134 results about "Pichia" patented technology

The invention belongs to the technical field of biological detection, and discloses a kit and a method for quantitatively detecting residual quantity of host cell protein of pichia pastoris in order to solve the problems of limit of quantitation and low detection sensitivity of an existing detection method. According to the present invention, the empty plasmid without the recombinant human serum albumin gene is introduced into the pichia pastoris, and purification is performed by combining the Protein A chromatographic column and the host cell protein coupling affinity column, such that the prepared polyclonal antibody can be effectively used for the quantitative detection of the residual amount of the pichia pastoris host cell protein in the biological product; the kit formed by the polyclonal antibody has good accuracy and repeatability on the quantitative detection of the host cell protein residues in the pichia pastoris source biological products, the quantification limit is 0.2 ng / mL, and the kit provided by the invention can meet the quantitative detection of the host cell protein residues in all the pichia pastoris source biological products.

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection method of a porcine epidemic diarrhea virus antibody SIgA, and belongs to the technical field of animal antibody detection. In order to solve the problems of limited detection range and single immune evaluation dimension of a porcine epidemic diarrhea virus detection method in the prior art, the invention provides an indirect ELISA detection method of a porcine epidemic diarrhea virus antibody SIgA, and the detection method adopts a pichia pastoris eukaryotic secretion platform to prepare a high-activity PEDV S1 antigen; meanwhile, a secreting type IgA antibody is used as a detection target; the method has the advantages of high detection sensitivity, low background noise, better variant compatibility and practical popularization value, and provides a clinical and industrial-level solution for maternal immunity monitoring, piglet passive protection evaluation and vaccine immunity strategy optimization.

The invention provides recombinant III-type collagen and application thereof, and relates to the technical field of genetic engineering. According to the invention, the yield of the collagen is improved by optimizing the nucleotide sequence of the target gene and optimizing the culture conditions of pichia pastoris, so that the collagen is efficiently expressed in engineering bacteria. The recombinant engineering bacterium shows strong potential in the aspect of improving the yield of protein with high economic value, and is suitable for large-scale industrial production. The recombinant III-type collagen is high in expression quantity, simple in preparation process and suitable for preparation of biological materials for medical cosmetology, wound repair and the like, and has a wide application prospect.

The invention discloses a fuzzy intelligent control method and device for a pichia pastoris fermentation process. The fuzzy intelligent control method comprises the following steps: (1) integrating an online sensor in a bioreactor and monitoring state parameters of fermentation liquor in real time; (2) inputting the monitored parameters into a central control system, and processing the parameters through a fuzzy intelligent control algorithm; and (3) according to an output instruction of the fuzzy intelligent control algorithm, cooperatively regulating and controlling fermentation operation parameters in real time. According to the method disclosed by the invention, a fuzzy intelligent control algorithm is developed by monitoring dissolved oxygen, relative concentration of dissolved carbon dioxide, methanol concentration and pH in fermentation liquor in the fermentation process in real time, so that the fermentation process is accurately regulated and controlled by multi-parameter coordination, and the method is applied to process regulation and control of pichia pastoris fermentation expression recombinant protein. The expression quantity and the production efficiency of the recombinant albumin can be remarkably improved, the production period is shortened, and the method is suitable for industrial large-scale production.

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.

This patent application discloses a recombinant human type II collagen, its preparation method, and its uses. The recombinant human type II collagen of this patent application is a truncated protein of natural human type II collagen, and its amino acid sequence is shown in SEQ ID No: 1. The recombinant human type II collagen of this invention can be expressed in commercial expression systems such as the commercial Pichia pastoris expression system with high expression yields. Furthermore, its tested biological activities (promoting cell proliferation and wound healing) are significantly superior to those of natural human type II collagen, and also significantly superior to other stages of natural human type II collagen.

The application discloses a kind of multi-effect repair type collagen and its application in the technical field of synthetic biology, the application selects transdermal peptide TD-1, the coding gene of the transmembrane region of human XVII type collagen, extracellular sixteenth non-collagen region and extracellular fifteenth collagen region and His tag is connected in series, and gene sequence is optimized by pichia pastoris codon selection preference, then obtains T-COL17R3 by construction and expression. Through efficacy experiment, it is verified that T-COL17R3 compared with similar products on sale, not only excellent transdermal performance, also has more optimal ability of promoting cell proliferation, migration, anti early glycation product ketone amine and anti late glycation product dicarbonyl compound, better free radical scavenging ability and anti-elastase ability, and can reduce the synthesis of melanin in B16-F10 cell, therefore, T-COL17R3 has excellent application potential in the development of drug composition or skin care product with repair, anti-wrinkle firming or whitening effect.

The application discloses a method for promoting transport and improving retinal synthesis, which is obtained by introducing an exogenous ABC transport protein Pdr5 into a host fungus and adding a copy of beta-carotene-15, 15'-monooxygenase BLH; wherein the host fungus is a genetically modified Pichia pastoris strain PP-BCCIV. The application further verifies the retinal production performance of the recombinant strain by screening different surfactants, and further improves the ability of Pichia pastoris to produce retinal. At the same time, all the retinal is extracted into the extracellular, and all the carotenes are left in the intracellular, so as to achieve the co-production of carotenes and retinal. The recombinant Pichia pastoris of the application has a simple construction method, can better promote the synthesis of retinal and other vitamin A, and can make the engineering strain utilize glucose and methanol to efficiently synthesize retinal through a 5 L fermentation tank, which is beneficial to industrial production.

The application discloses a recombinant nanobody against swine influenza virus of H1N1 subtype, and a preparation method and application thereof, and belongs to the field of biological medicines. A VHH sequence of the nanobody against swine influenza virus of H1N1 subtype is screened from a nanobody bacterial library of an immunized llama, the VHH sequence is directionally cloned into a pPIC9K vector to construct a recombinant eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid is transformed into a Pichia pastoris expression system to screen an expression engineering strain, and the recombinant nanobody is obtained after induction culture of the expression engineering strain. It is proved by in-vitro and in-vivo experiments that the recombinant nanobody developed in the application can neutralize CA / 04 and has a remarkable therapeutic effect. Therefore, the neutralizing nanobody provided in the application has considerable potential in treating CA / 04. The findings will provide experimental data for developing nanobody preparations against swine influenza virus of H1 subtype and provide a new strategy for preventing and controlling swine influenza.

The present invention relates to a method for the expression of genes belonging to different organisms (bacteria, fungi, plants, animals and humans) in P. pastoris cells, comprising the following processing steps;i. Transfer of the gene to be expressed into the plasmid carrying the alcohol oxidase 1 (AOX1) promoterii. Cloning of the plasmid carrying the geneiii. Transfer of the recombinant plasmid carrying the AOX1 promoter and gene into the expression hostiv. Induction of the AOX1 promoter with farnesol and consequent expression of the heterologous gene.The present invention will be used in the field of recombinant protein production.

The invention provides recombinant II-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The recombinant II-type collagen monomer is obtained by splicing Gly-X-Y repetitive gene sequences of multiple sections of triple helix regions in human II-type collagen, immunodominant epitopes, unstable amino acid sequence 3 conjunctions and glycosylation sites are avoided during selection, then the recombinant II-type collagen monomer is repeated for multiple times, and the recombinant II-type collagen monomer is obtained. The recombinant II type collagen with the molecular weight of 40-50 kDa is obtained; the recombinant II-type collagen is beneficial to expression of pichia pastoris, has the effects of promoting adhesion and migration of cartilage cells, can also promote differentiation into cartilage cells, and has good practicability.

The invention discloses an efficient preparation and application method of antibacterial peptide special for livestock and poultry, and belongs to the technical field of bioengineering and livestock and poultry breeding. Aiming at the problems that the existing antibacterial peptide preparation process is complicated, the yield is low, the targeting property is poor, the antibacterial peptide is easily degraded by gastrointestinal tracts of livestock and poultry in application, and the balance of intestinal flora is damaged, the pichia pastoris modified by genetic engineering is adopted as an expression host, and the efficient secretory expression of the antibacterial peptide is realized by optimizing a two-stage fermentation process and a culture medium formula. Complex purification steps are not needed, the yield of the antibacterial peptide reaches 85% or above, the activity retention rate is increased by 40%, the intestinal pathogenic bacteria of livestock and poultry can be specifically inhibited, no damage is caused to probiotics, the antibacterial peptide can be prepared into feed additives, oral preparations and the like to be applied to livestock and poultry breeding, antibiotic dependence is reduced, the breeding cost is reduced, and remarkable economic and ecological benefits are achieved.

This application provides a method and apparatus for optimizing the codon sequence of human albumin, belonging to the field of data processing technology. Addressing the current problems of low synthesis efficiency and scarcity of human albumin drugs, it allows for the grouping of large datasets of human albumin amino acid sequences. An improved genetic optimization algorithm is then used to optimize the corresponding messenger RNA item by item, ultimately obtaining the optimal second target messenger RNA. When using this second target messenger RNA for protein expression to obtain the target recombinant human albumin, the expression efficiency of the host Pichia pastoris cells can be maximized, significantly improving the current synthesis efficiency of recombinant human albumin and greatly alleviating the problem of human albumin drug scarcity.

The invention relates to the technical field of biology, in particular to a recombinant pichia pastoris engineering bacterium, a construction method of the recombinant pichia pastoris engineering bacterium and application of the recombinant pichia pastoris engineering bacterium in secretory expression of ovotransferrin. The heterologous expression of ovotransferrin is effectively realized by optimizing ovotransferrin genes and utilizing molecular chaperone genes to strengthen the regulation and control capability of hosts, and meanwhile, by optimizing signal peptides and constructing high-copy recombinant pichia pastoris expression strains, the low-copy expression condition is verified and compared by utilizing fermentation expression, so that the heterologous expression of ovotransferrin is realized. According to the high-copy recombinant pichia pastoris engineering bacteria constructed by the invention, the ovotransferrin expression quantity is obviously improved and reaches 140 mg / L, the secretory expression efficiency of the ovotransferrin is effectively improved, and theoretical guidance is provided for heterologous expression and industrial production of the ovotransferrin.

The present invention relates to a method for constructing a host bacterium with high expression of human serum albumin, which method comprises: S100, designing an optimized gene encoding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector into a Pichia pastoris host bacterium. The optimization design of a full-length sequence of a target gene is performed at the molecular level to obtain a target gene with the most suitable expression in Pichia pastoris; at the cellular level, the latest international Pichia pastoris expression system host bacterium CBS7435 is introduced for the gene expression of human serum albumin; and Pichia pastoris transformants harboring high-copy-number exogenous genes are selected by means of using a G418 resistance gene for the construction of an engineering bacterium with high expression of recombinant human serum albumin.

The invention relates to pichia pastoris with high yield of Brazilian sweet protein and application of the pichia pastoris, and belongs to the technical field of fermentation. The invention finds that two molecular chaperones ERV29 and CPR5 from saccharomyces cerevisiae jointly strengthen the expression of the Brazilian sweet protein so as to significantly improve the yield of the Brazilian sweet protein, the Pichia pastoris provided by the invention is subjected to fermentation culture, the yield of the Brazilian sweet protein in a shake flask reaches 1.8 g / L, the yield of the Brazilian sweet protein in a 5-L fermentation tank reaches 11.3 g / L, which is the highest yield of the Brazilian sweet protein in the existing strain, and the Pichia pastoris provided by the invention has a wide application prospect. The method has a wide application prospect in industrial production of Brazilian sweet protein.

The invention discloses a Pichia pastoris indirect surface display system based on a TRAP protein scaffold, which is characterized in that Sed1p protein from saccharomyces cerevisiae is used as anchoring protein, and tetrapeptide repeat affinity protein TRAP is fixed on the anchoring protein on the surface of a Pichia pastoris cell to obtain the Pichia pastoris indirect surface display system based on the TRAP protein scaffold. The target protein with the carboxyl end fused with the peptide MEEVV or the peptide MRRVW is indirectly displayed on the surface of the pichia pastoris cell through specific binding with the tetrapeptide repeat affinity protein TRAP. The xylanase and the beta-xylosidase are indirectly displayed on the surface of the pichia pastoris cell by utilizing the surface display system, so that the pichia pastoris surface display system has the catalytic capabilities of the xylanase and the beta-xylosidase, can perform a synergistic effect to degrade the zelkova xylan, and has a good application prospect.

The invention relates to the technical field of biology, in particular to a pagrosomus major antibacterial peptide Pm-NK-lysin as well as a preparation method and application of the pagrosomus major antibacterial peptide Pm-NK-lysin. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. The minimum inhibitory concentration of the antibacterial peptide to vibrio anguillarum is not higher than 8 [mu] g / ml, and / or the minimum inhibitory concentration of the antibacterial peptide to escherichia coli is not higher than 4 [mu] g / ml. The antibacterial peptide is prepared through a pichia pastoris expression system. The antibacterial peptide is applied to preparation of an antibacterial preparation. According to the invention, efficient secretory expression of recombinant Pm-NK-lysin is successfully realized by creatively utilizing a pichia pastoris eukaryotic expression system, and a protein purification process is optimized, so that sufficient sources of high-activity recombinant protein are guaranteed. The Pm-NK-lysin provided by the invention has an excellent killing effect on aquatic pathogenic bacteria. The antibacterial peptide Pm-NK-lysin is used for replacing or reducing the use of antibiotics to control the harm of aquatic bacterial diseases, has wide popularization and application values, and certainly has far-reaching significance for promoting the green and healthy development of the aquaculture industry.

The invention relates to the technical field of traditional Chinese medicine preparations, and discloses a novel pill for treating diabetes mellitus and a preparation method, and the novel pill comprises the following raw materials in parts by mass: 30-40 parts of Chinese yam, 10-15 parts of poria cocos, 20-25 parts of radix polygonati officinalis, 25-35 parts of guava, 30-35 parts of radix rehmanniae recen, 28-32 parts of radix puerariae, 30-35 parts of corn stigma, 20-30 parts of radix trichosanthis, 25-30 parts of acanthopanax and 20-30 parts of rhizoma polygonati. The preparation method comprises the following steps: S1, screening, cleaning, crushing and homogenizing; s2, performing protein denaturation treatment, performing multi-stage enzymolysis, performing enzyme inactivation and filtration, and performing ultrafiltration membrane separation; s3, carrying out gene fusion to construct a fusion gene expression vector, transferring into pichia pastoris for expression, and purifying; s4, wrapping the target peptide fragment with the human albumin fusion protein; and S5, by taking the hydroxypropyl methylcellulose aqueous solution as an adhesive, molding in a pill pan, pelleting, shaping and drying.

Provided are a high-stability recombinant type I collagen and a use thereof, relating to the technical field of collagens. By means of the directional selection of existing amino acid sequences, potential enzyme cutting sites and unstable sequence fragments are avoided during sequence design, enabling efficient expression in Pichia pastoris; and in addition, after being purified, the recombinant type I collagen still has high stability. The recombinant type I collagen takes into account factors such as related biological active sites and hydrophilicity, has related biological activity while maintaining high stability, and has good practicability.

The invention provides a coding gene of canine serum albumin, an expression vector, a recombinant engineering bacterium, and a preparation method and application of the recombinant engineering bacterium of canine serum albumin, the coding gene of canine serum albumin comprises a co-translation translocation chimeric signal peptide sequence and a canine serum albumin mature peptide sequence, the co-translation translocation chimeric signal peptide sequence comprises an Ost1 signal peptide pre-peptide region and an alpha-factor signal peptide pro-peptide region of saccharomyces cerevisiae, and the Ost1 signal peptide pre-peptide region and the alpha-factor signal peptide pro-peptide region are chimeric to obtain the co-translation translocation chimeric signal peptide sequence. According to the coding gene of the dog serum albumin provided by the invention, the efficient secretory expression of the dog serum albumin in pichia pastoris cells is realized by utilizing the synergistic effect of an Ost1 signal peptide pre-peptide region and an alpha-factor signal peptide pro-peptide region in a co-translation translocation chimeric signal peptide sequence.

The application discloses IV type recombinant collagen protein and a preparation method and application thereof, and relates to the technical field of genetic engineering. The recombinant collagen protein comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is obtained by repeating a collagen monomer fragment as shown in SEQ ID NO. 2 for 2-10 times. The application designs and obtains a recombinant collagen amino acid sequence which can be stably and efficiently expressed in Pichia pastoris and has excellent biological activity by accurately screening and optimizing the functional region sequence of human natural IV type collagen. The sequence is 100% homologous to the corresponding region of human natural IV type collagen, thereby ensuring low immunogenicity of the product and excellent biocompatibility with the human body from the source.

The present application relates to a Pichia pastoris indirect surface display system based on SpyC / T, construction and application thereof, and relates to the technical field of bioengineering. The Pichia pastoris indirect surface display system uses SED1 protein from Saccharomyces cerevisiae as an anchor protein, and fixes SpyC from Streptococcus pyogenes on the anchor protein on the surface of Pichia pastoris cells. The present application uses SED1 protein from Saccharomyces cerevisiae as an anchor protein, displays SpyC on the surface of Pichia pastoris, fixes various target proteins containing SpyTag on the cell surface through strong interaction of SpyC / T, so as to indirectly display the target proteins containing SpyTag on the surface of Pichia pastoris, exert the function of the target proteins containing SpyTag, and improve the stability of the target proteins.

The invention provides a recombinant mutant dextranase from chaetomium graciliforme as well as a construction method and application thereof, and belongs to the technical field of mutant enzymes. The recombinant mutant dextranase provided by the invention is CgDEXA289V, and the amino acid sequence of the recombinant mutant dextranase is as shown in SEQ ID NO: 21. Non-conservative site mutation is carried out on the basis of a Chaetomium gracile-sourced dextranase coding gene, a high-enzyme-activity mutant is preliminarily screened out through escherichia coli and is expressed in a pichia pastoris expression system, the high-performance mutant enzyme CgDEXA289V is obtained, the enzyme is endo-dextranase and has good substrate affinity for a substrate, and the mutant enzyme CgDEXA289V can be used for preparing a high-performance mutant enzyme CgDEXA289V. Due to higher enzymatic reaction rate and better stability, the enzymolysis preparation efficiency can be improved, the production cost is reduced, and the method has a better application prospect.

The invention relates to the technical field of genetic engineering, in particular to a recombinant pichia pastoris strain for expressing six tandem antibacterial peptides MPX as well as a construction method and application of the recombinant pichia pastoris strain. Aiming at the technical problems that the expression quantity of MPX in pichia pastoris is low, MPX is easily degraded by endogenous protease, the product activity is insufficient and the like, the invention constructs a recombinant pichia pastoris strain for expressing the six-tandem antibacterial peptide MPX, and a construction method comprises the following steps: designing and constructing a coding sequence of the six-tandem antibacterial peptide MPX; and a recombinant expression vector pPICZ alphaA-6MPX for expressing the six-tandem antibacterial peptide MPX is constructed. A linearized recombinant vector is introduced into pichia pastoris GS115 through a chemical conversion method, a positive transformant is screened through a bleomycin-containing selective culture medium, and a recombinant strain for stably expressing six tandem MPX is obtained by combining PCR and Westernblot identification. The recombinant strain can be used for inhibiting growth of pathogenic bacteria and has application potential as an antibacterial active preparation.

The present application relates to the technical field of recombinant protein, and particularly relates to screening of a recombinant keratin K35 mutant, construction of a Pichia pastoris expression vector and protein expression. The recombinant keratin K35M has an amino acid sequence as shown in SEQ ID NO. 2. The wild-type keratin K35 is subjected to sequence optimization through site-directed mutation, and the mutation site is determined as M237L / S243N, and part of the amino acids at the N-terminal and C-terminal are removed, and the central alpha helix rod-shaped region is reserved. The binding capacity of the protein to calcium ions is improved through the above modification, and the stability, functional activity and expression level of the protein itself are improved, which lays a molecular foundation for the application of the protein in tissue repair, oral cavity repair and the like. The technical scheme can solve the problems of the existing recombinant keratin K35, such as insufficient calcium chelation capacity, low recombinant expression level and disadvantages of a prokaryotic expression system, and significantly improves the application value and industrialization potential of the recombinant keratin.

The invention discloses a method for expressing trastuzumab in pichia pastoris, and belongs to the technical field of genetic engineering. The preparation method comprises the following steps: optimizing heavy chain and light chain genes of trastuzumab, constructing a recombinant plasmid containing double expression cassettes, linearizing the plasmid, and integrating the plasmid into a pichia pastoris genome to obtain an initial strain; and respectively integrating Sch9, Bmh2, PDI and MPDI gene expression cassettes at neutral sites of the genome by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats) technology to obtain engineering bacteria. The engineering bacterium solves the problems of chain ratio imbalance, low assembly efficiency and the like by co-expressing a molecular chaperone, ensures that the antibody is correctly folded and assembled, the expression quantity is remarkably improved compared with that of an initial strain, and a product only has a heavy chain containing N-glycosylation modification and is stable in activity, so that a low-cost and high-efficiency trastuzumab production platform is provided, and the engineering bacterium is suitable for large-scale industrial application.

The invention provides pichia pastoris for producing a honey truffle sweetening agent as well as a construction method and application thereof, and relates to the technical field of microorganisms. According to the invention, high-efficiency expression of a natural sweet protein honey truffle sweetening agent is realized in pichia pastoris cell engineering, a screened signal peptide C terminal is connected with a Pro region of an alpha mating factor to obtain a fusion signal peptide, and heterologous expression of a honey truffle sweetening agent coding gene is realized through the fusion signal peptide. The pichia pastoris capable of efficiently secreting and expressing the target sweetening agent in an extracellular mode is successfully constructed, the pichia pastoris can be used for efficiently producing the honey truffle sweetening agent, the shake flask yield reaches 44 mg / L, and an efficient and feasible new strategy is provided for large-scale microbial production of the honey truffle sweetening agent.

The application discloses a recombinant membrane-targeting type 17 collagen protein and application thereof. The application realizes gene fusion of a COL17A1 key function domain and an independent membrane-targeting structure domain for the first time, constructs a novel "activity-targeting" integrated recombinant membrane-targeting type 17 collagen protein (M-C17a1), effectively integrates function activity and cell targeting property through ingenious structure design, and exhibits excellent performance in resisting skin aging, tissue repair and promoting hair growth. The application also proposes a complete preparation method based on a Pichia pastoris expression system. The expression system has high-density fermentation capacity and secretory expression characteristics, significantly simplifies a purification process, and has relatively low production cost, so that the expression system is suitable for large-scale production of the recombinant protein and has good industrial application prospect.

The application discloses a pichia pastoris bioengineering bacterium for relieving degradation of recombinant collagen, a preparation method and application thereof. Through bioinformatics analysis, gene knockout and function verification screening, three protease genes capable of relieving degradation of collagen in a pichia pastoris host are obtained, namely, chr1-4_0611, chr1-4_0362 and chr2-2_0380. Knocking out the protease chr1-4_0611 can make the total protein yield accumulation increase to 3.5 times of that of a control bacterium, and the proportion of a target band is greater than 50%, which can provide good theoretical and technical guidance for effective expression of other recombinant proteins in the pichia pastoris host.