305 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 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 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.

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 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 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.

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.

Disclosed is a method for purifying single-stranded RNA (ssRNA). More specifically disclosed is a method of purifying an ssRNA-containing sample containing double-stranded RNA (dsRNA) as an impurity using two different chromatography steps. The method includes steps of: (a) subjecting a sample containing ssRNA and containing double-stranded RNA (dsRNA) as an impurity to primary purification using affinity chromatography; and (b) subjecting the sample to secondary purification using at least one chromatography selected from the group consisting of size-exclusion chromatography (SEC), anion-exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC). The method has a high mRNA recovery and a dsRNA removal rate of 95% or more, and is useful because it can minimize or appropriately control the dsRNA content in an mRNA sample, so that the mRNA can be developed into a drug regardless of the type of sequence, and can also maximize patient safety and drug efficacy.

The invention relates to the technical field of aquatic animal immune regulation and control, in particular to rHoLL-like stepped lectin-like recombinant protein as well as a preparation method and application thereof. The recombinant protein has pathogen-related molecular pattern recognition and combination capabilities, can promote aggregation of pathogens in a body fluid environment, and activates an aquatic animal complement system, so that the non-specific immune defense capability is enhanced. The preparation method comprises the following steps: amplifying a HoLL-like mature peptide coding sequence, constructing a recombinant expression vector, and converting the recombinant expression vector into an escherichia coli expression host for induced expression to obtain rHoLL-like lectin-like recombinant protein existing in an inclusion body form; and carrying out affinity chromatography purification on the inclusion body protein, and carrying out renaturation treatment in a manner of gradient reduction of denaturant concentration to obtain the rHoLL-like stepped lectin-like recombinant protein. The rHoLL-like stepped lectin-like recombinant protein has good biological safety, and can be applied to aquaculture as an aquatic animal immunopotentiator or a related biological product.

Disclosed are affinity chromatography devices comprising a fibrillated heat treated polymeric membrane containing inorganic particles having a spherical shape and a particle size distribution with D90 / D10 less than or equal to 3. A blend or composition of spherical inorganic particles having a nominal particle size of about 5 microns to about 20 microns can be used. Further, the devices have a hydraulic permeability of about 200 (X 10 ‑12 cm 2 ) to about 700 (X 10 ‑12 cm 2 ). The affinity chromatography devices have a dynamic binding capacity (DBC) at 10% breakthrough of greater than 35 mg / ml at a residence time of 20 seconds. Additionally, the affinity chromatography devices have a cycle durability of at least 100 cycles at an operating pressure of no more than 0.3 MPa. Also disclosed are collectors comprising a plurality of affinity chromatography devices and a plurality of collectors in parallel configuration.

The invention discloses a polyclonal antibody of panda Rbp7 protein as well as a preparation method and application thereof, and belongs to the technical field of antibodies. The first to nineteenth amino acid sequences of the panda Rbp7 protein are used as antigen polypeptides, and are specifically shown as SEQ ID No.1. After chemical synthesis or exogenous expression purification, a Balb / C mouse is immunized, and the polyclonal antibody is obtained through repeated immunization, serum titer detection and affinity chromatography purification. The titer of the antibody is not lower than 1 / 128000, the panda Rbp7 recombinant protein can be efficiently and specifically detected through WB and ELISA, the blank of a protein detection tool is filled, and reliable technical support is provided for research on the functional mechanism of the panda Rbp7 protein.

This invention discloses a method for preparing the multifunctional bioactive peptide Aj-UBL from sea cucumber, relating to the fields of biotechnology and biomedicine. Using the Aj-UBL coding sequence from sea cucumber as a template, the method involves PCR amplification, recombinant expression vector construction, engineered bacterial induction expression, bacterial cell disruption, affinity chromatography purification, and identification to obtain the target peptide possessing triple activities of antibacterial, antiprotozoal, and antitumor activity. This invention employs an optimized coupling process of genetic engineering and affinity chromatography to achieve high-purity, high-quality, and stable product preparation, while being environmentally friendly and free from drug resistance risks. It not only solves the problem of coordinated disease control in sea cucumber farming but also expands the high-value application of sea cucumber resources in the biomedical field, filling the technological gap in the large-scale preparation of multifunctional bioactive peptides from sea cucumber and the biological control of ciliates, possessing significant theoretical and practical application value.

The invention discloses a bispecific antibody coupling drug targeting CD24 and HER2 and a preparation method and application thereof.The bispecific antibody coupling drug comprises an anti-CD24 heavy chain, an anti-CD24 light chain, an anti-HER2 heavy chain, an anti-HER2 light chain, a tetrapeptide linker and a topoisomerase I inhibitor exatan derivative, the DAR value of the bispecific antibody coupling drug is 9.25, and the anti-CD24 heavy chain, the anti-HER2 light chain, the tetrapeptide linker and the topoisomerase I inhibitor exatan derivative are different. The compound has strong cytotoxicity and internalization ability on breast cancer cells, not only can specifically target tumor tissues, but also can release drugs to kill target cells and adjacent tumor cells, and can significantly inhibit growth of breast cancer tumors. An antibody part DACH023 in the bispecific antibody coupling medicine is the same as a traditional IgG molecule, a traditional monoclonal antibody structure is reserved to the maximum extent, due to the existence of an Fc fragment, a common Protein A column affinity chromatography method can be adopted for purification, and large-scale production and purification are facilitated.

The invention discloses a purification method of a bispecific antibody capable of removing fragments, which comprises the following steps: using an eluent containing PEG (polyethylene glycol) and CaCl2 in affinity chromatography, and matching with a high-pH impurity washing solution containing arginine and hydrophobic chromatography for fine purification to obtain the bispecific antibody with few impurities and high purity. In the purified eluent obtained through two-step chromatography, the proportion of the bispecific antibody is increased to 98% or above, the proportion of fragments is reduced to about 0.5%, fragment impurities generated in the preparation process of the bispecific antibody are effectively removed, and the purification method is efficient and has good application prospects.

The application discloses a kind of bionic transmembrane protein affinity chromatography column and its preparation method and application.The system (iSTAC) realizes the in-situ construction of transmembrane protein in highly bionic dynamic microenvironment by integrating cell-free protein synthesis, functional mesoporous silica modification and amphiphilic (AH) peptide stabilized planar lipid bilayer technology.The core is to use long-chain PEG-24 crosslinking agent to retain lipid bilayer on the surface of silica gel, provide sufficient conformational dynamic space for multi-transmembrane protein, and introduce AH peptide to repair membrane defects, ensure that the receptor realizes directional embedding while maintaining natural functional attributes.The preparation cycle is shortened from 168 hours to 5 hours, which significantly improves the efficiency of targeted drug screening and in-situ analysis of binding sites.Using this platform, 5-HT 1A Receptor agonists crocin I and crocin II with anti-insomnia and neuroprotective effects are successfully screened from saffron, and the action site is accurately depicted.

The invention discloses an affinity peptide ligand for separation and purification of a VSV-G pseudotype lentiviral vector and application of the affinity peptide ligand. The affinity peptide ligand contains an amino acid sequence combined with a VSV-G pseudotype lentiviral vector envelope protein; the amino acid sequence is any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. A biological raw material solution containing VSV-G pseudotype lentivirus vector components flows into the affinity chromatography medium, and after adsorption and cleaning, a target vector can be collected through a mild elution step; the recovery rate of virus vector particles is 78% or above, the removal rate of Vero host cell impure protein is 90% or above, the double-stranded DNA residue is 48% or below, and the separation effect is very remarkable.

The invention relates to the technical field of medicinal chemistry, and particularly discloses application of echinacoside in preparation of a medicine for improving autism social and cognitive impairment, and the application comprises the following steps: constructing a recombinant saccharomyces cerevisiae strain, and knocking out a precursor competition pathway gene; performing metabolic flow reprogramming fermentation on the recombinant strain; centrifuging the fermentation liquor at 7000-9000 r / min and pretreating the fermentation liquor with an 8-12 kDa ultrafiltration membrane; carrying out dual-targeting affinity chromatography purification on the pretreatment liquid; the preparation method comprises the following steps: preparing echinacoside into a liposome containing pH sensitive poly-histidine; and adding a freeze-drying protective agent into the suspension to carry out gradient pre-freezing and vacuum freeze-drying. According to the application, a mode of combining recombinant saccharomyces cerevisiae strain construction and metabolic flow reprogramming fermentation is adopted, targeted enrichment of echinacoside is realized through affinity chromatography coupled with autism brain region specific double ligands, then the echinacoside is prepared into the liposome containing pH sensitive poly-histidine, the double ligands are modified, and the effects of precise targeting and efficient effect are achieved.

The invention belongs to the technical field of protein engineering, and particularly relates to an immunoglobulin binding protein mutant with high alkali stability and application of the immunoglobulin binding protein mutant. The immunoglobulin binding protein mutant with high alkali stability provided by the invention is obtained by mutating in a Z structural domain of an amino acid sequence of staphylococcus aureus protein A, such as SEQ ID NO: 1, wherein the amino acid sequence of the Z structural domain is shown as SEQ ID NO: 1, and mutation sites comprise the 3rd amino acid, the 6th amino acid, the 9th amino acid, the 11th amino acid, the 23rd amino acid, the 25th amino acid, the 28th amino acid, the 32th amino acid, the 33rd amino acid, the 40th amino acid, the 42nd amino acid, the 43rd amino acid, the 44th amino acid, the 52nd amino acid and the 53rd amino acid. The prepared immunoglobulin binding protein mutant is prepared into affinity chromatography filler, the alkali resistance of the affinity chromatography filler and the affinity to an antibody can be remarkably improved, and the affinity chromatography filler can tolerate online cleaning of a 0.5-1.0 mol / L NaOH solution and has a good application prospect.

The invention relates to the technical field of genetic engineering, in particular to a small yellow croaker IRE1 alpha protein polyclonal antibody as well as a preparation method and application thereof. The invention provides a preparation method of a small yellow croaker IRE1alpha protein polyclonal antibody. The preparation method comprises the following steps: cloning an open reading frame of a small yellow croaker IRE1alpha gene, and constructing a small yellow croaker IRE1alpha gene recombinant expression vector pET-30a (+)-IRE1alpha. Performing inducible expression and purification on the recombinant protein of the Larimichthys polyactis IRE1 alpha, and performing specific analysis on Western Blot; and preparing the polyclonal antibody of the small yellow croaker IRE1 alpha. According to the invention, a recombinant expression vector of the small yellow croaker IRE1alpha gene is constructed by adopting molecular biology and genetic engineering methods, purified recombinant protein is obtained through induced expression and affinity chromatography, the prepared polyclonal antibody can be used for expression detection of the small yellow croaker IRE1alpha protein, and an important material foundation is laid for research on functions and action mechanisms of the small yellow croaker IRE1alpha protein.

The application belongs to the field of marine biotechnology, and specifically discloses a preparation method of a long oyster insulin-like peptide receptor (ILPR) polyclonal antibody and application thereof. The method is characterized by the following steps: specific antigen epitopes of the long oyster ILPR protein are screened through bioinformatics analysis, a pET32a-ILPR recombinant plasmid is constructed and induced to express in Escherichia coli; the purified recombinant protein is used as an immunogen to immunize New Zealand white rabbits, and rabbit antisera are collected after four times of booster immunization; specific polyclonal antibodies are purified by using antigen affinity chromatography technology, and can specifically recognize the ILPR protein in long oyster tissues. The antibody prepared by the application has high specificity and sensitivity, and provides an important biological tool for studying the insulin signal transduction pathway, growth regulation and energy metabolism mechanism of long oysters.

The invention discloses a PEDV-S1 recombinant protein antigen as well as a preparation method and a vaccine thereof, and belongs to the technical field of recombinant protein vaccines. The CHO eukaryotic expression system is adopted, high expression and complete glycosylation modification of protein can be achieved, and therefore it is guaranteed that the vaccine has strong immunogenicity; through His tag fusion expression design, nickel column affinity chromatography is conveniently adopted for purification, the process is simple and convenient, and the protein recovery rate is also improved; in combination with a serum-free suspension culture technology, the method is suitable for large-scale production, and the stability among batches is good; the prepared vaccine is high in safety and few in side reaction; an immune challenge test further proves that the vaccine can induce a body to generate a high-level antibody and can effectively protect PEDV attack.