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Chinese housefly antibacterial peptides 1 and method for separating and purifying same

A technology for separation and purification of antimicrobial peptides, which is applied in the field of Chinese housefly antimicrobial peptide 1 and its separation and purification. It can solve the problems of complex peptide purification technology and difficult separation and purification of a single peptide technology, and achieve good repeatability.

Inactive Publication Date: 2011-01-05
周义文
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Different antimicrobial peptides have different molecular sizes and biological characteristics. Currently, the purification technology of peptides is relatively complicated, and it is difficult to separate and purify a single peptide.

Method used

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  • Chinese housefly antibacterial peptides 1 and method for separating and purifying same
  • Chinese housefly antibacterial peptides 1 and method for separating and purifying same
  • Chinese housefly antibacterial peptides 1 and method for separating and purifying same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 A Chinese fly antibacterial peptide 1

[0032] The Chinese fly antimicrobial peptide 1 was hydrolyzed by trypsin to obtain 5 enzymolyzed fragments. The enzymolyzed fragments were analyzed by Nano-ESI-MS / MS technology. The measured mass spectra were analyzed by the special software MassSeq of Micromass. The amino acid sequences of the 5 peptides are shown in SEQ ID NO: 1-5:

[0033] (1) YDNVNLDELLNSDR (TOF MSMS 840.47ES+, Query 4)

[0034] Tyr-Asp-Asn-Val-Asn-Leu-Asp-Glu-Leu-Leu-Asn-Ser-Asp-Arg

[0035] (2) YDLLNVDEEVRFR (TOF MSMS 826.95ES+, Query 5)

[0036] Tyr-Asp-Leu-Leu-Asn-Val-Asp-Glu-Leu-Val-Arg-Phe-Arg

[0037] (3) QPNLYYNK (TOF MSMS 520.30ES+, Query 2)

[0038] Gln-Pro-Asn-Leu-Tyr-Tyr-Asn-Lys

[0039] (4) PNNVYYTK (TOF MSMS 499.76ES+, Query 1)

[0040] Pro-Asn-Asn-Val-Tyr-Tyr-Thr-Lys

[0041] (5) DYPNNVYYTK (TOF MSMS 639.34ES+, Query 3)

[0042] Asp-Tyr-Pro-Asn-Asn-Val-Tyr-Tyr-Thr-Lys

[0043] After the mass spectrum is processed by Micromass' special software MaxEnt3...

Embodiment 2

[0044] Example 2 A method for separating and purifying Chinese domestic fly antibacterial peptide 1

[0045] It includes the following steps:

[0046] 1. Immune induction

[0047] The house flies were divided into test group and control group, and the control group was not treated with injury induction. The experimental group put a petri dish containing ether in a fly cage to anesthetize the fly, then pierced the body wall with a stainless steel needle stained with bacterial suspension, and immediately put it in another cage to resuscitate it. After stabbing, they were kept at a temperature of 28~30℃ for 24h. Induction of immunity by damage to infection, the housefly produces antimicrobial peptides. The bacteria are Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus,

[0048] 2. Tissue homogenate

[0049] Mix housefly 1.0g and 7g liquid nitrogen, quickly grind with a mortar, or freeze the larvae at -80℃ overnight, take out the mortar and grind into a fine foam in an i...

Embodiment 3

[0063] Example 3 Determination of the molecular structure of Chinese domestic fly antibacterial peptide 1

[0064] (1) Perform Tricine SDS-polyacrylamide gel electrophoresis on Chinese fly antimicrobial peptide 1:

[0065] Using a discontinuous system, the separation gel concentration is 18%, the interlayer gel concentration is 8%, the concentrated gel concentration is 4%, and the voltage is 1.5 V / cm. After electrophoresis for 80 minutes, change the voltage to 10 V / cm and electrophoresis for 120 minutes. Stain with 0.2% R-250 Coomassie Brilliant Blue methanol-ethanol mixture (50% methanol + 10% ethanol), and the decolorizing solution is methanol-ethanol mixture (50% methanol + 10% ethanol).

[0066] (2) Amino acid sequencing:

[0067] After decolorizing the PAGE gel block of antimicrobial peptides, place it in a vacuum desiccator to dry for 20 minutes, then add 5-1010mg / L trypsin solution, and place it in a refrigerator at 4°C for 40 minutes, then add 10μl 25mmol / L ammonium bicarbonat...

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Abstract

The invention discloses Chinese housefly antibacterial peptides 1 and a method for separating and purifying the same. The Chinese housefly antibacterial peptide 1 comprises five peptide segments. The method for separating and purifying the Chinese housefly antibacterial peptides comprises the following steps of: 1, performing immune induction of houseflies; 2, performing tissue homogenate of immune-induced houseflies to obtain sample solution containing antibacterial peptides; 3, performing dialysis concentration of the sample solution to obtain a concentrated sample; 4, performing ultra-centrifugal filtration of the concentrated sample to obtain the semi-purified antibacterial peptides; and 5, performing HPLC separation and purification of the semi-purified antibacterial peptides to obtain Chinese housefly antibacterial peptides with pure chromatograph. The Chinese housefly antibacterial peptide 1 of the invention is a micromolecular peptide newly found, the activity of the antibacterial peptide is higher, and the Chinese housefly antibacterial peptide 1 is a novel antibacterial medicament which has development potential and can resist multiple resistant bacteria.

Description

Technical field [0001] The invention relates to the field of biological antibacterial peptides, in particular to a Chinese domestic fly antibacterial peptide 1 and a method for separation and purification thereof. Background technique [0002] With the widespread application of antibiotics and the increasing number of drug-resistant strains and multi-drug-resistant strains, clinical anti-infection treatment has faced severe challenges. It is still a very arduous task to find broad-spectrum and efficient antibacterial and antiviral drugs. Flies mainly grow in harsh environments containing large amounts of bacteria, such as decay, feces, and garbage. Many pathogenic bacteria on their body surfaces can spread a variety of diseases. However, it does not cause death due to infection with pathogenic bacteria. This shows that flies should have strong immunity to pathogenic microorganisms and produce powerful antibacterial substances. [0003] Biological antibacterial peptides have broad-...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/20
Inventor 周义文姬尚义申萍涂植光尹一兵
Owner 周义文
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