69 results about "Secretion expression" patented technology

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 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.

The invention relates to the technical field of bioengineering and environmental biology, and particularly discloses high-temperature-resistant polylactic acid depolymerases and application thereof in degradation of polylactic acid plastics. The nucleotide sequence of the coding gene of the high-temperature-resistant polylactic acid depolymerases B01 is as shown in SEQ ID NO: 1, and the amino acid sequence of the coding gene is as shown in SEQ ID NO: 2. The enzyme is derived from metagenome mining, the sequence similarity of the enzyme and the known PLA depolymerases PAM is only 34.45%, and the enzyme has remarkable novelty. According to the present invention, the secretory expression and the purification of the B01 are successfully achieved in the bacillus subtilis SCK6; an enzymatic property research shows that the optimal reaction temperature of the B01 is 65 DEG C, the half-life period at 60 DEG C is as long as 150 minutes, and the B01 shows excellent thermal stability; and the optimum pH value of the strain is 9.0. The invention provides a novel enzyme preparation with important application potential for high-temperature biological recovery treatment of PLA plastic waste.

The present application relates to the field of biosynthesis and microbial fermentation engineering technology, in particular to a kind of secretion expression human lactoferrin Yarrowia lipolytica and its construction method and application.The present application provides a kind of secretion expression human lactoferrin recombinant Yarrowia lipolytica, the secretion expression of human lactoferrin in the recombinant Yarrowia lipolytica is guided by secretion signal peptide;The expression of chaperone HAC1, Cne and Pdi1 is simultaneously enhanced.The present application realizes the secretion expression of human lactoferrin in Yarrowia lipolytica, effectively promotes the folding and secretion expression of human lactoferrin by the cooperation of chaperone, significantly improves the yield of human lactoferrin, and has important application value in the industrial scale production of human lactoferrin.

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 invention discloses a method for secretory expression of recombinant mechanical structural protein by using escherichia coli and application, and relates to the field of bioengineering. According to the method, signal peptides are introduced to the N end of recombinant mechanical structural proteins, and the signal peptides, namely OmpA, PelB, PhoA, DsbA and STII, capable of guiding different recombinant mechanical structural proteins to secrete and express are screened from six natural signal peptides from escherichia coli and other species. Meanwhile, the yield of extracellular protein is further improved by combining an amino acid tRNA supplement strategy. According to the method, the recombinant escherichia coli strain capable of efficiently secreting and expressing the mechanical structure protein can be obtained, the downstream purification process is simplified, and the method has important application value.

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 application relates to a recombinant type I collagen and application thereof, which utilizes the characteristics of Pichia pastoris capable of secreting and expressing recombinant proteins, secretes the recombinant type I collagen on the culture medium supernatant, and reduces the difficulty of protein purification. In addition, the integrin site RGD is inserted into the collagen to form a recombinant type I collagen with high stability and high expression. According to the relative proliferation of cells and the relative adhesion experiment of cells, it can be seen that the recombinant type I collagen has the effects of promoting the proliferation of skin fibroblasts, improving the skin, promoting wound healing, promoting the adhesion of fibroblasts in the skin layer and the like. Therefore, the recombinant type I collagen has great potential applications in the fields of skin care and beauty, medical treatment and the like.

The application provides a method for detecting secretion expression of a target protein, which comprises the following steps: fusing the target protein with a fluorescence-enhanced nanobody to obtain a fusion protein; adding the obtained fusion protein into a system containing a fluorescent protein and a fluorescence-inhibited nanobody to perform a reaction; and measuring the fluorescence intensity of the system after the reaction is completed, and calculating the secretion expression amount of the target protein according to the change amount of the fluorescence intensity. In the application, the GBP1 is fused with the target protein, and the expression interference of the GBP1 on the target protein is reduced by adding a connecting peptide, so that the secretion expression amount of the target protein is determined. The method of the application can detect a wide concentration range of the target protein, has a high signal response multiple, and can be applied to a shake flask, a well plate and a droplet microfluidic screening system.

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 invention relates to the technical field of gene engineering, in particular to high-activity peptide ligase and application thereof in synthesis of tilpotide. The high-activity peptide ligase provided by the invention shows catalytic efficiency far higher than that of an existing enzyme in a specific ligation reaction aiming at an acyl donor and an acyl receptor of a precursor fragment of the tilpoitide; by systematically transforming a host strain, the high-activity peptide ligase which can be secreted and expressed at high level and high fidelity and has correct folding and complete activity is obtained, and meanwhile, the degradation of a product is reduced to the maximum extent; and an optimal expression element combination of the high-activity peptide ligase is obtained through high-throughput optimization of an expression system. The method is finally integrated into a complete new process for synthesizing the tilpoitide by the chemical enzyme method, which can realize kilogram-level production, and is suitable for popularization and application.

The application discloses a method for increasing 2-pyrrolidone synthesis and belongs to the technical field of bioengineering. tacm The CoA transferase from Butyricicoccus faecihominis is expressed in the coryneform bacterium glutamicum, and the CoA transferase is secreted and expressed by means of P The promoter and the NS signal peptide strengthen the secretory expression of the CoA transferase, so that the constructed strain can ferment 2-pyrrolidone in a glucose carbon source and in a pH 7.0 environment, and the yield of the 2-pyrrolidone can reach 12.13 g / L in 96 h of flask fermentation.

This invention relates to bioengineering, specifically disclosing a method for preparing recombinant humanized type III collagen with a triple-helix structure, comprising the following steps: S1, constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment, and transforming the recombinant expression plasmid into yeast; S2, screening high-copy transformant strains from the strain and performing shake-flask level induction expression tests; S3, conducting high-density fermentation in a fermenter to obtain a large amount of secretible target protein. This invention, by constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment and electroporating it into a yeast expression system, screens for yeast strains capable of expressing recombinant humanized type III collagen fragments, and conducts high-density fermentation in a fermenter, followed by purification after fermentation, yields pure recombinant collagen with a triple-helix structure, which can be applied in skincare products, functional skincare products, medical devices, and biomedical materials.

The present application relates to a kind of mediated zymolytic enzyme of brown algae and application of double transcription unit recombinant bacteria of oxyprotein and brown algae, belong to genetic engineering technical field, the recombinant engineering bacteria is composed of two transcription units, one transcription unit is GAP-alpha-Vgb-AOX1TT, another transcription unit is AOX-alpha-102C300C-AOX1TT, two transcription units are expressed in tandem in pPICZ alphaA plasmid.The present application also provides the preparation method and application of the recombinant bacteria.The recombinant bacteria of the present application can improve the secretion expression efficiency of brown algae zymolytic enzyme, especially can be expressed in low-oxygen environment.

The invention relates to an mRNA nucleic acid molecule of an avian reticuloendotheliosis virus and application, and belongs to the technical field of immunology and biologica.According to the nucleic acid molecule, a T7 promoter, a 5 '-untranslated region (5' UTR), a gp90 antigen coding gene, a 3 '-untranslated region and poly (adenosine monophosphate) are used for constructing a recombinant plasmid. According to the invention, a gp90 gene sequence and polyadenylic acid are optimized to obtain the mRNA nucleic acid molecule of the reticuloendotheliosis of poultry, and the mRNA nucleic acid molecule is prepared into the mRNA vaccine; the constructed candidate mRNA vaccine is expressed in a 293T cell and is subjected to extracellular secretory expression.

The invention relates to the field of genetic engineering and fermentation engineering, and particularly discloses a method for exogenous expression of COQ3 by recombinant pichia pastoris engineering bacteria. According to the invention, a target gene COQ2 is inserted into a pichia pastoris secretory expression vector ppicZA to construct a recombinant expression plasmid. The plasmid is transformed into a pichia pastoris GS115 host bacterium to obtain a recombinant pichia pastoris engineering bacterium capable of expressing COQ3. Through methanol-induced fermentation, the engineering bacterium can accumulate COQ3 in cells. The method provided by the invention has the advantages of low cost, easiness in large-scale production and the like, and provides a new way for realizing industrial production of COQ3 and other ubiquinone substances.

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 invention relates to a method for improving the secretory expression level of expansin in bacillus subtilis, and aims to provide a method capable of promoting the bacillus subtilis to secrete expansin (microorganism-derived expansin BsEXLX1 and plant-derived expansin LeEXP2). The method comprises the following steps: firstly, screening a promoter capable of efficiently starting expansin to construct an engineering strain, and then performing overexpression of a secretory cofactor in the strain to improve the limitation of target protein in the aspects of secretory transport, folding maturation, stability and the like, thereby improving the extracellular secretion level of the two expansin. Amplification verification is carried out through a 5L fermentation tank, the secretion level of expansin is further improved, the expression quantity of BsEXLX1 protein is improved to 1.3 g / L, and the secretion quantity of LeEXP2 is improved to 43.7 mg / L. And a process basis and industrialization potential are provided for subsequent large-scale fermentation production and application development.

The invention discloses an antibacterial peptide as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. According to the invention, the antibacterial peptide with high antibacterial activity is designed through an AI technology, a multi-copy (four-copy) recombinant plasmid expression system (pHBM905M-Eel-AMP-4C) based on a pHBM905M vector is constructed, and the antibacterial peptide is efficiently secreted and expressed in pichia pastoris; besides, the antibacterial peptide has excellent thermal stability and pH tolerance, and has a remarkable inhibition effect on common pathogenic bacteria (such as aeromonas hydrophila and vibrio parahaemolyticus) in aquaculture, so that the antibacterial peptide has an important application value in green prevention and control of aquaculture diseases.

The application belongs to the technical field of biology, and discloses a ferritin expression and purification method in bacillus subtilis, wherein the food safety level strain bacillus subtilis WB600 is used as a host, and the synergistic regulation of expression element (promoter, signal peptide) screening, bottom strain (dltD gene knockout, csaA molecular chaperone gene overexpression) modification and induction condition (induction time, temperature, IPTG concentration) optimization is used to successfully realize the high-efficiency secretory expression of Japanese capsule prawn ferritin (MjFer) and H-2 type soybean ferritin (H-2). The transmission electron microscope and dynamic light scattering analysis prove that the two proteins both retain the typical cage structure of ferritin, and the iron oxidation precipitation and reduction release experiment proves that the two proteins retain the basic iron oxidation precipitation and reduction release characteristics of ferritin, which is helpful for the large-scale production of ferritin in the food industry.

This invention discloses a porcine CD40L trimer protein, a recombinant Bacillus subtilis strain expressing this protein, and its applications, belonging to the fields of microbial genetic engineering and livestock and poultry biological products technology. This invention utilizes Bacillus subtilis WB800N as a host and pHT43 as a vector to successfully construct a recombinant strain that efficiently secretes and expresses porcine CD40L trimer protein. Soluble expression of the porcine CD40L trimer protein was achieved. The amino acid sequence of the porcine CD40L trimer protein expressed by the recombinant strain is shown in SEQ ID NO:1. Immunological activity verification using this porcine CD40L trimer protein revealed that the CD40L trimer protein expressed in this invention can simultaneously activate humoral and cellular immunity, possessing practical application value as a novel vaccine adjuvant.

The application discloses a pig IZUMO2 eukaryotic protein and a preparation method and application of a monoclonal antibody of the pig IZUMO2 eukaryotic protein. The full-length sequence of pig IZUMO2 nucleotide CDS is verified, a research method for removing the transmembrane structure of the pig IZUMO2 nucleotide CDS and only expressing the extramembrane sequence of the pig IZUMO2 nucleotide CDS in eukaryotes is designed, and the IZUMO2 protein is expressed and secreted by CHO-K1 cells. The protein expressed by the method is close to natural protein, has biological activity and is simple to operate. The qPCR and ELISA tests verify that the IZUMO2 protein can reduce the expression of inflammatory factors in RAW264.7 cells induced by LPS, and has a certain anti-inflammatory capacity. It is found through experiments that the IZUMO2 protein can promote the capacitation of boar sperm, and it is proved that the protein can be applied to the preparation of a reagent for promoting the capacitation of boar sperm.

The application discloses a novel coronavirus vaccine based on controllable secretion expression of attenuated salmonella, a preparation method and application thereof, and is used for preventing the novel coronavirus. The application constructs controllable, stable and secretory expression plasmids of different antigen domain proteins of the novel coronavirus and attenuated salmonella expression strains of the different antigen domain proteins; a plurality of attenuated salmonella antigen presentation strains which can controllably and secretively express in antigen presenting cells are mixed, and through an oral route, the plurality of different antigen proteins can be efficiently secretively expressed in the antigen presenting cells by means of the unique secretion system of the attenuated salmonella after administration. The secretively expressed antigenic proteins can be effectively processed and presented by the antigen presenting cells, and finally the immune system is activated / regulated, higher titer antibodies are generated, and the vaccine plays a role.

Provided are a cell membrane-expressed IL-15 fusion protein and a use thereof in cell therapy. The provided fusion protein comprises an IL-15Rα signal peptide, IL-15, an IL-15Rα sushi domain, and a transmembrane region; and a cell modified with the fusion protein can achieve efficient expression of IL-15 on the surface of a cell membrane, can effectively avoid side effects caused by the secretion and expression of IL-15, and is safe and controllable. An immune cell modified with a chimeric antigen receptor containing the membrane-expressed IL-15 fusion protein has a stronger tumor killing function, higher IFN-γ secretion, and a greater proliferative capacity; after treatment with lactic acid and TGF-β which are abundant in a tumor microenvironment, the immune cell retain a strong tumor killing function; and the immune cell can activate the anti-tumor function of immune cells other than the immune cell. The constructed immune cell can improve the therapeutic effect against tumors, prolong the duration of disease remission, and improve overall survival in patients.

The invention discloses a recombinant protein as well as a coding gene and application thereof, and belongs to the technical field of bioengineering. The invention designs a recombinant protein which is composed of two subunits with different functions, and design and transformation are carried out on the basis of a conserved sequence of an alpha herpes virus protein with an immune escape function, so that the secretory expression of the recombinant protein is promoted, and the defects of inclusion body formation, overhigh host toxicity and high target protein purification difficulty are overcome. Experimental results show that the recombinant protein provided by the invention has a remarkable effect of inhibiting innate immunity of cells, and can effectively inhibit interferon generated by innate immunity of cells, so that the titer of cultured viruses is improved.

This invention belongs to the field of human Ly6d protein secretion expression technology, and particularly relates to a recombinant expression plasmid, recombinant cells, and construction method for extracellular expression of human Ly6d protein. The recombinant expression plasmid includes a basic plasmid pcDNA3.1 and a target sequence for expressing recombinant human Ly6d protein inserted at the restriction enzyme sites of the basic plasmid. When this recombinant expression plasmid is transformed into EXPI293F cells to construct recombinant cells, it can highly express human Ly6d extracellularly, reaching an expression level of 255.67 mg / 1000 mLCells, which is highly beneficial for the purification and collection of this recombinant protein, providing assistance for subsequent research and production.

This invention relates to the field of vaccine technology and provides a CHO cell line for exocrine expression of PEDV GIIb subtype S protein and its application. The CHO cell line integrates a codon-optimized PEDV GIIb S protein encoding gene carrying the TPA signal peptide into its chromosome. The nucleotide sequence of the PEDV GIIb S protein encoding gene is shown in SEQ ID NO.1. This invention uses currently prevalent GIIb strains as the antigen source to ensure vaccine strain matching; it constructs a secretory expression structure, thereby significantly improving the exocrine expression efficiency of the S protein; it precisely truncates the transmembrane region while retaining key structural domains, maintaining the protein's native conformation and immunogenicity; and it obtains a CHO cell line with stable gene integration and long-term passage to maintain expression levels through optimized transfection conditions and an antibiotic selection system.

The application discloses a sweet protein secretion expression system and a construction method and application thereof, belongs to the technical field of genetic engineering, and specifically discloses an engineered bacterium for expressing thaumatin, an expression vector, a molecular chaperone and an expression method. The thaumatin expressed by the engineered bacterium has a sweet taste after artificial trial, has higher purity and thermal stability compared with natural extraction thaumatin from African canes, the biosynthesis method is more moderate than a chemical extraction method, has higher yield, and is suitable for industrialization.

The invention discloses a bacillus subtilis genetic engineering strain of high-yield 4-alpha-glycosyltransferase (4GT) as well as a construction method and application of the bacillus subtilis genetic engineering strain. The engineering bacterial strain is constructed by taking bacillus subtilis DB403 as a host and introducing a recombinant plasmid pHTN13 / PspovG-PspovG-SPsacB-4GT, and the engineering bacterial strain is constructed by taking bacillus subtilis DB403 as the host. According to the invention, the powerful constitutive tandem promoter PspovG-PspovG is combined with the efficient signal peptide SPsacB, and is combined with the 4GT gene, so that the secretory expression efficiency of 4GT in bacillus subtilis is remarkably improved. According to the engineering strain, under the optimized fermentation condition, the extracellular 4GT enzyme activity of shake flask fermentation can reach 88.53 U / mL and is improved by 96.21% compared with that of an original strain. The constructed engineering strain can be used for producing the 4GT enzyme preparation with low cost and high efficiency, and has important application value in the fields of starch modification, functional food development and the like.

The invention is related to methods of producing rod-derived cone viability factor (RdCVF). This invention also relates to the treatment of an ocular disease in a mammal using RdCVF. Also provided are expression vectors for high secreted expression of RdCVF of using nucleotide sequences encoding heterologous signal proteins and optionally markers for furin cleavage.