147 results about "Exogenous protein" patented technology

This invention provides an in vitro cell-free protein synthesis system, its reagent kit, and its applications, belonging to the field of protein synthesis technology. This invention improves an in vitro cell-free protein synthesis system based on Kluyveromyces lactis by adding exogenous L-arabinose, significantly enhancing the system's protein synthesis capacity. Furthermore, it provides a more efficient and higher-throughput in vitro protein synthesis reagent kit and a method for synthesizing exogenous proteins. The improvements provided by this invention can be achieved without molecular modification, are simple and convenient to operate, and save costs.

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing a foreign protein based on an NC048604-1 site in a CHO cell genome. A site for stably expressing protein in the CHO cell genome is located in the 94142000 to 94148000 basic group range of the CHO cell genome NC048604-1, and the nucleotide sequence of the site is as shown in SEQ ID NO: 1. According to the invention, different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

The application provides an in-vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and application thereof, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system containing exogenous magnesium ions adopts magnesium aspartate as a novel magnesium ion source, and especially in a eukaryotic cell-free system, compared with a traditional magnesium ion source, can significantly improve the protein synthesis efficiency and protein expression amount. A more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of exogenous proteins are also provided, and the kit has the advantages of simplicity, convenience and low cost.

The invention discloses a novel plasmid vector capable of increasing CHO foreign protein expression quantity and construction and application thereof, and belongs to the technical field of gene engineering. The plasmid vector pCHO6GS belongs to a brand-new plasmid system, and the expression quantity of a common monoclonal antibody and recombinant protein (such as H5HA trimer) difficult to express in CHO can be increased by at least more than one time, so that the expression quantity of foreign protein is increased beyond expectation through the novel plasmid vector; and the method also plays an extremely important role in reducing cost and improving efficiency of various protein products taking CHO as a cell factory.

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The invention provides a preparation method of a recombinant cell, the recombinant cell and application of the recombinant cell, and relates to the technical field of biology. A target protein expressed by the recombinant cell contains one, two, three or four polypeptide chains; the preparation method comprises the following steps: integrating exogenous polynucleotides for coding a target protein into a genetic material of a starting cell in a site-specific manner, wherein each exogenous polynucleotide for site-specific integration contains coding genes of all polypeptide chains of the target protein; each exogenous polynucleotide contains at least four expression cassettes, and each polypeptide chain is encoded by at least one expression cassette. The preparation method is simple to operate, coding genes of all polypeptide chains can be integrated into genetic materials of starting cells at a time, and the expression quantity of foreign proteins of the recombinant cells obtained through the preparation method is high.

To provide a new method for efficiently purifying an exogenous protein expressed in a plant.SOLUTION: A method for purifying a foreign protein expressed in a plant, comprising the step of adding an acid to an extract of a tissue of a plant in which a foreign protein is expressed to degrade contaminating proteins derived from the plant.SELECTED DRAWING: None

The invention discloses a protein sequence analysis method, equipment, a medium and a product, and relates to the technical field of combination of biotechnology and artificial intelligence. The method comprises the steps that a first protein sequence is obtained, corresponding first embedding expression is constructed, and the first protein sequence is an exogenous protein sequence which is expressed in Escherichia coli or is to be expressed in Escherichia coli and is not from Escherichia coli; inputting the first embedded representation into a pre-trained proteotoxicity analysis model to obtain a prediction result aiming at whether the first protein sequence is toxic to escherichia coli or not; the model is a convolutional neural network model based on a channel attention mechanism and a space attention mechanism. According to the technical scheme provided by the embodiment of the invention, the problem that a method for analyzing the toxicity of the escherichia coli by aiming at the protein sequence cannot give consideration to both efficiency and accuracy is solved, and the toxicity of the endogenous or exogenous protein sequence expressed in the escherichia coli to the escherichia coli can be efficiently, stably and accurately predicted and analyzed.

According to the method for efficiently identifying adulteration of the dairy products through the endogenous nanometer characteristics, natural nanometer particles existing in endogenous sources in the dairy products are detected, multiple nanometer characteristic indexes are integrated to form discrimination parameters, an artificial learning-machine algorithm is utilized to construct a discrimination model, the discrimination model is optimized, the dairy products to be detected are discriminated, and the adulteration of the dairy products to be detected is identified. The dairy product adulteration can be efficiently identified on the premise that adulterants are not known. According to the method, various dairy products including bulk milk and rare camel milk can be identified, a judgment model needs few optimization parameters, the fitting performance is good, the equation fitting degree can reach 0.98 or above, and the prediction effect is good. Experiments prove that the method provided by the invention has a relatively high discrimination rate on common adulterants such as foreign protein, starch and water in dairy products, and the discrimination rate can reach 90% or above.

The invention discloses a PVX expression vector pWX for efficiently expressing foreign protein and application of the PVX expression vector pWX. In the construction process of optimizing potato X virus infectious clone, after a single base mutation NruI-gene sequence (CCGCGA) is inserted into an upstream regulation region of a PVX coat protein subgene promoter, a novel PVX expression vector capable of promoting foreign protein expression is obtained, and the novel PVX expression vector is named as pWX. Experiments prove that compared with a control vector, the accumulation amount of GFP (Green Fluorescent Protein) generated by the pWX vector through transient expression of agrobacterium in tobacco is obviously increased. The invention further applies a pWX vector to successfully express an important target protein HPV16L1 researched and developed by an HPV vaccine, and the expression quantity of the important target protein HPV16L1 in tobacco is 5.25 micrograms per gram of fresh heavy leaves. The invention provides a novel vector based on a PVX genome for high-efficiency expression of a plant source target protein, and the vector has a good application prospect in preparation of plant source vaccines.

The invention discloses gluten-free rice bread and a preparation method thereof, and relates to the technical field of rice bread preparation, and the gluten-free rice bread comprises rice flour, glutinous rice flour, water, white granulated sugar, milk powder, butter, yeast and edible salt. According to the gluten-free rice bread, any complex modifying agent such as hydrophilic colloid, foreign protein or enzyme preparation does not need to be added, and the quality of the gluten-free rice bread is improved under the extremely simple formula.

The present application relates to the field of microbial technology, and particularly relates to a gene editing method of propionibacterium acnes VB208 and application thereof. Propionibacterium acnes ) on February 20, 2023, and is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.26576. The propionibacterium acnes screened by the present application has the characteristics of skin surface occupation, high conversion efficiency, gene modification potential and efficient expression of exogenous proteins, and can be used as an engineering strain. Furthermore, a genetic operation system suitable for the strain can be established by using the propionibacterium acnes, and the application prospect is wide.

The present invention discloses a VHH chain of an anti-APP nanobody and its application. The anti-APP nanobody of the present invention has the following beneficial effects: (1) small molecule and drug delivery. (2) easy to manufacture and express. (3) high affinity and strong specificity. (4) stable performance and good plasticity. (5) low immunogenicity and good metabolic characteristics. The APP nanobody of the present invention retains the ability of the VVH chain antibody and avoids the introduction of non-human exogenous proteins as much as possible, taking into account the function of effectively binding to anti-APP and the lowest possible heterology. In addition, the reduction of heterology can not only reduce hypersensitivity reactions but also prolong its half-life in the body and improve metabolic characteristics.

The invention provides an in-vitro cell-free synthesis method and kit of foreign protein, and belongs to the technical field of protein synthesis. According to the in-vitro cell-free synthesis method of the foreign protein, a water-phase in-vitro cell-free protein synthesis system and a nucleic acid template for coding the foreign protein are provided, a hydrophobic interface in surface contact with the water-phase in-vitro cell-free protein synthesis system is provided, incubation reaction is carried out, the foreign protein is synthesized, and the in-vitro cell-free synthesis method of the foreign protein is obtained. The protein synthesis efficiency and the protein expression quantity of the cell-free system are improved. The invention further provides an in-vitro protein synthesis kit which is more efficient and higher in throughput. The invention provides a simple and convenient hydrophobic interface optimization method which is simple and convenient to operate and saves cost.

The invention relates to a combined additive, a CHO cell culture medium, a preparation method and a culture method. The combined additive comprises cyclohexyl alanine and 3-(2-naphthyl)-L-alanine. The CHO cell culture medium comprises amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates and a combined additive, the preparation method is used for preparing the CHO cell culture medium. The culture method uses a CHO cell culture medium to culture CHO cells. The combined additive is obtained through structural domain virtual docking and hydrophobicity regulation and control screening, G1 / S conversion can be mildly delayed, cell apoptosis can be inhibited, and an expression window can be prolonged. The culture medium contains amino acids, vitamins, trace elements and the like, and preferably selects a functional additive to replace serum and foreign protein functions. According to the application, high proliferation of CHO cells and efficient target protein expression can be realized under the conditions of no serum and no foreign protein, meanwhile, the accumulation of lactic acid and ammonia is reduced, the cell caking phenomenon is reduced, and the application has remarkable technical advantages.

The invention belongs to the technical field of chemiluminescence detection, and particularly relates to a kit for detecting a bovine leukemia virus IgG antibody as well as a detection method and application of the kit. A sample treatment solution in the kit is prepared from Protein A, disodium hydrogen phosphate dodecahydrate, monopotassium phosphate, potassium chloride, sodium chloride, casein, mercaptoethanol, Tween-20 and Proclin; and the mark of the magnetic bead is sealed by combining PEG4000 and ethanolamine. Compared with traditional single protein sealing, the sealing mode provided by the invention has the advantages that interference of foreign protein is removed, non-specific binding in a sample is reduced, the background is reduced, and the stability, sensitivity and specificity of detection are improved; compared with a traditional large-proportion diluted sample, the sample treatment liquid has the advantages that excessive IgG in the sample is removed, dilution treatment of an equipment dilution module is not needed in the detection process, manual pre-dilution treatment is also not needed, and the detection efficiency is improved.

The present disclosure provides a nucleic acid molecule comprising a 3' type II intron fragment, a 5' type II intron fragment, and a protein coding region or non-coding region, which can obtain a circular RNA with biological activity. The present disclosure also provides a vector comprising the nucleic acid molecule, and applications thereof. The self-splicing system constructed and optimized by the present disclosure determines that other type II intron self-splicing systems can be used to produce circular RNA and express exogenous proteins in vivo and in vitro, and the system has high ring formation efficiency and protein expression amount.

Nanoparticulate pharmaceutical formulations and methods for co-delivery of two or more species of nucleic acids for simultaneous suppression and expression of target genes in a cell, are provided. The nanoparticles encapsulate two or more nucleic acid species. The first nucleic acid suppresses expression of a gene or product thereof, e.g., inhibitory nucleic acid, such as antisense, siRNA, miRNA, Dicer siRNA, piRNA, etc. The second nucleic acid increases expression of, or encodes, an endogenous or exogenous protein or polypeptide, e.g., an mRNA. The first and second nucleic acid species simultaneously target or affect the same or different cellular processes within a cell including communication, senescence, DNA repair, gene expression, metabolism, necrosis, and apoptosis.

The present invention discloses a recombinant Vibrio natriegens and a method for promoting the secretion and expression of exogenous proteins. The recombinant Vibrio natriegens is constructed by using the Vibrio natriegens with the preservation number of CICC 10908 as the chassis cell, knocking out the deoxyribonuclease gene on its genome, and inserting a combined expression cassette of T7 RNA polymerase and aminoglycoside adenyltransferase. The recombinant plasmid carrying the exogenous protein is transformed into the above-mentioned recombinant Vibrio natriegens, and the seed liquid is inoculated into the fermentation medium. After culturing to the mid-logarithmic growth phase, IPTG and NaCl are added, and then the culture is continued to obtain a fermentation broth containing the exogenous protein. The present invention solves the problem of secreting and expressing exogenous proteins by Vibrio natriegens at the fermentation level for the first time, with a short culture time, low cost, and certain universality, providing technical guidance for the research of Vibrio natriegens engineering bacteria in the fields of protein engineering and fermentation engineering.

The invention provides application of ursolic acid in improving transient expression efficiency of tobacco leaves, and belongs to the technical field of bioengineering. The invention creatively finds that when ursolic acid is applied to a tobacco transient expression system, the transient expression efficiency of tobacco leaves can be remarkably improved. Experiments prove that after a proper amount of ursolic acid is added for treatment, the yield of exogenous protein in the tobacco leaves is greatly increased, so that powerful support is provided for large-scale production and scientific research of high-value biological products; ursolic acid can be used as a universal transient expression enhancement means, and is suitable for various different biotechnology application scenes. As a natural product existing in nature, ursolic acid has the characteristics of good biocompatibility and low toxicity, and does not cause harm to experimental subjects. Meanwhile, the synthesis cost is relatively low, the material is easy to obtain, and the principle of sustainable development is met. Therefore, ursolic acid can be predicted to have a wide application prospect in the field of transient expression of tobacco.

The invention belongs to the field of biology, and discloses preparation and application of a non-methanol-induced expression H5 subtype avian influenza virus subunit antigen and a vaccine thereof, PDAK is applied to pichia pastoris expression of avian influenza virus H5 subtype HA protein, an HA protein multiple-insertion recombinant strain with a PDAK promoter is successfully constructed, and in the research process, it is found that the H5 subtype avian influenza virus subunit antigen and the vaccine thereof have the advantages that the H5 subtype avian influenza virus subunit antigen and the vaccine thereof can be used for preparing the H5 subtype avian influenza virus subunit antigen; by adopting the recombinant plasmid disclosed by the invention, multiple insertions can be generated in a pichia pastoris strain, the target protein expression quantity can be obviously improved, and the foreign protein expression efficiency of the recombinant plasmid is superior to that of a PAOX1 promoter and a PGAP promoter; according to the invention, the HA protein of the PDAK promoter is multiply inserted into the recombinant strain to express the HA protein to prepare the subunit vaccine, after the vaccine is immunized, a higher hemagglutination inhibition antibody level can be generated aiming at homologous strains, and the survival rate of experimental chickens after challenge is 100%.

Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., α-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1 protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

The application discloses a CMV virus-like particle for producing a VLP recombinant vaccine and a preparation method thereof, the CMV virus-like particle is obtained by cloning a C-terminal G4SLPETG modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector, the recombinant expression vector is transfected into E. coli BL21 (DE3), and expression is obtained through the recombinant E. coli BL21 (DE3); and the amino acid sequence of the C-terminal G4SLPETG modified CMV is SEQ ID NO. 1. Test proves that the recombinant strain constructed by the application is stable to expression of an exogenous protein. The recombinant protein expressed by the application is used for preparing a virus-like particle for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, the virus-like particle is not pathogenic to mice and other animals, and the safety evaluation is easy.

The invention discloses an inflammation regulation sequence of dairy cow mastitis and application thereof, and belongs to the technical field of biology, the inflammation regulation sequence is a regulation sequence responding to a gram-negative bacterium inflammation signal, the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 1, and the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 2. A regulatory sequence responding to a gram-positive bacterium inflammation signal, which is a promoter partial sequence of the NLRP3 gene and is as shown in SEQ ID NO: 2; the regulatory sequence responding to a gram-positive bacterium inflammation signal is a promoter reverse complementary sequence of the TLR2 gene and is shown as SEQ ID NO: 3; the endogenous inflammation regulation sequence provided by the invention is composed of genome elements of the dairy cow, and no exogenous coding gene is introduced, so that potential risks caused by exogenous proteins are avoided.

The present invention relates to compositions comprising yeast oleosomes and at least 30 wt% exogenous protein, based on the total solids content of the composition, to food products or beverages comprising such compositions, to the use of yeast oleosomes to improve the sensory properties of a composition comprising at least 30 wt% exogenous protein based on the total solids content of the composition and to a process for the preparation of a composition comprising at least 30 wt% exogenous protein based on the total solids content of the composition.

The invention relates to the technical field of bioengineering, and discloses a saccharomyces cerevisiae self-replication system and application thereof.The system comprises a recombinant plasmid, and the recombinant plasmid comprises upstream and downstream homologous arms of a saccharomyces cerevisiae delta site, a selection marker TRP1, an autonomous replication sequence ARS310, a G418 resistance gene and a resistance gene promoter; wherein the nucleotide sequence of the autonomous replication sequence ARS310 is as shown in SEQ ID NO. 3; the nucleotide sequence of the G418 resistance gene is as shown in SEQ ID NO. 4; the nucleotide sequence of the selection marker TRP1 is as shown in SEQ ID NO. 5. The invention provides a pressure-driven saccharomyces cerevisiae self-replicating system for high-efficiency expression of foreign protein (target gene), and the expression of the foreign target gene with high conversion efficiency, high stability and high copy number is realized by utilizing natural multiple copies of a delta site, the self-replicating capability of ARS and the screening pressure of a G418 resistance gene.

The application discloses a silk material with a cell proliferation function and application thereof, the functional material is expressed by a human type I collagen truncated fragment in a middle silk gland of a domestic silkworm, a sericin promoter is used as a tissue-specific promoter to drive specific expression of an exogenous gene in the middle silk gland, and finally the exogenous gene is combined with sericin protein and is secreted to outside of the body, the exogenous protein can be separated after a certain treatment of a cocoon, and the expression product can keep biological activity. The silk material is used to prepare a hydrogel, has a function of promoting wound healing, and can be applied to aspects of promoting skin regeneration and repair, bone graft substitutes, cartilage reconstruction, artificial skin and the like.

Provided are a protein for increasing the expression yield of an exogenous protein and the use thereof, wherein an ERV29 protein and a related protein, and a P180 protein and a related protein can be used as molecular chaperones to improve the expression yield of exogenous proteins such as brazzein, the ERV29 protein has an amino acid sequence as shown in SEQ ID No. 4, and the P180 protein has an amino acid sequence as shown in SEQ ID No. 6.

The application discloses a novel plasmid vector with improved CHO exogenous protein expression amount and construction and application thereof, and belongs to the technical field of genetic engineering. The plasmid vector pCHO6GS belongs to a novel plasmid system, and can at least improve the expression amount of general monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by more than 1 times, that is, the unexpected improvement of the exogenous protein expression amount is realized by the novel plasmid vector, and the novel plasmid vector has an extremely important role in cost reduction and efficiency increase of various protein products with CHO as a cell factory.