29 results about "Bovine lactoferrin" patented technology

The invention relates to the technical field of antibacterial peptides, and discloses a preparation method and application of a multi-segment fused antibacterial peptide fusion protein. The antibacterial peptide fusion protein is obtained by introducing OST signal peptide and collagen and optimizing the connection sequence of the OST signal peptide, the collagen, bovine lactoferrin peptide LFcinB, bovine lactoferrin N-leaf small peptide LFcin and bovine lactoferrin N-leaf derived peptide Lf. The antibacterial peptide fusion protein can simultaneously contain three segments of bovine lactoferrin active peptides, namely LFcinB, LFcin and Lf, and the yield of the fusion peptide is maximized. Experiments prove that the fermentation yield of the fusion protein comprising the OST signal peptide, the collagen, the bovine lactoferrin peptide LFcinB, the bovine lactoferrin N-leaf small peptide LFcin and the bovine lactoferrin N-leaf derived peptide Lf which are sequentially connected from the N terminal to the C terminal can reach 600mg / L.

The invention discloses bovine lactoferrin freeze-dried powder and a preparation method thereof, and belongs to the technical field of protein freeze-drying. The preparation method of the bovine lactoferrin freeze-dried powder comprises the following steps: adding a histidine buffer solution into a bovine lactoferrin solution with the mass concentration of 30-40%, and mixing to obtain a mixed solution; adding a freeze-drying auxiliary material accounting for 0.25-7.0% of the volume of the mixed solution into the mixed solution to obtain a bovine lactoferrin freeze-drying solution; performing spray freezing and freeze drying on the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder; the freeze-drying auxiliary materials comprise a surfactant, amino acid, D-chiro-inositol and calcium chloride. Compared with the traditional inositol, the D-chiral inositol in the freeze-dried auxiliary material can more effectively replace water molecules, maintain the natural conformation of protein and prevent the bovine lactoferrin structure from collapsing. The calcium chloride in the freeze-drying auxiliary material can stabilize the iron binding area of the bovine lactoferrin in the freeze-drying process, and the biological activity of the bovine lactoferrin after freeze-drying is guaranteed.

This invention provides an antimicrobial peptide spray, its preparation method, and its application in the prevention and treatment of peristomal skin infections. The spray comprises, by weight percentage, 0.05%-0.5% of a complex antimicrobial peptide, 0.1%-5% of a moisturizing and repairing film-forming matrix, 0.05%-0.2% of a buffer, 0.8%-1.2% of an isotonic regulator, 0.01%-0.5% of a stabilizer, and 0.005%-0.05% of a metal ion chelating agent, with the balance being water for injection. The complex antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a weight ratio of (3-5):(2-3):1. This spray possesses four core functions: broad-spectrum antibacterial activity, inhibition of bacterial biofilm, anti-inflammatory and soothing effects, and skin barrier repair. It is less likely to induce bacterial resistance, does not irritate damaged skin around the stoma, and can effectively prevent and treat peristomal skin infections.

The invention discloses a fusion protein expression system suitable for expressing long-chain helper protein in yeast, which is a gene expression cassette of fusion protein of helper protein and bovine lactoferrin, and comprises a double KREAEA structural domain + flexible Linker tandem helper protein gene and bovine lactoferrin gene, and the fusion protein assists in folding of bovine lactoferrin in cells, so that the expression of long-chain helper protein is realized. The fusion protein is excised in cells, and the secretion efficiency of correctly folded bovine lactoferrin is remarkably enhanced.

The application discloses a complex preparation containing bovine lactoferrin and lyticase and application thereof, and belongs to the technical field of biology. The preparation is composed of bovine lactoferrin and streptococcal lyticase, and both are prepared through a baculovirus-insect cell eukaryotic expression system. Researches show that the complex preparation has a significant synergistic antibacterial effect. The complex preparation not only can effectively inhibit a plurality of gram-positive bacteria, but also can overcome the limitation that single streptococcal lyticase has weak activity on gram-negative bacteria, and significantly enhances the inhibiting effect on escherichia coli and salmonella. Especially, the complex preparation still has clear in-vitro antibacterial activity on multi-drug resistant escherichia coli such as ciprofloxacin and doxycycline, thereby providing a new way for developing a new type of drug for resisting drug-resistant bacteria.

This invention discloses a Bacillus amyloliquefaciens strain expressing bovine lactoferrin, its construction method, and its applications, belonging to the field of bioengineering technology. This invention utilizes the highly efficient protein secretion capacity of Bacillus amyloliquefaciens NFΔSE and the constitutive expression vector pMA5 for recombinant expression of bovine lactoferrin. Further, extracellular secretory expression of lactoferrin is achieved through signal peptide screening, and efficient fermentation synthesis of lactoferrin is realized in a fermenter through strategies such as optimized combined nutrient conditions and enhanced iron supply. Compared to traditional extraction processes, this process can produce lactoferrin through fermentation using this recombinant strain, effectively improving lactoferrin production capacity and demonstrating good industrialization potential.

The present application relates to the Pichia pastoris fermentation medium and fermentation method for expressing recombinant bovine lactoferrin, and belongs to the technical field of biochemistry, which solves the technical problem of improving the expression amount of bovine lactoferrin in the process of industrialized and large-scale preparation of recombinant bovine lactoferrin. The components and their proportions in the Pichia pastoris fermentation medium aqueous solution for expressing recombinant bovine lactoferrin are as follows: yeast extract 10-30 g / L, phosphoric acid aqueous solution 25-35 g / L, calcium sulfate 1.0-3.0 g / L, potassium sulfate 18-22 g / L, potassium hydroxide 4.0-5.0 g / L, glycerol 30-50 g / L, magnesium sulfate heptahydrate 7-13 g / L, ammonium chloride 4-5 g / L, PTM1 trace element solution 4-6 ml / L, and defoaming agent 1-2 ml / L. The high-density fermentation method of the present application is realized by controlling the technical parameters such as the dissolved oxygen amount, pH value, temperature and feeding medium rate in the fermentation process, so that the expression amount of bovine lactoferrin is improved to 4-5 g / L.

The invention belongs to the field of biological materials, and particularly relates to a bovine lactoferrin complex and a preparation method thereof.The preparation method comprises the steps that bovine lactoferrin is subjected to deglycosylation, and deglycosylated bovine lactoferrin is obtained; carrying out dehydration condensation on the de-glycosylated bovine lactoferrin and chitosan to obtain a composite precursor; and compounding the compound precursor with chlorogenic acid to obtain the bovine lactoferrin compound. Compared with single bovine lactoferrin and chlorogenic acid, the bovine lactoferrin compound prepared by the preparation method disclosed by the invention can show a remarkable effect of promoting osteoblast proliferation under a very small concentration, and has an obvious synergistic interaction capability.

The invention discloses a construction method of a lactoferrin hydrolysate oligopeptide database, an identification system and application, and belongs to the cross technical field of bioinformatics, mass spectrum data analysis and milk protein deep processing. Aiming at the problems of low recall rate and insufficient accuracy of the existing general database and tool, the method comprises the following steps: firstly, inputting a bovine-derived lactoferrin full-length sequence, simulating to generate all possible 25 peptide sequences through programmed enzymolysis, and de-weighting; calculating the theoretical b / y ion fragment mass of each peptide fragment in the mass spectrum and the mass-to-charge ratio of each peptide fragment in different charge states; and finally constructing a special structured database containing sequence, molecular weight, state of charge and fragment ion information. Based on the database, the invention further provides a lactoferrin hydrolysate oligopeptide identification system. According to the method and the system, the identification efficiency and the coverage degree of the lactoferrin hydrolysate oligopeptide are remarkably improved, and reliable technical support is provided for active peptide screening and quality control of the lactoferrin hydrolysate oligopeptide in the fields of functional foods, medicines and the like.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The invention belongs to the technical field of biology, and particularly relates to bovine lactoferrin polypeptide and application thereof. According to the invention, two polypeptide sequences QADQFQGKW and LRIPSKVDSAL with antitumor activity are obtained by identifying polypeptide sequences obtained by digesting bovine lactoferrin through pepsin under an acidic condition and combining virtual screening and in-vitro and in-vivo activity experimental analysis. The related discovery of the invention lays a foundation for deep development of bovine lactoferrin and preparation of related products for improving immunity, resisting inflammation and bacteria and preventing and treating tumors.

The invention discloses a recombinant yeast engineering bacterium for secretory expression of recombinant bovine lactoferrin as well as a construction method and application of the recombinant yeast engineering bacterium, and belongs to the technical field of genetic engineering. According to the invention, a CRISPR / CAS9 gene editing technology is utilized to construct sgRNA expression vectors and bovine lactoferrin homologous recombination gene expression cassettes targeting multiple sites of yeast genomes AOX1, AOX2, FLD1, DAS1 and DAS2, and the sgRNA expression vectors and the bovine lactoferrin homologous recombination gene expression cassettes are transformed into yeast host bacteria to obtain recombinant yeast engineering bacteria with bovine lactoferrin genes inserted at multiple sites. The yield of the bovine lactoferrin of the recombinant yeast engineering bacterium is remarkably increased, heterologous secretion and efficient expression of the bovine lactoferrin in pichia pastoris are achieved, industrial production and construction of the bovine lactoferrin are promoted, and the recombinant yeast engineering bacterium has wide application value.

This disclosure provides expression systems comprising secretion signals that promote production and / or secretion of proteins of interest, as well as one or more polynucleotides encoding chaperone proteins (e.g., CRT and / or PDIA3) which, as demonstrate herein, enhance production and / or secretion of proteins. Moreover, genetically modified host cells comprising these expression systems are capable of producing high levels of protein of interest, such as bovine lactoferrin (bLF), bovine lactoglobulin (bLG), or ovalbumin (Ova).

The present invention provides a preparation method and renaturation method of bovine lactoferrin peptide inclusion bodies, which belong to the field of biotechnology. The preparation method includes: designing the coding DNA of bovine lactoferrin peptide Bl fFf 344 based on the preferred codon of Escherichia coli, then constructing an Escherichia coli inducible expression vector and transforming it into Escherichia coli to obtain genetically engineered recombinant expression Escherichia coli; inducing the genetically engineered recombinant expression Escherichia coli to efficiently express the bovine lactoferrin peptide Bl fFf344 and form inclusion bodies with isopropylthiogalactoside to obtain a bacterial mixture; centrifuging the bacterial mixture to collect the Escherichia coli cells, resuspending them and performing a cell wall breaking process, and then centrifuging to collect the bovine lactoferrin peptide inclusion bodies. The method can efficiently prepare bovine lactoferrin peptide inclusion bodies, and combined with the bovine lactoferrin peptide inclusion body separation, dissolution, purification and renaturation method established by the present invention, realizes genetic engineering and efficient production of bovine lactoferrin peptide.

The invention discloses a composite peptide premix feed capable of improving immunity of livestock and poultry aquatic products and a preparation method thereof, and relates to the technical field of feeds. In order to reduce the use amount of antibiotics in the livestock breeding process, a composite peptide component is added into the premix feed, and the use amount of the antibiotics is reduced by adding bovine lactoferrin peptide in the premix feed. Macrophages and natural killer cells are activated, immune globulin secretion is promoted, and the mucosal immune barrier function is enhanced, so that the autoimmunity of livestock is improved, and the disease resistance of the livestock is improved; and in order to avoid the defect that the feed is easy to mildew to generate mildew toxins, mildew-proof modified attapulgite clay is also prepared and added, so that the mildew speed after the premixed feed and the feed are mixed and watered is effectively slowed down, and the diarrhea rate of livestock is reduced.

The invention discloses pichia pastoris capable of producing bovine lactoferrin at high yield and application of the pichia pastoris, and belongs to the field of bioengineering. According to the present invention, after signal peptide screening, promoter engineering modification and codon optimization, the Pichia pastoris strain P.pastoris GS115 / PAOX1-T2S-UBLF5 / PAOX1-TEF1-T2S-UBLF capable of highly producing the bovine lactoferrin is obtained, such that the efficient expression of the bovine lactoferrin in the Pichia pastoris is achieved, and the shake flask fermentation yield is 550.9 mg.L <-1 >. The recombinant bacterium is subjected to high-density fermentation in a fermentation tank, the glucose feeding strategy is optimized, the yield of the bovine lactoferrin is as high as 8.7 g.L <-1 >, and about 6.5 g.L <-1 > of the bovine lactoferrin with the purity higher than 95% is obtained through enzyme digestion separation and purification and is the highest level reported publicly. According to the method, the resource limitation of extracting and obtaining the bovine lactoferrin from traditional milk is broken, and the preparation technology is suitable for industrial large-scale production.

The invention discloses a bovine lactoferrin mutant, a coding gene and application thereof, and belongs to the technical field of protein engineering. According to the invention, beneficial mutation sites N233S, N281A, T370N, T478A and N545Q are obtained by mutation aiming at bovine lactoferrin, so that the bovine lactoferrin mutant with high yield in a pichia pastoris system is obtained. The bovine lactoferrin mutant obtained by the invention has the characteristics of high expression yield of a pichia pastoris system and simplicity in preparation, has potential clinical application value, and also has wide application in medicine, feed and food industries.

The invention belongs to the technical field of aquaculture feed, and particularly relates to a feed composition for strengthening largemouth bass parents and application of the feed composition. Aiming at the physiological development characteristics of parents, functional substances such as arginine, lysine, vitamin E, organic selenium, manganese and zinc required for promoting gonad development are supplemented, the brood amount is further increased, and the gonad index is improved. In order to improve the health condition of parents and vertically spread the good health condition to the offspring, substances for healthy development of the parents and the offspring, such as biological fermentation fibers, beer yeast, astaxanthin and bovine lactoferrin peptide, are supplemented, so that the fertilization rate, the hatching rate, the emergence rate and the survival rate of offspring seeds are increased. According to the parent strengthening feed, the synergistic interaction effect of lecithin oil, vitamin E, organic trace elements and biosynthetic substances such as bovine lactoferrin peptide and astaxanthin on the reproductive capacity of aquatic animal parents is greatly exerted, and the application effect advantages in the aspect of improving the brood amount, the fertilization rate, the emergence rate and the filial generation survival rate of micropterus salmoides are obvious.

The application discloses a fermentation medium and a fermentation method for expressing bovine lactoferrin, and belongs to the technical field of fermentation. The fermentation medium comprises the following components: yeast extract 10-20 g / L, proteose peptone 20-30 g / L, glycerol or glucose 10-20 g / L, yeast nitrogen base without amino acid 12-15 g / L, dipotassium hydrogen phosphate 4-6 g / L, potassium dihydrogen phosphate 2-3 g / L, biotin 0.0002-0.0005 g / L, amino acid 4-46 g / L, ferrous salt 2-5 g / L, sugar material 5-10 g / L and sugar material derivative 5-10 g / L. The fermentation medium can meet the growth and metabolism requirements of the bacteria and improve the expression amount of bovine lactoferrin.

The invention discloses a recombinant engineering bacterium for efficiently expressing a bovine lactoferrin functional fragment as well as a construction method and application of the recombinant engineering bacterium, and belongs to the field of microbial engineering. The construction method of the recombinant engineering bacterium comprises the following steps: carrying out optimization synthesis on a coding gene of a bovine lactoferrin functional fragment BLF-C4 according to codon preference of pichia pastoris, and cloning the coding gene to a pichia pastoris protein expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into a pichia pastoris host cell to obtain a recombinant engineering bacterium capable of secreting and expressing a bovine lactoferrin functional fragment; the amino acid sequence of the bovine lactoferrin functional fragment BLF-C4 is as shown in SEQ ID NO. 7. The lactoferrin secreted and expressed by the recombinant engineering bacterium constructed by the invention is a part with biological activity in macromolecules of bovine lactoferrin, has theoretical molecular weight and conformation similar to those of natural lactoferrin, and remarkably improves the expression level of lactoferrin derived functional polypeptide.

The present application provides a dual-source lactoferrin composition and a formula food. The dual-source lactoferrin composition comprises bovine lactoferrin and camel lactoferrin. The dual-source lactoferrin composition can overcome the limitations of single-source lactoferrin in the prior art in terms of antibacterial effect, cost and functional suitability, and the formula food comprising the dual-source lactoferrin composition has excellent antibacterial property, solubility and stability, and has excellent market application prospect.

The present application belongs to the field of biotechnology, in particular, the present application relates to bovine lactoferricin polypeptide and its application. The present application obtains two polypeptide sequences QADQFQGKW and LRIPSKVDSAL with anti-tumor activity through identification of polypeptide sequence obtained by pepsin digestion of bovine lactoferricin under acidic conditions, combined with virtual screening and in vitro and in vivo activity experiment analysis. The related findings of the present application lay a foundation for in-depth development of bovine lactoferricin, preparation of related products for improving immunity, anti-inflammatory and antibacterial, and preventing and treating tumors.

The application discloses a recombinant Pichia pastoris genetic engineering bacterium with high yield of lactoferrin, and simultaneously satisfies the following three modifications: overexpression of a bovine lactoferrin expression gene, mutation of an alpha mating factor signal peptide at position E86 to F, and overexpression of a PDI molecular chaperone gene. The application also discloses a construction method of the recombinant Pichia pastoris genetic engineering bacterium, and application of the recombinant Pichia pastoris genetic engineering bacterium in production of bovine lactoferrin. The 5L fermenter test result of the bacterium strain shows that the yield of exogenous protein is significantly improved, and is increased from 10.04 mg / L in a flask fermentation to 95.78 mg / L, nearly 10 times. The application is based on the Pichia pastoris system and is not related to complex culture condition change, and is low in cost and easy to enlarge. The method is not only suitable for production of bovine lactoferrin, but also provides a new modification idea for high-efficiency expression of other heterologous proteins in the yeast system.

The invention discloses a compound preparation containing bovine lactoferrin and lyase and application of the compound preparation, and belongs to the technical field of biology. The preparation is composed of bovine lactoferrin and streptococcus lyase which are both prepared through a baculovirus-insect cell eukaryotic expression system. Researches show that the compound preparation has a remarkable synergistic antibacterial effect. The streptococcus lyase not only can effectively inhibit various gram-positive bacteria, but also can overcome the limitation that single streptococcus lyase has weak activity on gram-positive bacteria, and obviously enhances the inhibition effect on escherichia coli and salmonella. Particularly, the preparation still has definite in-vitro bacteriostatic activity on multi-drug-resistant escherichia coli such as ciprofloxacin and doxycycline, and a new way is provided for developing novel drugs for resisting drug-resistant bacteria.

The invention belongs to the technical field of bioengineering, and particularly relates to lactoferrin-producing pichia pastoris for enhancing iron ion supply and a construction method and application thereof. According to the invention, by utilizing a genetic engineering means, the pichia pastoris also expresses the iron transporter DZIP13 / HZIP13 gene while expressing lactoferrin, so that the iron ion supply in a secretion way is enhanced, and the correct folding of bovine lactoferrin is improved. Fermentation results show that the strategy can also significantly increase the content of lactoferrin in the supernatant of the fermentation broth, and the method can effectively promote expression and secretion of lactoferrin in pichia pastoris, and has good application prospects.

The present invention relates to a method of treating a disease or condition that is associated with abnormal TNF / TNFR1 signaling in a subject, comprising administering to the subject peptides derived from bovine lactoferrin, which target the conformationally active region of TNFR1 and inhibit its signaling. In particular, the peptide is FKCRRWQWRMKK (SEQ ID NO: 7) and the disease or condition that is associated with abnormal TNF / TNFR1 signaling is inflammation. Also disclosed are new peptides having improved potency to inhibit ligand induced NF-KB activation downstream of TNFR1 signaling. In a preferred embodiment, the new peptide is GKCLRWNWRFKK (SEQ ID NO: 3).

The invention provides a bovine lactoferrin-phosvitin compound and a preparation method thereof. The phosvitin provided by the invention contains a large amount of phosphorylated serine, so that the phosvitin has relatively high negative charges, and forms a compound with a structure taking LF as a core and PSV as a shell with bovine lactoferrin through the synergistic effect of electrostatic interaction and hydrogen-bond interaction, so that the stability coefficient of the phosvitin is increased, the denaturation temperature is increased by 1-16 DEG C, and the denaturation time is shortened by 1-16 DEG C; the adverse effect of heat treatment on the lactoferrin is reduced, the heat stability of the lactoferrin is enhanced, the oxidation resistance is not affected, and the application range of the lactoferrin is expanded.

The present invention discloses an amylase signal peptide variant mutant SEQ ID NO: 2 or 3 generated by a natural language model. The encoding gene thereof is fused with a bovine lactoferrin gene or a thaumatin gene and expressed in Aspergillus. The variant mutant can improve the secretion and expression of bovine lactoferrin and / or thaumatin by Aspergillus and enhance the production economy of bovine lactoferrin or thaumatin.

The application discloses a collagen short peptide mutant fusion protein and application thereof in preparation of lactoferrin peptides, and the amino acid sequence of the collagen short peptide mutant fusion protein is shown as SEQ ID NO:3. The application can better guide the expression of bovine lactoferrin peptides by modifying the collagen short peptide as a carrier protein, increase the expression amount, and improve the expression amount by 10-15 times. Moreover, the collagen short peptide and the connected acidolysis peptide can be degraded into amino acids by acid in the subsequent acidolysis process, and the lactoferrin peptides can be released. The lactoferrin peptides can be used in the fields of medicine, food and feed and the like.