Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K

A technology of antibacterial peptides and tandems, which is applied in the preparation of tandem antibacterial peptide Alloferon-1 and the application field of rAlloferon-1-K, can solve the problems of low natural expression yield and difficulty in meeting industrialization requirements, and is conducive to popularization and application , the effect of reducing the preparation cost

Inactive Publication Date: 2011-11-16
严杰
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, its low natural expression yield is difficult to meet the requirements of industrialization

Method used

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  • Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K
  • Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K
  • Preparation method of serial antibacterial peptide Alloferon-1 and application of rAlloferon-1-K

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Embodiment

[0024] The preparation method of the tandem antimicrobial peptide Alloferon-1 is:

[0025] 1) Obtain the amino acid sequence of natural Alloferon-1 from GenBank: N-His Gly Val Ser Gly His Gly Gln His Gly Val His Gly-C (Alloferon-1 sequence does not contain arginine and lysine);

[0026] 2) Design a pair of non-mirror-symmetrical single-stranded DNA with complementary cohesive ends; according to the reported amino acid sequence of Alloferon-1 and the codon preference of E. coli, while considering the isolation of Alloferon-1 expressed in tandem, the positive strand sequence: 5'-phosphorylation -CATGGCGTGAGCGGCCATGGCCAGCATGGCGTGCATGGCAAA-3', a trypsin cleavage site lysine codon is set at the 3' end of the positive strand DNA Alloferon-1 gene, the negative strand sequence: 5'-phosphorylation-GCCATGGCCGCTCACGCCATGTTTGCCATGCACGCCATGCTG-3'. Negative-strand DNA interleaved with positive-strand; entrust Invitrogen to synthesize.

[0027] 3) Formation of double strands of the target p...

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Abstract

The invention relates to a preparation method and application of an antibacterial peptide. The invention aims to provide a preparation method of an antibacterial peptide Alloferon-1, which has simple steps and high expression efficiency and keeps the in-vitro anti-tumor activity of the antibacterial peptide, and provide application of rAlloferon-1-K. The technical scheme is as follows: the preparation method of the serial antibacterial peptide Alloferon-1 comprises the following steps of: (1) designing a pair of terminal single-stranded DNAs (deoxyribonucleic acids); (2) generating a concatemer in a PCR (polymerase chain reaction) system; (3) carrying out electrophoretic screening on a 14 Alloferon-1 concatemer; (4) constructing a prokaryotic recombinant expression system E.coliBL21DE3pET42a-14Alloferon-1-K; (5) expressing and purifying recombinant protein 14rAlloferon-1-K; and (6) performing trypsin digestion on the 14rAlloferon-1-K, and purifying to obtain the rAlloferon-1-K.

Description

technical field [0001] The present invention relates to the establishment of a peptide tandem prokaryotic expression method, enzymatic cleavage, purification and application of recombinant tandem peptides, in particular to the tandem prokaryotic expression method of the antibacterial peptide Alloferon-1, and the recombinant peptide monomer rAlloferon-1 is obtained by enzymatic digestion with trypsin. 1-K, the antitumor activity of rAlloferon-1-K was tested. Background technique [0002] Antimicrobial peptides are non-antigenic short peptides that widely exist in a variety of organisms. They have broad-spectrum and high-efficiency inhibitory or killing activities against various microorganisms and protozoa. Some antimicrobial peptides also have anti-tumor effects and do not damage normal cells. Therefore, antimicrobial peptides are currently a research hotspot for new anti-infection and anti-tumor drugs. [0003] Alloferon-1 was first discovered in the blood of the red-heade...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/435A61K38/17A61P35/00
Inventor 孙琦孙爱华严杰
Owner 严杰
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