167 results about "Secretion expression" patented technology

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 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 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 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 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.

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.

The present invention discloses an engineered protein glutaminase mutant and its method for active secretion expression in Pichia pastoris. The present invention successfully improves the catalytic activity of FBPG through surface charge modification, and the specific activity of mutant F3 reaches 9.43 times that of the wild type. The secreted expression of FBPG is achieved in Pichia pastoris, and under the regulation of the P DH promoter, the active expression of FBPG is achieved by introducing the Kex2 cleavage site. After overexpressing the transcription factor Sko1, the yield of active FBPG reaches 2610 U / L in a 7 L bioreactor, providing a feasible technical solution for the application of FBPG in the food industry, and also providing new ideas and methods for the active expression of similar enzymes.

The application discloses a method for conformational signal peptide-mediated secretion and expression of heterologous proteins in Escherichia coli, and specifically comprises the following steps: simultaneously fusing at least two heterologous proteins to a conformational signal peptide (such as sfGFP) and constructing a heterologous recombinant protein expression vector, wherein the conformational signal peptide has a beta-barrel structure, and the fusion sites are at least two of the amino terminal end, the carboxyl terminal end and a plurality of loop rings of the conformational signal peptide; and then transforming the heterologous recombinant protein expression vector into Escherichia coli and culturing the obtained genetic engineering strain for expression. According to the method, the extracellular secretion and expression of the heterologous proteins in the Escherichia coli can be realized without the mediation of a classical signal peptide, the sfGFP as a fusion tag has no influence on the function of the target protein, and the corresponding target protein can be quickly expressed and purified; and the method expands the secretion and expression path of the protein, can realize the simultaneous expression of a plurality of heterologous proteins, and can effectively improve the expression amount of the target protein.

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 molecular biology, in particular to a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and application thereof, the recombinant human heparin N-deacetylase constructed in the expression process shows excellent stability and activity, has the ability of specific catalysis of deacetylation of acetyl group in heparin precursor, and can effectively generate deacetylated heparin precursor. This property significantly improves the conversion efficiency and reduces the generation of by-products. The present application optimizes the codon of the recombinant human heparin N-deacetylase, and uses the promoter P AOX1 or P GAP to regulate the expression, so that the expression level of the recombinant human heparin N-deacetylase in Pichia pastoris is significantly improved, and the obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin.

The invention discloses a preparation method of pichia pastoris expression secreting type deglycosylation alkaline phosphatase, and belongs to the technical field of biological engineering. According to the invention, the GS115-BIAP II pichia pastoris genetically engineered bacterium capable of secreting and expressing bovine intestine alkaline phosphatase BIAP II is prepared by using a conventional method. The method comprises the following steps: carrying out site-directed knockout on an ALG3 gene for coding alpha-1, 3-mannosyl transferase and an OCH1 gene for coding alpha-1, 6-mannosyl transferase by using a CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated Protein 9) technology to prepare a glycosylation gene defect type engineering bacterium GS115-delta ALG3-delta OCH1-BIAP II. The secretory expression BIAP II is deglycosylated alkaline phosphatase, and the specific activity of the secretory expression BIAP II is close to that of natural BIAP II. The deglycosylated alkaline phosphatase can improve the antibody coupling efficiency and marking uniformity under specific conditions. The method has remarkable advantages in chemiluminescence diagnosis, high-sensitivity immunodetection and development of multifunctional biological coupling reagents.

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 present invention relates to a recombinant bacterium producing Arctic-derived β-glucosidase and its application, belonging to the field of biotechnology. The target gene of the recombinant bacterium is β-glucosidase derived from Arctic bacteria Arc12, the nucleotide sequence of which is shown in SEQ ID No: 1, and the nucleotide sequence of the β-glucosidase with signal peptide is shown in SEQ ID No: 2, the signal peptide is Bacillus subtilis signal peptide CitH, and the expression vector is Escherichia coli-Bacillus shuttle plasmid pMA5, which contains constitutive promoter P HpaII The expression strain is Bacillus subtilis WB600. The present invention provides a method for secretory expression of the enzyme under specific promoter, signal peptide, and strain conditions. This method is simple to operate and can be achieved without the need for special inducers. Secretory expression of the enzyme eliminates the need for cell disruption in downstream processing, reducing production costs and possessing promising application prospects.

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 Trichoderma reesei engineering bacterium for secreting and expressing plastic degrading enzyme as well as a construction method and application of the Trichoderma reesei engineering bacterium, and belongs to the technical field of gene engineering. An expression plasmid of the plastic degrading enzyme is constructed, trichoderma reesei is converted, and the trichoderma reesei engineering bacterium for secreting and expressing the plastic degrading enzyme is constructed. In the Trichoderma reesei engineering bacteria, an endogenous protein coding gene cbh1 with the highest secretory expression quantity in Trichoderma reesei is replaced with a gene for coding the plastic degrading enzyme. The Trichoderma reesei engineering strain for secreting and expressing the plastic degrading enzyme is successfully constructed for the first time, and the Trichoderma reesei engineering strain constructed by the invention can significantly increase the productivity of the plastic degrading enzyme, and has potential to support future large-scale application.

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 invention belongs to the technical field of gene recombination, and particularly relates to a recombinant human III-type triple helix collagen, a recombinant yeast strain for expressing the recombinant human III-type triple helix collagen and a construction method of the recombinant human III-type triple helix collagen. The amino acid sequence of the recombinant human III-type triple-helix collagen is shown as SEQ ID NO. 1, and the recombinant human III-type triple-helix collagen has triple-helix conformation close to a natural biological structure of collagen and has good application value and potential. According to the recombinant yeast strain disclosed by the invention, human-derived III-type collagen and proline hydroxylase are co-expressed in yeast cells without YPS1 protease, and meanwhile, a gene beneficial to vesicle transport and protein folding is transferred, so that hydroxylation of the recombinant collagen can be realized, a natural triple helix conformation is formed, and the recombinant yeast strain can be used for preparing a recombinant collagen product. The degradation problem of the collagen in the expression process can be solved, and the extracellular secretion expression of the triple helix collagen is realized.

The invention belongs to the field of gene engineering and the technical field of molecular biology, and particularly relates to double promoters for expressing sucrose phosphorylase and application of the double promoters. The double promoters comprise any one of the following sequences: a nucleotide sequence as shown in SEQ ID NO.1 or a complementary sequence of the nucleotide sequence; 1, and a nucleotide sequence as shown in SEQ ID NO. 2 or a complementary sequence thereof. The sucrose phosphorylase is synthesized in bacillus subtilis by adopting a double-promoter expression system, the effect of the sucrose phosphorylase is obviously superior to the expression effect of a single promoter P43, the extracellular enzyme activity is improved by 403%, and efficient production is realized. Meanwhile, the DNA fragment with the promoter and RBS functions provided by the invention can realize secretory expression of protein genes under the condition of not adding an inducer, provides an effective means for secretory expression of protein by bacillus subtilis by applying the DNA fragment to expression of sucrose phosphorylase, and has a very good development prospect.

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 present invention discloses a genetically engineered bacterium and a method for producing PHB. The present invention also discloses a fusion gene, which comprises a nucleotide encoding the Ncgl1289 signal peptide and the phaC gene, and the sequences of the nucleotide encoding the Ncgl1289 signal peptide and the phaC gene are respectively shown as SEQ ID NO:6 and SEQ ID NO:4. The genetically engineered bacterium is a Corynebacterium glutamicum engineered bacterium, which comprises the fusion gene and secretes and expresses PHA synthase, can efficiently produce PHB, and can improve the yield of poly-3-hydroxybutyrate produced using substrates derived from lignocellulose.

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.