210 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 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 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 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 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 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 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 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 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.

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.