409 results about "Antibody Affinity Chromatography" patented technology

The invention relates to the technical field of biology, and particularly discloses a recombinant collagen III and application thereof in preparation of gel. The recombinant collagen III is designed by optimizing functional area sequences of human I-type and III-type collagen, the amino acid sequence is shown as SEQ ID No.2, and the recombinant collagen III has the characteristics of high stability, good hydrophilicity and low immunogenicity. The preparation method comprises the steps of expression vector construction, escherichia coli induced expression, affinity chromatography purification and renaturation. The recombinant collagen III can be prepared into a gel dressing and comprises sodium alginate, methylparaben and other components. Experiments show that the gel can effectively promote cell proliferation and has no cytotoxicity; in a mouse skin injury model, the collagen can relieve inflammation, accelerate wound healing and inhibit scar formation, and the effect of the collagen is superior to that of natural human III-type collagen. The invention provides a safe and efficient novel material for wound repair, and is suitable for medical dressings and tissue engineering.

The application discloses a method for recovering active glycoprotein Patatin from potato starch processing waste juice, and belongs to the technical field of protein extraction. The method is characterized in that the active Patatin protein is efficiently separated by using the combination of ammonium sulfate fractionation precipitation, anion exchange chromatography, Con A affinity chromatography and gel chromatography, and the obtained Patatin protein retains the original protein structure and biological function activity. Compared with the prior art, the method has better separation purity and higher efficiency, and the obtained Patatin protein has complete protein higher structure and function activity, and has a better application scene.

The invention provides a bispecific antibody and a purification method thereof. The purification method comprises the following steps: a) loading a sample containing a bispecific antibody to a affinity chromatographic column, and leaching and eluting; the biomacromolecules in the sample are adsorbed on the affinity chromatography column in the leaching stage, the biomacromolecules comprise antibodies, host cell proteins and protein aggregates, and the antibodies comprise bispecific antibodies and homodimers; separating the antibody from the affinity chromatography column in the elution stage, and collecting the eluate in the elution stage; b) loading the eluent to a hydrophobic membrane material, carrying out flow-through, and collecting a flow-through liquid containing the purified antibody; and the flow-through liquid used for flow-through contains 0.73 to 0.77 M of (NH4) 2SO4. The method can solve the problem that homodimers in bispecific antibodies are difficult to efficiently remove in the prior art, and is suitable for the field of antibody purification.

The present invention provides a heparin affinity chromatography medium and a preparation method thereof. The heparin affinity chromatography medium comprises a porous polymer microsphere matrix modified with a polyamide-amine dendrimer to obtain a modified matrix; heparin is coupled to the modified matrix to obtain the heparin affinity chromatography medium. The heparin affinity chromatography medium of the present invention has a high adsorption capacity for target biomolecules, significantly improving separation efficiency; and also exhibits high mechanical strength.

The invention discloses an influenza C virus HEF protein polyclonal antibody preparation method, which comprises: carrying out signal peptide prediction, antigen epitope screening and hydrophobicity analysis through bioinformatics, constructing a recombinant prokaryotic expression vector pET32a-HEF by using a seamless cloning technology, and efficiently expressing the HEF protein in Escherichia coli to obtain the influenza C virus HEF protein polyclonal antibody. The inclusion body protein yield is improved by optimizing conditions; and purifying by nickel column affinity chromatography to obtain high-purity recombinant protein, emulsifying the high-purity recombinant protein and a Freund's adjuvant, immunizing a Japanese white rabbit, immunizing for three times, and collecting high-titer serum. The method breaks through detection limitation caused by antigen tag concealment in a eukaryotic expression system, the obtained polyclonal antibody is high in specificity, the eukaryotic expression HEF protein can be accurately recognized, and a key tool is provided for ICV virus-like particle identification, vaccine research and development and diagnosis technologies. Compared with a traditional method, the scheme is simple and convenient to operate, low in cost and high in antibody titer, and has remarkable application value.

The invention relates to the technical field of biology, in particular to a method for producing vitamin B12 through fermentation of propionibacterium shermangii, which comprises the following steps: inoculating propionibacterium shermangii into a seed culture medium to culture a seed solution; inoculating the seed solution into a fermentation culture medium for fermentation; after fermentation is finished, thalli are removed in a centrifugal separation or filtration mode, a fermentation solution is subjected to ultrafiltration concentration, a concentrated solution is purified through ion exchange chromatography or affinity chromatography, and a vitamin B12 crude product is obtained; and further purifying by adopting a crystallization method to obtain a high-purity vitamin B12 finished product. According to the method for producing the vitamin B12 through fermentation of the propionibacterium shermannii, the yield and the purity of the vitamin B12 are remarkably improved and the production cost is reduced by accurately controlling fermentation conditions, optimizing culture medium components, introducing an intelligent regulation and control system and adopting a specific purification process, so that the method is suitable for industrial production. The problems of low yield, unstable purity, difficult production process control and the like of the vitamin B12 in the prior art are solved.

The invention relates to the technical field of protease separation, in particular to a novel earthworm protease separation and purification process. According to the present invention, the ultra-high speed separation, the molecular weight spectrum comparison extraction and the affinity chromatography purification scheme are adopted, such that the different biological macromolecules in the earthworm can be effectively separated, the earthworm protease-containing cell lysis solution can be concentrated, and most of the impurities can be removed so as to obtain the high-purity protease solution, the method is simple in operation and does not depend on complex chemical reactions, contact between protein and chemical reagents can be reduced, meanwhile, damage of high temperature to earthworm protease is avoided through a low-temperature centrifugation scheme, the enzymatic activity of earthworm protease is kept, high-purity and high-activity earthworm protease is obtained through affinity chromatography purification, operation is easy, and the method is suitable for industrial production.

The invention discloses preparation and application of grouper enterospora sporowall protein SWP26 and a polyclonal antibody of the grouper enterospora sporowall protein SWP26, and belongs to the field of animal quarantine, the grouper enterospora sporowall protein SWP26 is obtained by amplifying grouper enterospora genes by using a PCR (Polymerase Chain Reaction) method, shearing a target band and transferring the target band into a pET-32a vector to construct a recombinant plasmid, and then transforming escherichia coli BL21 (DE3) for induced expression. A Ni-NTA affinity chromatography method is used for protein purification, animal immunization and polyclonal antibody purification are carried out on the purified SWP26 recombinant protein, western blot detection is carried out on the purified antibody, an obvious signal appears at about 43kDa, and it is proved that the anti-SWP26 polyclonal antibody can be subjected to a specific reaction with the SWP26 purified protein. The invention clones and identifies the high-abundance spore wall protein SWP26 positioned on the surface of the enterosporidium of the grouper for the first time, belongs to a specific protein of the enterosporidium of the grouper, and can be used as a drug target for treating enterocytozoonosis of the grouper.

The invention discloses a cod bone-derived high-activity calcium chelating peptide and a preparation method thereof, and belongs to the technical field of functional biological product preparation. The calcium chelating peptide provided by the invention is derived from a cod bone collagen enzymatic hydrolysate and is obtained through affinity chromatography separation of hydroxyapatite, the calcium chelating activity is 4.9-5.2 mu g / mg, and the calcium chelating peptide contains aspartic acid and glutamic acid enriched peptide fragments, through deep processing of cod bones, high-valued utilization of cod bone resources is realized, the high-activity calcium chelating peptide is prepared, and the calcium chelating peptide has a wide application prospect. And a high-quality raw material is provided for the development of a novel calcium supplement.

The invention provides a preparation method of thermal-stability T7RNA polymerase and the thermal-stability T7RNA polymerase, belongs to the field of biology, and can solve the problems that an existing T7RNA polymerase preparation process is complex, the expression culture period is long, multi-step purification treatment is needed, liquid change treatment needs to be carried out independently before storage, and the production cost is high. The method comprises the following steps: transferring plasmids containing a T7RNA polymerase gene sequence into expression host bacteria M15, culturing, screening, cloning, adding glycerol for strain preservation, and establishing a strain three-level library; the method comprises the following steps: taking glycerol bacteria, carrying out streak resuscitation on a flat plate, selecting monoclone, inoculating the monoclone into an LB culture medium, culturing, carrying out enlarged culture, carrying out IPTG induced expression, centrifugally collecting thalli, cleaning, adding a lysis buffer solution, resuspending, carrying out ultrasonic cell disruption, centrifuging, and collecting supernate; purifying by nickel column affinity chromatography, removing impure protein by an impure washing buffer solution, eluting target protein, purifying by matching with anions and cations, and collecting the purified protein; and mixing the purified protein with a storage buffer solution, and subpackaging and storing. The method can be applied to rapid preparation of the thermal-stability T7RNA polymerase.

The invention provides a method for screening a hyaluronidase (HAase) inhibitor, and belongs to the technical field of medicines. The method comprises the following steps: firstly, rapidly identifying a natural product with HAase inhibition activity by adopting a fluorescence sensing technology according to the principle that carbon quantum dots (CDs) are used as a fluorescence probe, hyaluronic acid (HA) modified gold nanoparticles are used as a quencher, and the fluorescence of the CDs can be quenched by the quencher; when HAase exists, HA can be enzymatically decomposed to release CDs, and fluorescence recovery is caused; when a screened sample has HAase inhibition activity, CDs fluorescence recovery can be prevented. And 2, capturing components in a sample capable of inhibiting HAase activity by virtue of an affinity chromatography technology, immobilizing HAase on a carrier material by virtue of a method, eluting by virtue of a solvent to obtain components capable of being combined with HAase, and identifying the components by virtue of a liquid chromatography-mass spectrometry technology. According to the method, the HAase inhibitor can be rapidly and accurately screened from natural products.

The invention discloses an escherichia coli-bacillus subtilis shuttle plasmid and application, and belongs to the field of gene engineering. The shuttle plasmid comprises the following elements: a promoter sequence, a coding sequence of bacillus subtilis signal peptide, a replication initiation sequence of bacillus subtilis, a replication initiation sequence of escherichia coli and an antibiotic resistance gene. According to the plasmid design, a modular structure is adopted, and quick replacement of key elements such as a promoter, a signal peptide and RBS is supported through optimized enzyme cutting site layout; a 6 * His tag introduced at the tail end of a multiple cloning site (MCS) can be adapted to nickel column affinity chromatography, and the purification process of the recombinant protein is simplified. The plasmid has the characteristics of small molecular weight and convenience in operation, can be efficiently cloned and constructed in escherichia coli and then directly converted into bacillus subtilis for protein expression, is particularly suitable for development of a gram-positive bacterium secretory expression system, and provides an efficient tool for large-scale production of industrial enzyme preparations.

The current invention reports a method for purifying an antibody by reducing the content of a host cell protein. The method employs a wash step with a low conductivity aqueous solution in an affinity chromatography.

The invention relates to the technical field of agricultural fertilizers. More specifically, the invention relates to an extraction method of tenebrio molitor antibacterial peptide and application of the tenebrio molitor antibacterial peptide in preparation of tea tree anthracnose resistant fertilizer. According to the preparation method, the colletotrichum camelliae liquid is taken as an induction factor, the ultrasonic induction technology is utilized to strengthen the absorption of tenebrio molitor to the induction factor, meanwhile, a supercritical / ethanol cosolvent extraction process and a bionic affinity chromatography purification process are combined, the efficient extraction of the antibacterial peptide is realized, and the obtained antibacterial peptide has a relatively good inhibition effect on the colletotrichum camelliae. A tea tree anthracnose resisting leaf fertilizer is further prepared through the tenebrio molitor antibacterial peptide, the leaf fertilizer has a good prevention and treatment effect on tea tree anthracnose caused by camellia colletotrichum spinosus, meanwhile, tea growth can be promoted, and the yield reduction caused by anthracnose and nutrition deficiency of tea tree crops can be effectively reduced.

The invention discloses a PD-L1 / TGF-beta / STING triple targeting antibody coupling drug as well as a preparation method and application thereof, and belongs to the field of tumor immunotherapy. The ADC structure is Ab-[L-D] n, Ab is a bispecific antibody YM101 targeting PD-L1 and TGF-beta, L is a cleavable connexon, and D is an STING agonist. The preparation method comprises the following steps: carrier molecule deprotection, linker functionalization, linker-load coupling, antibody sulfhydrylation modification, coupling reaction and affinity chromatography purification. The ADC can increase the number of CXCL16 < + > macrophages, dendritic cells and CXCR6 < + > T cells infiltrated in tumors, effectively reverses the drug resistance of a TGF-beta * PD-L1 bispecific antibody, has a strong anti-tumor effect and no obvious toxic or side effect, and has a good application prospect in preparation of anti-tumor drugs.

The invention belongs to the field of protein purification, and relates to a method for preparing bevacizumab, which comprises the steps of affinity chromatography, virus inactivation, deep filtration, composite anion exchange chromatography, cation exchange chromatography and the like.

Provided herein are fusion proteins and methods for purifying nucleic acids from a composition. The disclosed compositions and methods allow for faster, more efficient purification of nucleic acids compared to traditional affinity chromatography.

The utility model discloses a novel affinity chromatography column which comprises a chromatography column body, the positioning part comprises side plates, a supporting frame, a supporting disc, a positioning semi-ring, a limiting ring, a sliding block, an inserting ring, an inserting groove, a sliding groove and a drainage tube, the drainage tube is connected to the bottom of the chromatographic column body, the side plates are connected to the lower portions of the two sides of the outer surface of the chromatographic column body, the sliding groove is formed in each side plate, and the sliding blocks are movably connected to the interiors of the sliding grooves; a supporting frame is connected to one side of the outer surface of the sliding block, a positioning semi-ring is connected to the lower portion of one side of the supporting frame, and a supporting disc is connected to the top of the drainage pipe. The utility model relates to an affinity chromatography column, and aims to solve the problems that a drainage tube needs to be mounted at the bottom of the chromatography column to drain extracted fish Igm monoclonal antibodies during actual use of an existing affinity chromatography column, a supporting structure is not arranged between the drainage tube and the chromatography column, the drainage tube and the chromatography column are loosened after long-time drainage, the drainage tube even falls off under serious conditions, and the service life of the chromatography column is influenced. And the stability is poor.

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating active components in tobacco based on liquid-phase segmented separation, SPR, affinity chromatography enrichment and mass spectrometry detection, a product and application. According to the method, liquid chromatography separation, SPR detection screening, affinity chromatography enrichment and mass spectrum identification technologies are fused, and active small molecules are screened and enriched from a complex system by virtue of high sensitivity of SPR and accuracy of mass spectrum. The method comprises the following steps: firstly, carrying out liquid chromatography segmented separation on extract components to improve SPR detection precision and affinity enrichment efficiency; by means of specific binding of Ni Sepharose FF (NTA) resin and a His tag, more target proteins are fixed, and the micromolecule enrichment effect is further enhanced; and finally, identifying the molecular structure by using mass spectrum, and locking the bioactive small molecule combined with the target protein. According to the strategy, efficient enrichment of small molecule ligands is achieved, and a new way is provided for early-stage development of small molecules in drug discovery. 6.

Embodiments described herein relate to novel chromatography media for removing anti-A and / or anti-B antibodies from a sample, as well as methods of using the same. The media described herein have several advantages over previously described media including, acid and alkaline stability.

The present disclosure is directed to methods of performing affinity chromatography. The injection elution methods disclosed herein afford rapid and robust elution of a target analyte from an affinity chromatography column, therefore improving workflow efficiency. Further, the single injection elution methods result in highly concentrated samples, mitigating the need for sample concentration or manipulation.

The present invention discloses a recombinant porcine circovirus type 3 trimer protein, a preparation method and an application thereof. The present invention uses bioinformatics methods and resources to predict the B cell antigen epitopes and T cell antigen epitopes of the PCV3Cap protein. Taking into account the stability and immunogenicity of the antigen epitopes, a recombinant porcine circovirus type 3 trimer protein based on the PCV3Cap protein antigen epitope is designed, and its efficient soluble expression in Escherichia coli is achieved. The expressed recombinant porcine circovirus type 3 trimer protein can be mass-produced and purified by Ni-NTA affinity chromatography. The purified recombinant porcine circovirus type 3 trimer protein can assemble into a stable trimer structure. The subunit vaccine prepared using the recombinant porcine circovirus type 3 trimer protein can induce experimental pigs to produce a high level of antibodies and has a significant protective effect on the experimental pigs. The recombinant porcine circovirus type 3 trimer protein designed by the present invention provides a new idea for the development of PCV3 vaccine.

The invention provides a chromatographic detection method capable of accurately detecting the saccharification level of an antibody, the method is used for detecting through a boric acid affinity column, factors influencing the accuracy of a boric acid affinity chromatography (BAC) method of the monoclonal antibody are deeply discussed, reasons for inaccurate saccharification detection of the BAC method of the monoclonal antibody are clarified, an improved BAC method is successfully developed, and the method can be used for detecting the saccharification level of the monoclonal antibody. The saccharification level of infliximab can be measured more accurately, and the accuracy of the method is confirmed through a rapid multi-attribute method based on mass spectrum. In addition, by testing different types of antibody drugs, the wide applicability of the method in conventional antibody drug saccharification detection is proved. The method provides a new rapid and accurate technical scheme for quality control of antibody drugs. The research results are helpful for promoting the improvement of related technical platforms and the updating of concepts, so that the product development and quality control capabilities are enhanced.

The utility model discloses a kind of device for separating and purifying cosmetic functional raw materials using affinity chromatography technology, it is related to chromatography column field, including fixed plate, chromatography column, heat preservation shell and connecting assembly, chromatography column is by effluent bucket, glass shell and top cover composition, fixed plate fixedly sets in effluent bucket outer wall, effluent bucket and top cover are with glass shell and are formed sealed cavity by thread connection, heat preservation shell is clamped by two half shells and is wrapped in chromatography column outer wall, connecting assembly is installed in fixed plate lower surface, connecting assembly is used for the fixation and separation of two half shells, the utility model is split by heat preservation shell, so that scale can be completely removed in cleaning process, improve heat transfer efficiency, ensure the uniformity of chromatography column internal temperature distribution, conducive to maintaining the stability and separation effect of chromatographic process.

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

The invention discloses an efficient purification process of a bispecific antibody and a special chromatography system thereof. The efficient purification process of the bispecific antibody comprises the following steps: pretreatment: performing 0.22 mu m microfiltration on a cell culture supernatant to remove cell debris, and performing deep filtration to reduce turbidity to be less than or equal to 1NTU so as to reduce subsequent chromatography medium pollution; carrying out improved Protein A affinity chromatography, namely carrying out affinity chromatography by adopting an agarose medium modified by a recombinant Protein A ligand, taking 20mM Tris-HCl as a loading buffer solution, and taking the pH value of the loading buffer solution as 7.4, taking 100mM citric acid as an elution buffer solution, and taking the pH value of the elution buffer solution as 3.2-3.5; performing mixed mode chromatography: adopting a composite mode medium containing a benzamidine functional group, taking 50mM sodium phosphate as a buffer solution, adjusting the pH value to 6.0-6.5, adjusting the NaCl concentration to 0.1-0.3 M, and specifically removing a double-antibody polymer and a mismatched isomer by utilizing a hydrophobic effect and an ion exchange synergistic effect; and performing nanoscale filtration and terminal treatment: removing viruses by adopting a virus filtration membrane with the aperture of 30nm, and performing 0.22 mu m sterile filtration to obtain a final product. According to the process, the purification efficiency, the yield and the product purity are remarkably improved, and the process is suitable for efficient purification of the bispecific antibody.

The application discloses a preparation method of a recombinant ternary fusion protein dressing with epidermal cell activation function. The dressing is a fusion protein designed based on the research on the functions and properties of COL3A1 and IFN-kappa proteins by international peers and the laboratory. The specific method is that the fusion gene fragment of the covalent connection of COL3A1 and IFN-kappa with GGSGG as a linker is obtained by using overlap extension PCR technology, and a 6×His label is added. The target gene is cloned into a prokaryotic expression vector pET30 (modified in the laboratory) to obtain the fusion protein COL3A1-IFN-kappa with an MBP label. After expansion culture, the MBP-IFN-kappa-COL3A1 with a purity of more than 90% can be obtained through Ni-NTA column affinity chromatography. We verified the resistance of the fusion protein to salmonella in THP-1 cells, and verified the effect of the fusion protein on promoting wound healing and resisting salmonella infection on mice. Finally, it is proved that the MBP-IFN-kappa-COL3A1 has good uniformity, is easy to purify, has high activity, has no side effects, and is very suitable for being used as an epidermal cell activation dressing.

The present invention provides a nickel ion metal chelate affinity chromatography medium and a preparation method thereof. The nickel ion metal chelate affinity chromatography medium uses porous dextran gel microspheres as a matrix, and allyl glycidyl ether is bonded to the surface of the dextran gel microspheres. The allyl glycidyl ether is connected to ethylenediaminetetraacetic acid, and the ethylenediaminetetraacetic acid is complexed with nickel ions. The nickel ion metal chelate affinity chromatography medium uses the ethylenediaminetetraacetic acid to bond with allyl glycidyl ether, and the ethylenediaminetetraacetic acid can bind nickel ions. 2+ Carry out complexation and effectively improve the stability of chromatography media.

This invention provides a method for screening β-galactosidase inhibitors, belonging to the field of pharmaceutical technology. The first step of this method uses fluorescence sensing technology to rapidly identify natural products with β-Gal inhibitory activity. The principle is based on the fluorescence quenching property of p-nitrophenol (PNP) on sulfur quantum dots (SQDs); β-Gal catalyzes the hydrolysis of p-nitrophenyl-β-D-galactopyranoside (PNPG) to generate the specific product PNP; in the presence of β-Gal, PNPG can be enzymatically catalyzed to generate PNP, quenching the fluorescence of SQDs; when screened for β-Gal inhibitory activity, the fluorescence of SQDs can be restored. The second step uses affinity chromatography to capture components in samples with β-Gal inhibitory activity. This method involves immobilizing β-Gal on a support material, eluting with a solvent to obtain components that can bind to β-Gal, and then using liquid chromatography-mass spectrometry (LC-MS) to identify the components. This method can rapidly and accurately screen β-Gal inhibitors from natural products.

The present disclosure provides methods for analyzing a mixture of proteins using affinity chromatography. In some embodiments, the proteins are heterodimeric bispecific antibodies or antigen-binding fragments thereof. In some embodiments, guanidinium salts are used in the mobile phase of the affinity chromatography. This disclosure provides protein analysis methods that provide superior resolution and lower machine maintenance than other methods.