temperature is 25-40 DEG C, and the incubation time is 4-12 hours; then adding a formaldehyde solution until the final concentration is 180-220 mu g / ml, and inactivating for 68-76 hours under the condition that the temperature is 35-39 DEG C, so as to obtain an inactivated vaccine; and finally, washing and filtering the inactivated solution to remove TritionX-100 and formaldehyde in the sample. According to the technical scheme, the recovery rate of the antigen in the inactivation solution is greatly improved, and the residual quantity of TritionX-100 and formaldehyde is small.

Methods are disclosed for producing high-titer recombinant adeno-associated virus (rAAV) using a modified adenovirus-based helper nucleic acid. One aspect provided herein is a human adenovirus 5 (hAd)-based nucleic acid that does not contain one or more of (a) an E4 region having E4-ORF6 / 7, (b) a viral associated (VA) RNA region, (c) an E2A region having L4-22K and L4-33K, (d) at least one packaging protein, (e) at least one structural protein, (f) a major late promoter (MLP), (g) an E1 region, and / or (h) an E3 region.

The application discloses a kind of disposable bioreactor and method for preparing adenovirus harvest, belong to biological preparation technical field, the bioreactor includes reaction bag and magnetic stirring mechanism, the magnetic stirring mechanism includes stirring shaft, stirring vane, the number of the stirring vane is greater than 1 and is installed on stirring shaft in layers, the stirring vane includes second stirring vane and first stirring vane, the second stirring vane is fixed on stirring shaft, the first stirring vane is installed on stirring shaft by collar, further include linkage module and shear force transmission device, the linkage module is used to drive shear force transmission device action, realize shear force transmission device and collar connection and disengage.The method is realized based on the bioreactor.Adopting the present scheme, the stirring consistency of disposable bioreactor can be effectively guaranteed in the process of being used for expansion culture.

The present invention relates to a method for purifying viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and / or molecules having a diameter of 5 nm or more by filtration of supported polyalkylene glycol precipitation, and a filtration module that can be used in the method.

The present invention relates to a production method of an inactivated SARS-CoV-2 vaccine, the method including: a step of bringing a SARS-CoV-2 containing solution or an inactivated SARS-CoV-2 containing solution into contact with a cellulose sulfate ester gel at a pH of 8 or more and 10 or less to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel; then removing impurities; and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

The application discloses a production method of a high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus, and belongs to the technical field of biological pesticide production. 8 The virus yield of the high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus is 6*10 The virus loss rate is reduced by 1.4% by adding 0.1% detergent before centrifugal purification of the virus, and the high yield of the virus is further ensured.The insecticidal effect of the high-efficiency strain produced by the method is increased by more than 64.1% than that of an ordinary strain, and the high-efficiency property is continuously maintained.The method has a very beneficial effect on the production of the high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus, and provides a solid guarantee for popularization and application of the high-efficiency strain.

A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.

Methods of characterizing biomolecular formulations are provided. In some examples, there is provided a method of characterizing a viral particle formulation, the method comprising subjecting the viral particle formulation to analytical ultracentrifugation under sedimentation velocity conditions, measuring sedimentation of the viral particle at a plurality of points in time, determining a friction ratio function c (s, ff0) and a sedimentation coefficient for the viral particle formulation, mapping the sedimentation coefficient (S) with the c (s, ff0) to obtain c (s, ff0), and characterizing the c (s, ff0). Ff0) distribution, and characterizing the presence and / or relative composition of viral particles comprising a totiviral genome, a partial viral genome, an over-packaged viral genome, and / or an empty viral particle in the viral particle formulation.