777results 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.

The invention provides a nano antibody and application thereof, a variable region in an amino acid sequence of the nano antibody comprises a complementarity determining region CDR and a framework region FR, the complementarity determining region CDR comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, the CDR3 is RWPDXY family, and binding sites of the CDR3 and AAV capsid are W99, D101 and Y103. The nano antibody provided by the invention has very high affinity and activity, and can specifically recognize and combine various serotypes of AAV, and an adsorbent prepared from the nano antibody has very strong adsorption capacity on AAV, can be applied to AAV affinity chromatography, and is beneficial to industrial application of an AAV affinity chromatography column. In addition, the method can also be applied to the field of AAV detection, and can detect empty capsid and virions together.

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.

The invention relates to the field of microorganisms, and particularly discloses a highly pathogenic gosling plague virus and a preparation method of an egg yolk antibody of the highly pathogenic gosling plague virus. The invention relates to a gosling plague virus, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), is named as the gosling plague virus RD2311B-E21, is preserved on April 22, 2024, and has the preservation number of CGMCC NO.45964. The gosling plague virus is named as the gosling plague virus RD2311B-E21. The preparation method of the egg yolk antibody comprises the following steps: proliferating and inactivating gosling plague virus RD2311B-E21 to prepare an immunogen; respectively injecting the immunogen into the laying hens for four times to obtain four-immunized eggs; the preparation method comprises the following steps: soaking a tetra-immunized egg in a bromogeramine solution, performing spray fumigation with glutaraldehyde, then separating yolk, treating the yolk with disodium hydrogen phosphate to obtain a turbid solution, and performing acidification extraction, degreasing with octanoic acid, degerming filtration, virus removal by ultrafiltration, ultrafiltration concentration and subpackaging to obtain a finished product. The compound has the advantages of high purity, high titer and good treatment, prevention and control effects.

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 provides a salmonella bacteriophage and a preparation method and application thereof, the salmonella bacteriophage is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 10, 2021, and the preservation number is CGMCC No.23090. The invention also provides a preparation method of the salmonella bacteriophage, which comprises the following steps: carrying out combined fermentation on salmonella host bacteria from multiple species and salmonella bacteriophage seeds to obtain fermentation liquor, and centrifugally filtering the fermentation liquor to obtain the salmonella bacteriophage. According to the invention, multi-strain combined fermentation of bacteriophages of salmonella of different provenances and species is realized. According to the combined fermentation process, combined fermentation of multiple strains of bacteriophages of salmonella of different provenances and species is achieved, it is guaranteed that the bacteriophage of each strain of host bacteria can be successfully fermented, and the production cost and the production cycle of bacteriophage fermentation of a single strain of salmonella are reduced.

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.

According to the lysogenic phage community and the extraction method and application thereof, bacterial and phage communities are separated from in-situ soil in a medium proportion through a tangential flow technology, single bacterial communities and single phage communities are obtained through enrichment and concentration, meanwhile, mitomycin C is added into the single bacterial communities for lysogenic induction, and the lysogenic phage communities are obtained. And then filtering and enriching by using the tangential flow system to obtain a high-flux lysogenic phage community, and finally obtaining a single bacterial community, a bacterial-phage mixed community and a bacterial-lysogenic phage mixed community. The phage transplantation technology can promote indigenous microorganisms to participate in the plant growth and development process, and the transplanted lysogenous phage can participate in host plant hormone anabolism by optimizing a microbial network structure and injecting auxiliary metabolism genes. The method provides a long-acting environment-friendly microorganism synergistic growth promoting technology for green agriculture development in China.

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.

One aspect of the present invention relates to a method for producing a purified liquid having an increased purity of a separation target substance, or a purification kit for purifying a separation target substance, and the method for producing a purified liquid having an increased purity of a substance α is a method for producing a purified liquid having an increased purity of the substance α from a sample solution containing a separation target substance (substance α) or a substance containing the substance α (substance β), the method including: (1) the following step 1 and step 2-1; or (2) the following step 1 and step 2-2 (here, the substance α in the substance β is a protein, a nucleic acid, a lipid, or a saccharide), in which a container (i) including a first chamber having an opening in an upper portion and a second chamber partitioned from the first chamber by a filter and having an opening in an upper portion is used. Step 1: a step of obtaining a container (ii) having a carrier to which the substance α or the substance β in the sample solution is adsorbed, in the first chamber. Step 2-1: a step of obtaining a purified liquid having an increased purity of the substance α by bringing a liquid capable of releasing the substance α from the carrier into contact with the carrier and releasing the substance α from the carrier, in the container (ii). Step 2-2: a step of obtaining a purified liquid having an increased purity of the substance α by bringing a liquid capable of releasing the substance α from the carrier into contact with the carrier and releasing the substance α from the carrier. (here, the filter does not allow the carrier to pass therethrough.)

The present inventions provide covalently surface modified adeno-associated viruses can comprise gene of interest (GOIs) and advantageously can be targeted to certain cell and tissue types for preventative and therapeutic purposes. The present inventions further provide systems and methods for engineering adeno-associated virus (AAV) to create covalently surface modified adeno-associated viruses, and methods of purifying such covalently surface modified adeno-associated viruses. The inventions further include covalently surface modified adeno-associated viruses and preparations and products comprising such covalently surface modified adeno- associated viruses.

To provide methods of producing and purifying AAV viral vectors having low empty capsids, low host cell proteins, and / or low contaminating DNA, while retaining high potency; and to provide a pharmaceutical composition containing the vector.SOLUTION: A method of manufacturing an AAV viral vector comprises: a. culturing adherent cells; b. transfecting the adherent cells with one or more plasmids to enable production of the AAV viral vector; c. lysing the adherent cells to isolate the AAV viral vector; d. acidifying and clarifying the cell lysate of c; e. purifying the product of d using cation exchange chromatography; f. filtering the product of e using tangential flow filtration; g. ultracentrifuging the product of f in a cesium chloride buffer; h. collecting the AAV viral vector from the product of g; and i. formulating and filling the AAV viral vector collected in h. to produce a final pharmaceutical product.SELECTED DRAWING: None

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.

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for efficiently producing BaEV enveloped lentivirus. Comprising the following steps: inoculating 293T cells into a culture dish for culturing; the preparation method comprises the following steps: uniformly mixing plasmids pGFP, pLP1, pLP2 and plp-BaEV with a DMEM complete medium, and incubating at room temperature to obtain a plasmid medium mixture; uniformly mixing the PEI solution with the DMEM complete culture medium to obtain a PEI culture medium mixture; adding the PEI culture medium mixture into the plasmid culture medium mixture, and uniformly mixing to obtain a plasmid PEI mixture; adding the plasmid PEI mixture into a culture dish for culturing, and transfecting 293T cells; after 5-6 hours of transfection, replacing the culture medium with an equivalent DMEM complete culture medium for continuous culture; and after transfecting for 36-72 hours, harvesting a 293T cell culture solution, and filtering the 293T cell culture solution to obtain the lentivirus. The lentivirus yield is effectively increased.

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).

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.