465results 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 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.

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 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.

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.

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 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.

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.

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 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.

Herein is reported a method for producing a recombinant adeno-associated viral particle preparation (rAAVp), the method comprising the steps of culturing a mammalian cell comprising an expression cassette directed against: a non-adeno-associated viral gene interposed between two AAV inverted terminal repeats (ITRs), and thereby producing the rAAVp; an adeno-associated virus rep gene; an adeno-associated virus cap gene; an adeno-associated virus E1A gene; an adeno-associated virus E1B gene; an adeno-associated virus E2A gene; an adeno-associated virus E4orf6; and an adeno-associated virus VA RNA gene, wherein the culturing is carried out at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value. The yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is higher than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2 and including a terminal value, and the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is lower than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2. And the rAAVp produced by the culture at a pH value in the range of pH 7.4 to pH 7.6 and including an endvalue has a higher percentage of intact particles than the rAAVp produced by the culture at a pH value in the range of pH 7.0 to pH 7.2 and including an endvalue.

The invention mainly relates to a bovine rotavirus inactivated vaccine and a preparation method thereof, and according to the preparation method of the bovine rotavirus inactivated vaccine, ultrasonic emulsification conditions are as follows: the ultrasonic frequency is 24-28 kHz, the power density is controlled within the range of 0.6-0.8 W / cm, the single pulse time is 8-12 seconds, the ultrasonic time is 60-120 seconds, ultrasonic emulsification is carried out in a refrigerant bath at the temperature of 2-5 DEG C, the temperature fluctuation is + / -0.5 DEG C, and the ultrasonic time is 60-120 seconds. The virus content in the inactivated vaccine is greater than or equal to 5 * 10 < 5.0 > TCID50 / ml, and the neutralizing antibody titer within 21-49 days after secondary immunization is greater than or equal to 900. The prepared bovine rotavirus inactivated vaccine is subjected to ultrasonic emulsification, a generated neutralizing antibody can completely neutralize an RVA / Bovine-tc / CHN / SHH2023001 / 2023 / G10P [11] virus strain, the titer is high (the titer can reach 1700 or above at most), the duration time is long, the G10P [11] type bovine rotavirus is the most common virus infected in a dairy cow group, and the G10P [11] type bovine rotavirus can be used for preparing the vaccine. Therefore, the vaccine can be used as an alternative vaccine for preventing calves from being infected with bovine rotavirus.

The application belongs to the technical field of biology, and discloses a kind of Myzus persicae densovirus, amplification and connector method and its application, Myzus persicae densovirus name is Myzus persicae densovirus 2, abbreviated as MpDV2, preserved in China typical culture preservation center, and the preservation number is V202281.The Myzus persicae densovirus MpDV2 provided in the application is a virus strain that has not been isolated and identified before and has pathogenicity to Myzus persicae, and the full-length sequence of the genome of the Myzus persicae densovirus MpDV2 (including the complete terminal inverted repeat sequence) is disclosed for the first time, and the influence of the virus infection on the growth and development of Myzus persicae and the population dynamics / growth.The Myzus persicae densovirus MpDV2 of the application significantly reduces the intrinsic growth rate (r), net reproduction rate (R0) and week-long growth rate (lambda) of Myzus persicae population, and prolongs the average generation time (T) of the population, and the MpDV2 infection has a significant influence on the growth and development of Myzus persicae and population growth.

A method for producing a target protein, comprising: providing a host cell, a vector comprising a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and harvesting the culture to obtain the target protein from the harvested culture, wherein the cells of the culture are not passaged before the culture is harvested.

The present invention provides: a culture medium composition which contains a pyruvate dehydrogenase kinase inhibitor and is used for promoting the production of a virus having a target gene mounted thereon when culturing a virus-producing cell into which the target gene has been introduced; a culture medium composition for promoting the production of a virus or a virus-like particle when culturing a cell that produces the virus or the virus-like particle; and the like.

A method of purifying a composition comprising a Group B adenovirus, for example comprising the following purification step: diafiltration of a composition comprising the Group B adenovirus using a diafiltration buffer having a conductivity of at least 180 mS cm ‑1 , for example a conductivity of 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 or 290 mS cm ‑1 . Also provided are compositions obtained using the purification methods disclosed herein.

The present invention provides a cryopreservation solution for Koi herpesvirus, as well as its preparation method and application, relating to the field of virus preservation technology. The solution comprises 2-8 w / v% hydroxyethyl starch, 3-5 w / v% sucrose, and 1-3 w / v% bovine serum albumin. The Koi herpesvirus has an activity of ≥90% after resuscitation, can withstand five freeze-thaw cycles, can be stably frozen at -20°C, and has a shelf life of up to two years. This solution solves the technical problems in the prior art, such as the need to store cryopreservation solutions at ultra-low temperatures, the risk of osmotic pressure imbalance and viral envelope rupture, and low activity after cryopreservation and resuscitation.

The invention discloses a method for efficient targeted separation of bacteriophage based on a modified medium and application, the method comprises the following steps: taking cellulose sponge as a medium, reacting the medium with APTES in an anhydrous reaction system built by inert gas, and converting surface activated hydroxyl into amino; terminating the reaction, and removing APTES; 1 mM of a cross-linking agent EDC is smeared on the surface of the medium, and pretreatment of the medium is completed; coating the thalli on the surface of a modified medium by using a coating rod for cross-linking; and filtering the water sample to be separated by adopting the treated medium. According to the method, cellulose sponge is used as a medium, the surface of the cellulose sponge is modified, so that different types of strains can be immobilized, and bacteria are immobilized on the medium based on the specific binding characteristic of the bacteria and the bacteriophage, so that targeted separation of the bacteriophage is realized; the method is suitable for bacteriophage targeted separation of water bodies with different physicochemical properties, and the bacteriophage grabbing success rate is high.

The present invention relates to a method for producing and purifying a viral vector (V) wherein the viral vector (V) is produced from cells in a fluid (F) and wherein the fluid (F) comprising the viral vector (V) is conducted through a tangential flow filtration device (2) for purification, whereby the viral vector (V) is purified in the fluid (F). It is proposed that the cells and cell debris from the cells are at least partially separated from the viral vector (V) by a fluidized bed centrifuge (5) before purification by the tangential flow filtration device (2), which cells and cell debris are contained in a fluid (F) containing the viral vector (V) after production of the viral vector (V).

The present disclosure provides a method for producing and purifying viral vectors.In particular, the present disclosure provides an improved method for producing lentiviral vectors from host cells grown in suspension.More specifically, the present disclosure provides an improved large-scale lentiviral production method, which includes growing cells to an appropriate number, transfecting with lentiviral packaging plasmid, transfer plasmid and transfection agent; treating with endonuclease; harvesting and clarifying suspension culture supernatant; capturing and concentrating lentiviral vectors using affinity chromatography or cation exchange chromatography; filtering concentrated lentiviral vectors; ultrafiltering and diafiltering lentiviral vectors; formulating lentiviral vectors; and sterile filtering formulated bulk lentiviral vectors.

A method for separating an oligonucleotide or nucleic acid biopolymer for analysis includes loading a sample containing a plurality of oligonucleotides to a chromatographic column; flowing a mobile phase formed by combining the first solution and the second solution through the chromatographic column to elute the plurality of oligonucleotides; at least one of the first oligonucleotide and the second oligonucleotide is analyzed using a mass spectrometer. The plurality of oligonucleotides includes a first oligonucleotide and a second oligonucleotide. The first solution includes an ion pair reagent. The ion pair reagent comprises a primary amine or a secondary amine. The ratio of the first solution and the second solution in the mobile phase varies over time to separate the first oligonucleotide from the second oligonucleotide.