256 results about "Secretion expression" patented technology

The invention discloses recombinant human XVII type collagen as well as a preparation method and application thereof. The recombinant human XVII type collagen is obtained by repeating an amino acid sequence from natural human XVII type collagen. The amino acid sequence of the recombinant human XVII type collagen is derived from natural human XVII type collagen, and the recombinant human XVII type collagen does not generate adverse immune response when being used for a human body. The recombinant human XVII type collagen can be secreted and expressed in a commercial yeast expression system, the expression quantity is greater than 10g / L, the preparation method is simple, and the high-yield recombinant human XVII type collagen can be obtained at low cost; in addition, the cell adhesion promoting activity of the recombinant human XVII type collagen is obviously superior to that of human collagen.

The invention provides a construction method and application of a pichia pastoris strain capable of efficiently secreting and expressing recombinant human-derived protein, and relates to the technical field of synthetic biology. According to the invention, pichia pastoris is used as a chassis strain, a ku70 gene and / or a pep4 gene in a pichia pastoris genome are / is knocked out through a gene editing technology, and a new signal peptide O factor signal is introduced, so that the secretion efficiency of the pichia pastoris for secreting and expressing recombinant human-derived protein can be remarkably improved, the problem of protein degradation is effectively improved, and the recombinant human-derived protein can be obtained. And meanwhile, a high-copy pichia pastoris strain is efficiently obtained, so that the expression quantity of recombinant human protein is improved, and powerful support is provided for research and development of related drugs and biological manufacturing.

The present invention discloses a recombinant sheep β-defensin 2 protein expressed by Pichia pastoris and its application. The recombinant sheep β-defensin 2 gene designed according to the CDS of the sheep β-defensin 2 gene is integrated into the genome of Pichia pastoris GS115, thereby realizing the secretory expression of the target gene on Pichia pastoris. The recombinant sheep β-defensin 2 protein expressed by Pichia pastoris eukaryotic cells has a strong antibacterial effect and has broad application prospects in the development of new antibiotic alternative products and antimicrobial peptides for sheep feed.

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 discloses a trichoderma reesei engineering bacterium for secreting and expressing neutral cellulase and a construction method of the trichoderma reesei engineering bacterium, and belongs to the technical field of genetic engineering. In a trichoderma reesei engineering bacterium, two endogenous acidic cellulase genes cbh1 and cbh2 in the trichoderma reesei are sequentially replaced by genes for coding neutral cellulase, and the trichoderma reesei is converted into the engineering bacterium for secreting and expressing the neutral cellulase; the amino acid sequence of neutral cellulose is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2. The trichoderma reesei engineering strain for secreting and expressing the neutral cellulase is successfully constructed and has a wide pH application range, and a new strain and method are provided for production of the neutral cellulase in the fields of textile and the like.

The invention provides a genetically engineered bacterium and a construction method of the genetically engineered bacterium, and the genetically engineered bacterium is characterized in that a green fluorescent protein sfGFP is used for mediating protein glutaminase, PG (protein glutaminase), PGF (protein glutaminase), PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF and PGF; eC 3.5. 1.44) is efficiently secreted and expressed in engineering bacteria, and belongs to the technical field of biological engineering. The method comprises the following steps: an obtained sfGFP gene is derived from an NCBI database (GenBank numbering: CP035486.1), an obtained PG zymogen gene PP (Propeptide-Protein glutaminase) is derived from a Chryseobacterium prion prgA gene (GenBank numbering: AB046594.1) in the NCBI database, a recombinant plasmid pHT01 / sfGFP-PP is introduced into bacillus subtilis WB800N to obtain an engineering bacterium, extracellular secretion expression of PP is realized, the sfGFP-PP is activated by trypsin to obtain PG, and the PG is subjected to enzyme activation to obtain the recombinant bacillus subtilis. The expression quantity and the secretion efficiency of the PG in the bacillus subtilis are greatly improved. The recombinant PG produced by the method can generate 23.5 U / mL enzymatic activity under activation of trypsin, can be used for improving protein functional characteristics, and has relatively high industrial production and application values.

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

The invention belongs to the technical field of gene engineering, and particularly relates to a strain for efficiently expressing beta-lactoglobulin as well as a construction method and application of the strain. By utilizing the beta-lactoglobulin expression vector disclosed by the invention, beta-lactoglobulin is guided to be secreted out of a host cell by an exogenous signal peptide, and efficient secretory expression of the beta-lactoglobulin in the host cell is realized by integrating a multi-copy expression cassette through a genome and co-expressing ATP enzyme Ssa4 participating in protein folding and coping with pressure; the amino terminal and the carboxyl terminal of the secreted and expressed beta-lactoglobulin do not contain redundant amino acids, and do not contain animal-derived components. Meanwhile, the invention provides a construction method and a fermentation culture method of the engineering strain, compared with a single-copy genetically engineered strain, the expression quantity of the genetically engineered strain in shake-flask fermentation is increased by 2.47 times, and the genetically engineered strain has a good industrial application prospect.

The present invention discloses an antibacterial peptide cecropin A mutant, a recombinant expression vector, a Bacillus subtilis engineering bacterium and applications thereof. By modifying the antibacterial peptide cecropin A, the present invention screened mutants with antibacterial activities superior to those of the original cecropin A, and through the construction of a recombinant expression vector, heterologous expression was carried out using the biosafe strain Bacillus subtilis. Compared with the original antibacterial peptide cecropin A, the antibacterial activities of the mutants of the present invention were increased by 60% and 80% respectively. The present invention uses Bacillus subtilis as a secretion expression system to express the antibacterial peptide cecropin A mutant, which is conducive to large-scale industrial production and cost savings.

The invention provides a recombinant engineering bacterium for preparing recombinant human albumin based on a transport secretion way and application, and relates to the technical field of gene engineering. One or more of endoplasmic reticulum related genes SRP54 and ERJ5, vesicle transport related genes YOS1 and SLY1 and Golgi apparatus related genes GOS1 and YIP1 are integrated into a recombinant engineering bacterium genome through a gene recombination technology, so that efficient secretory expression of recombinant human albumin is realized. According to the method provided by the invention, the expression quantity of the recombinant human albumin is remarkably improved, and the method has a wide application prospect.

The invention discloses an application of knockout of PEP4 and BAR1 genes in reduction of degradation of Kluyveromyces marxianus on lactoferrin. The invention provides application of inhibiting expression of PEP4 protein and BAR1 protein in any one of the following aspects: (A1) reducing degradation of Kluyveromyces marxianus on lactoferrin; and (A2) reducing the degradation of the fermentation supernatant of the kluyveromyces marxianus on lactoferrin. Experiments prove that after the encoding gene of the PEP4 protein and the encoding gene of the BAR1 protein are knocked out in yeast cells, the lactoferrin degradation amount can be reduced, the complete protein amount is remarkably increased, and the method has important significance on increasing the secretory expression yield of the lactoferrin in kluyveromyces marxianus.

The invention provides recombinant VII type collagen with a whitening effect as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. Research finds that the recombinant VII type collagen with a whitening effect, the expression quantity of the recombinant VII type collagen is improved by taking a genetic engineering technology as a means, and expression production of the recombinant VII type collagen is carried out by using a pichia eukaryotic expression system; the recombinant VII type collagen disclosed by the invention is expressed in pichia pastoris, can be efficiently secreted and expressed outside cells, and is not easy to degrade in a purification stage, so that the purification difficulty is reduced; the recombinant VII type collagen has an excellent whitening effect, is suitable for sensitive skin, does not cause skin irritation, itching and scorching hot, and has good practicability.

The invention discloses a novel recombinant human XV type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human XV type collagen is as shown in SEQ ID No. 1. The recombinant human XV type collagen is formed by splicing disconnected peptide fragments from a triple helix region of an alpha1 chain of the natural human XV type collagen, and the recombinant human XV type collagen can be secreted and expressed in a commercial pichia pastoris expression system at high yield and has good biological activity of resisting cancer and inhibiting proliferation and migration of vascular endothelial cells.

The invention relates to the technical field of biology, in particular to recombinant bacillus for heterologous synthesis of ovotransferrin as well as a construction method and application of the recombinant bacillus. According to the invention, bacillus (bacillus subtilis and bacillus amyloliquefaciens) is taken as a host, and the OVT gene is heterologously expressed by using a genetic engineering technology, so that heterologous secretory expression of OVT1 and OVT2 from different sources in chassis cells is realized; meanwhile, the invention also provides a method for more economically and efficiently increasing the expression level of ovotransferrin. Three different molecular chaperones PrsA, DnaK and CasA are further selected for co-expression with the ovotransferrin, correct folding of the ovotransferrin in host cells is promoted, the soluble secretory expression quantity of the ovotransferrin is increased, and the expression efficiency of the ovotransferrin is improved.

The invention discloses an expression vector for secretory expression of bovine lactoferrin, bacillus subtilis and application thereof, and belongs to the technical field of genetic engineering. The expression vector is constructed by taking pMA5 as a skeleton, the expression vector sequentially contains a promoter and an optimized bovine lactoferrin gene, a coding gene of a signal peptide is further connected between the promoter and the optimized bovine lactoferrin gene, and the signal peptide is SPywbN, SPamyE, SPPhoB or SPpus. The optimized bovine lactoferrin gene is obtained through optimization according to codon preference of a host strain, bovine lactoferrin can be smoothly expressed in the host strain, and no toxic effect is generated on the host strain. The signal peptide coding gene can realize secretory expression of the bovine lactoferrin and improve the secretory expression quantity of the bovine lactoferrin, so that the subsequent separation and purification process of the bovine lactoferrin is simplified, and the production cost is saved.

The invention relates to the technical field of biological fermentation, in particular to a recombinant yeast cell and application thereof in production of thaumatin. The recombinant yeast cell contains nucleic acids corresponding to thaumatin, PDI and MSN4 genes, and can functionally express the thaumatin, PDI and MSN4 genes. The invention provides the recombinant yeast cell for co-expressing the chaperonin PDI and the transcription factor MSN4 to improve the secretory expression of the thaumatin, and accidentally, under the synergistic effect of the chaperonin PDI and the transcription factor MSN4, the secretory expression quantity of the exogenous thaumatin is remarkably increased by 3.25 times.

The invention discloses a collagenase mutant, a gene segment, a recombinant plasmid, a recombinant expression system and application thereof, and a method for preparing the collagenase mutant and collagen tripeptide, and belongs to the field of gene engineering and fermentation engineering. The invention provides a collagenase mutant KT231 capable of realizing high-efficiency secretory expression in streptomyces S.lividans TK24 and a preparation method of the collagenase mutant KT231. The collagenase mutant KT231 can be used for efficiently carrying out enzymolysis on the skin of the narrow gadus at 60 DEG C, has excellent heat resistance and higher enzyme activity, and can be used for efficiently preparing the collagen tripeptide from the skin of the narrow gadus. The collagen peptide is compounded with commercially available papaya chymotrypsin, so that the proportion of collagen tripeptide in the fish skin enzymatic hydrolysate can be remarkably increased, and the fish skin enzymatic hydrolysate has no bitter taste. The method has wide application prospects in preparation of products, tissue engineering materials and wound healing accelerators for resisting wrinkles, preserving moisture, repairing damaged skin and improving bone and joint health.

The invention relates to the technical field of genetic engineering biology, and particularly discloses a GLP-1 analogue fusion protein as well as a preparation method and application thereof. According to the application, fGLP-1 and a mutant thereof are respectively fused with FSA, IgG4-Fc and a mutant thereof through connecting peptides to form a GLP-1 analogue fusion protein with the effects of high biological activity, long half-life period, high stability and the like, and meanwhile, after pichia pastoris codon is optimized and modified, an efficient pichia pastoris methanol-induced secretory expression system is utilized for expression; the problems of short half-life period, low biological activity, poor thermal stability, potential immunogenicity, complex production process, high purification and preparation cost and the like of the GLP-1 analogue prepared by the existing expression system and related biotechnology are solved.

The invention relates to the technical field of protein engineering or genetic engineering, and discloses a method for improving the secretory expression efficiency of sweet protein in pichia pastoris. The invention discloses pichia pastoris for secretory expression of sweet protein. The pichia pastoris simultaneously expresses a sweet protein gene, a secretory signal peptide gene and a gene expression regulatory element. The pichia pastoris is subjected to two-stage fermentation culture, the sweet protein can be successfully prepared, and the yield is 4g / L or above. The technical scheme provided by the invention has an extremely high effect of improving the secretory expression efficiency of the sweet protein.

The invention belongs to the field of gene engineering. In particular to high-stability recombinant XVII type collagen as well as a preparation method and application thereof. The recombinant XVII type collagen is obtained by repeating an amino acid sequence (as shown in SEQ ID NO.1) from a natural human XVII type collagen peptide for 2-5 times. The recombinant XVII type collagen can also be secreted and expressed in a pichia pastoris expression system, the preparation method is simple, and the high-yield recombinant XVII type collagen can be obtained at low cost. The cell proliferation, migration and adhesion promoting activities of the recombinant XVII type collagen are obviously superior to those of commercially available XVII type collagen, a triple helix structure can be formed at room temperature, a 1% aqueous solution has high-temperature stability and moist heat sterilization stability, and the recombinant XVII type collagen can be widely applied to the fields of cosmetics and medical instruments.

The invention relates to the technical field of recombinant proteins, and relates to a recombinant XII type collagen as well as a preparation method and application thereof. The recombinant XII type collagen is formed by connecting 5-30 repetitive units in series, and the amino acid sequence of each repetitive unit is as shown in SEQ ID NO: 1. The nucleic acid molecule is used for coding the recombinant XII type collagen; a vector comprises the nucleic acid molecule. According to the invention, a nucleotide sequence for coding the amino acid sequence is inserted into a vector to obtain a recombinant plasmid vector, then the recombinant plasmid vector is introduced into a host cell for culture, and then separation and purification are carried out to obtain the recombinant XII type collagen. The recombinant XII type collagen provided by the invention is easy to obtain, can realize efficient secretory expression, and meets the requirements of industrial production; the biological activity is good, and the proliferation and migration of human immortalized cartilage cells can be promoted.

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a polypeptide for promoting exocrine expression of a target protein, a biological material and application thereof, an antibacterial peptide fusion protein and a recombinant genetic engineering bacterium. As a signal peptide, the polypeptide provided by the invention further improves the exocrine expression efficiency and yield of the target protein; a contrast experiment verifies that compared with the existing signal peptide, the optimized and modified signal peptide has the advantage that the efficiency of exocrine expression of target protein of a constructed lactococcus lactis expression system is improved by 2.1-13.6 times. The recombinant genetically engineered bacterium constructed by the invention successfully realizes efficient exocrine expression of the antibacterial peptide by the lactococcus lactis, has the probiotic property of lactic acid bacteria and the antibacterial effect of the antibacterial peptide, can be directly orally taken or used as an additive to be applied to food and animal feed, and greatly saves the production and preparation cost of the antibacterial peptide.

The invention discloses recombinant human VII type collagen as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. The recombinant human VII type collagen comprises a plurality of amino acid residue repetitive units shown as SEQ ID NO.1. Compared with natural VII type collagen, the recombinant human VII type collagen obtained by repeating the repetitive units for multiple times has the advantages that the molecular weight is smaller, the sequence is more easily and efficiently secreted and expressed in a pichia pastoris system after optimization, and the recombinant human VII type collagen can be used for preparing the recombinant human VII type collagen. The method has the characteristics of high yield, low cost, simplicity in operation and easiness in large-scale production. Meanwhile, the anti-oxidation activity is excellent, the skin moisturizing and repairing capacity and the wound healing capacity are promoted, due to the fact that the collagen is derived from human collagen, the risks of allergy and rejection reaction can be remarkably reduced, and the safety is extremely high. Therefore, the recombinant collagen can be used as a core active component to be widely applied to the fields of cosmetics, medicines and the like, and has a good application prospect.

The invention discloses a trichoderma reesei engineering bacterium for secreting and expressing egg white lysozyme and a construction method of the trichoderma reesei engineering bacterium, and belongs to the field of molecular biology and biotechnology. The invention firstly provides an egg white lysozyme mutant, and the amino acid sequence of the egg white lysozyme mutant is shown as any one of SEQ ID NO.2-4. The construction method of the trichoderma reesei engineering bacterium for secreting and expressing the egg white lysozyme comprises the following steps: by taking trichoderma reesei as a starting strain, transforming an expression module into the trichoderma reesei to construct the trichoderma reesei engineering bacterium; the expression module comprises a promoter, a secretory peptide gene, an egg white lysozyme gene and a terminator which are connected in sequence. The coding gene of the egg white lysozyme is transformed into a trichoderma reesei genome, so that a trichoderma reesei engineering strain for secreting and expressing the egg white lysozyme is successfully constructed. Meanwhile, different egg white lysozyme variants can bring different effects, and when the trichoderma reesei RUT-C30 is used as an original strain, the effect is superior to that of QM6a.

The invention relates to the technical field of oligosaccharide preparation through enzyme catalysis, in particular to a method for preparing a worm oligosaccharide product through cascade catalysis of glycosyltransferase. The method comprises the following steps: secreting and expressing truncated beta 1, 4-N-acetamino galactosyl transferase and alpha 1, 3-fucosyl transferase E by using an insect cell baculovirus expression system to obtain GalNAcT delta TM and FucT-E delta TM; gn [beta] GlyFmoc and Gn2Man3Gn2 [beta] AsnFmoc are used as substrates, and continuous catalysis is carried out, so as to prepare an oligosaccharide intermediate containing LDN epitopes and an oligosaccharide product containing LDN-F epitopes. The method solves the problem of high production cost of an LDN-F epitope-containing oligosaccharide product.

The invention discloses a saccharomyces cerevisiae engineering bacterium for producing heme-binding protein as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. The construction method comprises the following steps: (1) by taking Saccharomyces cerevisiae CEN.PK2-1C as an original strain, optimizing a metabolic pathway, enhancing the supply of cytoplasm alpha-ketoglutaric acid (alpha-KG) and succinyl coenzyme A, and realizing the efficient accumulation of 5-aminolevulinic acid (ALA); (2) regulating and controlling the heme synthesis gene cluster in a time-sharing manner, and cooperatively expressing heme binding protein, so as to improve the assembly efficiency of heme (Heme); and (3) a secretion system is optimized, the secretion bottleneck of eukaryotic protein is broken through, and transmembrane transport and extracellular accumulation of the heme-binding protein are realized. Through the transformation of the combination strategy, an engineering strain for efficiently producing the heme-binding protein is obtained, and the yield of the bean hemoglobin in the saccharomyces cerevisiae is remarkably improved. Under the fermentation condition of a 5L fermentation tank, the secretory expression quantity of the bean hemoglobin reaches 286.6 mg / L.

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 present disclosure relates to chimeric antigen receptor cells targeting ROBO1, in particular, enhanced CAR-T cells and CAR-NK cells targeting ROBO1, and preparation and application thereof. The cells can stably expressing CAR elements, while secreting extracellular domain molecules expressing PD-1 protein or mutants thereof, and thus may block PD-11PD-L1 molecular interaction. It has been found through animal experiments that the cells have very good anti-tumor effects, and the above-mentioned cells can significantly reduce tumor recurrence and improve the survival rate compared with the conventional ROBO1-targeted CAR modified cells.

Provided are a bifunctional recombinant protein GIK for regulating the glycolipid metabolism and resisting aging, and a preparation method therefor. The recombinant protein GIK comprises an N-terminus, a middle section and a C-terminus. The N-terminus is an optimized human GLP-1 polypeptide having an amino acid sequence of SEQ ID NO: 1; the middle section is an Fc region of human IgG4 having an amino acid sequence of SEQ ID NO: 2; and the C-terminus is an s-klotho polypeptide having an amino acid sequence of SEQ ID NO: 3. The N-terminus is connected to the middle section via a connecting peptide 1 having an amino acid sequence of SEQ ID NO: 4; the middle section is connected to the C-terminus via a connecting peptide 2 having an amino acid sequence of SEQ ID NO: 5; and a signal peptide of human serum albumin having an amino acid sequence of SEQ ID NO: 7 is introduced at the N-terminus, such that a bifunctional recombinant protein that can be secreted and expressed in mammalian cells is obtained, and the bifunctional recombinant protein has an amino acid sequence of SEQ ID NO: 8.

The invention discloses a high-biological-activity human-derived I-type collagen variant, a composite material based on the high-biological-activity human-derived I-type collagen variant and a preparation method. According to the invention, the COL109 fragment which can be efficiently secreted and expressed in pichia pastoris and has a triple helix structure is obtained through screening; an adhesion domain (GPP) 10 is further fused to improve the mechanical strength, and a variant COL109 (GPP) 10 is obtained; and finally, compounding with polycaprolactone (PCL) according to an optimized ratio (1: 1) to obtain the composite material with high biological activity (promoting osteogenic differentiation and wound healing) and excellent mechanical properties. The material is suitable for bone tissue engineering scaffolds, skin regeneration dressings and the like.