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Antimicrobial peptide fusion protein and its preparation method and application

A fusion protein and antibacterial peptide technology, used in the preparation of antibacterial drugs, antibacterial peptide fusion protein and the field of preparation thereof, can solve the problem that antibacterial activity needs to be improved, and can ensure the later purification and renaturation and fusion protein activity, widely used Prospect, the effect of large expression

Active Publication Date: 2019-03-12
杭州皓阳生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] But at present, most of the antimicrobial peptides exist alone to exert their antibacterial function, and there are still certain limitations in the bactericidal ability against different strains, and the antibacterial activity also needs to be improved.

Method used

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  • Antimicrobial peptide fusion protein and its preparation method and application
  • Antimicrobial peptide fusion protein and its preparation method and application
  • Antimicrobial peptide fusion protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Construction of recombinant expression vector

[0037] 1. Design and synthesis of the target fragment

[0038] Obtain the thanatin nucleic acid sequence (as shown in SEQ ID NO.1) by NCBI database, find shark liver peptide orf_91 gene (as shown in SEQ ID NO.2) in the shark liver small peptide cDNA database of this laboratory, orf_91 The shark liver peptide is a small peptide translated from the shark liver peptide orf_91 gene with an isoelectric point of 10.72 that may have potential biological activity screened from the regenerated liver cDNA library of the striped bamboo shark. Three nucleotide sequences are added in the middle The tandem segment (Gly+Ser+Ala) encoding gene (as shown in SEQ ID NO.3) forms thanatin+3GSA encoding segment+shark liver peptide orf_91 fusion gene (hereinafter referred to as tha-orf_91 fusion gene, as shown in SEQ ID NO.4 shown) and shark liver peptide orf_91+3GSA coding fragment+thanatin fusion gene (hereinafter referred to as...

Embodiment 2

[0046] Example 2: Induced expression and purification of fusion protein

[0047] 1. Induced expression of recombinant fusion protein

[0048] After the sequencing was correct, the plasmid was extracted, and the recombinant plasmid was transformed into BL21(DE3) Escherichia coli for induced expression.

[0049] The two successfully constructed engineering bacteria and the existing strain pET-duet-His-SUMO-thanatin in the laboratory were inoculated in 100mL LB culture medium, and cultured on a constant temperature shaker at 37°C until A 600 The value is about 0.6, then placed in a shaker at 16°C to pre-cool for 20 minutes, then added IPTG to a final concentration of 0.4mmol / L, induced at 16°C for 18 hours, centrifuged at 12000rpm to collect the bacteria, washed twice with PBS, and the induced recombinant Bacteria were sonicated for 30 min, sonicated for 3 s, stopped for 3 s, then centrifuged at 12000 rpm for 25 min, supernatant and precipitate were taken respectively, and the e...

Embodiment 3

[0056] Embodiment 3: Oxford cup method detects antibacterial activity

[0057] 1. Antibacterial activity detection of recombinant protein tha-orf_91 and recombinant protein orf_91-tha

[0058] In the ultra-clean bench, divide the flat plate containing Escherichia coli and the flat plate of Bacillus subtilis into four areas, put an Oxford cup in each area, add 250 μL of Amp (1mg / mL), ddH 2 O, undialyzed and digested recombinant protein and concentrated and digested recombinant protein. After culturing overnight at 37°C, observe the size of the inhibition zone to determine the antibacterial activity of the sample.

[0059] 2. Detection of antibacterial activity of thanatin protein

[0060] The protein concentration is adjusted by the recombinant protein concentration and the number of amino acids in the protein, so that the molar concentration of thanatin is the same as that of the recombinant protein (Table 1), TG1 is used as a gram-negative test bacterium, and Bacillus subti...

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Abstract

The invention provides antimicrobial peptide fusion protein. The protein comprises antimicrobial peptide thanatin protein and orf-91 shark hepatic protein. The antimicrobial peptide thanatin protein and the orf-91 shark hepatic protein are connected through 3GSA flexible peptide segments. The fusion protein is prokaryotic expression soluble recombinant protein obtained after gene horizontal fusion of an antimicrobial peptide thanatin protein encoding gene, an orf-91 shark hepatic encoding gene and a 3GSA flexible peptide encoding gene between the antimicrobial peptide thanatin protein encoding gene and the orf-91 shark hepatic encoding gene, and the protein size is 21 KD. Antibacterial activity research shows that the antimicrobial peptide fusion protein has antibacterial activity, a construction system solves the problems that natural antimicrobial peptide expression quantity is low, harm to a host is large, and natural antimicrobial peptides are difficult to obtain through a genetic engineering means, and application prospects are wide.

Description

technical field [0001] The invention relates to the technical field of biotechnology and pharmaceutical engineering, in particular to an antibacterial peptide fusion protein and a preparation method thereof, as well as the application of the fusion protein in the preparation of antibacterial drugs. Background technique [0002] Antimicrobial peptides (AMPs) are a class of small molecular polypeptides with antibacterial activity induced by organisms when they are attacked by pathogenic microorganisms. They are the main component of innate immunity and generally consist of 20-60 amino acid residues. Many antimicrobial peptides can not only effectively inhibit the growth of various pathogenic microorganisms such as bacteria, viruses, fungi, and protozoa at low concentrations, but can even kill tumor cells, but have little effect on normal eukaryotic cells without pathological changes. Since the discovery of cecropin, more than 1,700 antimicrobial peptides have been isolated fro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P31/00
CPCA61K38/00C07K14/43563C07K14/461
Inventor 吕正兵吴佳葛英花张文杰舒建洪
Owner 杭州皓阳生物技术有限公司