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Bovine lactoferricin-human lysozyme fused protein, gene and application thereof

A bovine lactoferrin peptide and fusion protein technology, applied in the field of bovine lactoferrin peptide-human lysozyme, can solve the problems of high purification cost and unhelpful product functions, achieve broad antibacterial spectrum, strong antibacterial activity, and improve piglet growth performance Effect

Active Publication Date: 2018-11-13
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fusion protein used in these two methods does not contribute to the final product function, and the obtained product needs post-processing, and the purification cost is too high

Method used

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  • Bovine lactoferricin-human lysozyme fused protein, gene and application thereof
  • Bovine lactoferricin-human lysozyme fused protein, gene and application thereof
  • Bovine lactoferricin-human lysozyme fused protein, gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Fusion expression of bovine lactoferrin peptide-human lysozyme hybrid protein (LfcinB-hLY) in Pichia pastoris

[0028] Pig myofibrillar protein antioxidant peptide (SEQ ID NO.1), DPNG linking sequence, bovine lactoferrin peptide (SEQ ID NO.4), NGDPE linking sequence, chicken ovalbumin antioxidant peptide (SEQ ID NO.2), The bovine lactoferrin peptide- Human lysozyme hybrid protein (SEQ ID NO.6) ( figure 1 Middle a). According to Pichia pastoris codon-optimized LfcinB-hLY hybrid protein gene coding sequence SEQ ID NO.8, the whole gene synthesis was carried out by Gene Synthesis Company.

[0029] The DNA sequence of SEQ ID NO.8 is cloned on the plasmid pGAPZα ( figure 1 In middle b), after using the restriction endonuclease BlnI to linearize the plasmid, electrotransform Pichia pastoris (P. pastoris) GS115 strain (Invitrogen Company) to obtain a recombinant bacterium containing the LfcinB-hLY hybrid protein gene. Take 100 μL of the recombinant bacteria liquid...

Embodiment 2

[0036] Example 2 Separation and purification of fusion protein LfcinB-hLY

[0037] (1) After centrifugation, the fermentation broth obtained in Example 1 was purified by ion exchange resin SP Sepharose FastFlow with a column volume of 20 mL and UV detection wavelengths of 220 nm and 280 nm. The column was equilibrated with 5mM acetic acid-sodium acetate buffer (pH 4.5) at a flow rate of 1.5mL / min. 50 mL of the supernatant of the fermentation broth was loaded. After sample loading is complete, re-equilibrate the column with the above buffer. After equilibration, 50 mM acetic acid-sodium acetate buffer (pH 4.5) containing 0.2 mol / L NaCl was used as the eluent for elution at a flow rate of 1.5 mL / min. After all the elution peaks were collected, the antibacterial activity was measured to obtain a fusion protein LfcinB-hLY sample with antibacterial activity. The elution peak with antibacterial activity obtained after cation exchange chromatography was lyophilized and concentrate...

Embodiment 3

[0039] Example 3 Hydroxylamine Hydrochloride Cleavage of Fusion Protein LfcinB-hLY

[0040] Hydroxylamine lysis buffer is 25.80g hydroxylamine hydrochloride and 4.8g Tris dissolved in 100mL double distilled water, adjusted to pH 9.0 with 4M NaOH and fixed to 200mL.

[0041] A cleavage site (Asn-Gly) was designed in the fusion protein sequence. Add twice the volume of hydroxylamine lysis buffer to the fusion protein LfcinB-hLY sample after cation exchange chromatography in step (1) of Example 2, and after 4 hours in a water bath at 45°C, use hydrochloric acid to adjust the pH value of the reaction solution to about 7 and Lower the reaction temperature to room temperature to terminate the cleavage reaction. Hydroxylamine and small peptides were subsequently removed using 1KD dialysis bags. The permeate after the dialysis treatment was purified by circulating preparative chromatography, and the operation was consistent with that in Example 2. Such as Figure 4 As shown, there...

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Abstract

The invention discloses bovine lactoferricin-human lysozyme fused protein. The bovine lactoferricin-human lysozyme fused protein is characterized by comprising porcine myofibrillar protein antioxidative peptide, bovine lactoferricin, hen egg white protein antioxidative peptide, porcine muscle myosin antioxidative peptide, flexible peptide and human lysozyme which are connected in series. Through anionic antioxidative peptide derived from fusion expression animals, the positive charge of antimicrobial peptide is neutralized, and inhibition to host bacteria by antimicrobial peptide is reduced; antioxidative peptide is beneficial to the improvement of storage stability of the antimicrobial peptide, the antibacterial activity to escherichia coli (ATCC 25922) by the fused protein is only reduced by 7.7 percent after the fused protein is laid for 20d at 4 DEG C, and the loss of activity to the escherichia coli (ATCC 25922) by the fused protein is only reduced by 15.4 after the fused proteinis laid for 30d.

Description

(1) Technical field [0001] The invention relates to bovine lactoferrin peptide-human lysozyme, gene and application thereof. (2) Background technology [0002] The abuse of antibiotics in the field of medical treatment and animal breeding has caused the prevalence of drug-resistant bacteria, posing a serious threat to human health. There is an urgent need to develop new antibacterial drugs or feed additives to replace antibiotics. Antimicrobial peptides and lysozymes are a class of natural peptides and proteins with bactericidal activity, which have been widely used in clinical medicine and food industry as antibiotic substitutes. [0003] The antibacterial mechanism of antimicrobial peptides is that positively charged antimicrobial peptides bind to negatively charged bacterial cell membranes, causing bacterial death by causing membrane perforation or inhibiting the physiological metabolic process of bacteria. The N-terminus of bovine lactoferrin contains two antibacterial...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/36C12N15/62A23K50/60A23K50/30A23K50/75A23K20/147A23K20/189
CPCA23K20/147A23K20/189A23K50/30A23K50/60A23K50/75C07K14/4716C07K14/77C07K14/79C07K2319/00C12N9/2462C12Y302/01017
Inventor 孙杰汪钊魏春
Owner ZHEJIANG UNIV OF TECH
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