Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof

A silkworm antimicrobial peptide and antimicrobial peptide technology, applied in recombinant DNA technology, fermentation, etc., can solve the problems of low purity, single purification method of recombinant antimicrobial peptide, and high purification cost, and achieve simple operation, tumor growth inhibition, and short time Effect

Inactive Publication Date: 2010-06-02
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the current situation that the purification method of recombinant antimicrobial peptides is relatively simple, the purity is not high, and the purification cost is relatively high

Method used

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  • Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof
  • Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof
  • Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation Method 1 of Recombinant Xinjiang Silkworm Antimicrobial Peptide

[0039] The fermentation broth was centrifuged at 12000rpm for 5min to remove the precipitate, then centrifuged at 100°C for 30min at 12000rpm for 10min to remove the precipitate, and then freeze-dried and stored at -20°C for later use.

[0040] The first step, ion exchange chromatography: dissolve 40 mg of the fermented dry powder collected above in 10 mM Na 2 HPO 4 -NaH 2 PO 4 pH 6.0 buffer solution, centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was taken, and then filtered with a 0.45 μm filter membrane. Loaded in 10mM Na 2 HPO 4 -NaH 2 PO 4 The Q Sepharose Fast Flow anion exchange column balanced with pH 7.0 buffer, the length of the exchange column is 10mm, the diameter is 5mm, washed by 2.5 column volumes of the same buffer until the flow-through peak is completely eluted. with 10 mM NaCl containing 2.0 M NaCl 2 HPO 4 -NaH 2 PO 4 Gradient elution wi...

Embodiment 2

[0043] Embodiment 2: Preparation method two of recombinant Xinjiang silkworm antimicrobial peptide

[0044] The fermentation broth was centrifuged at 12000rpm for 5min to remove the precipitate, then centrifuged at 100°C for 30min at 12000rpm for 10min to remove the precipitate, and then freeze-dried and stored at -20°C for later use.

[0045] The first step, ion exchange chromatography: dissolve 40 mg of the fermented dry powder collected above in 100 mM Na 2 HPO 4 -NaH 2 PO 4 pH9.0 buffer solution, 12000rpm, centrifuge at 4°C for 10min, take the supernatant, and then filter with a 0.45μm filter membrane. Loaded in 10mM Na 2 HPO 4 -NaH 2 PO 4 The Q Sepharose Fast Flow anion exchange column balanced with pH 7.0 buffer, the length of the exchange column is 160mm, the diameter is 20mm, washed by 2.5 column volumes of the same buffer until the flow-through peak is completely eluted. with 100 mM NaCl containing 2.0 M 2 HPO 4 -NaH 2 PO 4 Gradient elution with pH9.0 b...

Embodiment 3

[0048] Embodiment 3: Preparation method three of recombinant Xinjiang silkworm antimicrobial peptide

[0049] Preparation of raw materials as in the above examples.

[0050] The first step, ion exchange chromatography: dissolve 40 mg of the fermented dry powder collected above in 50 mM Na 2 HPO 4 -NaH 2 PO 4 pH8.0 buffer solution, centrifuged at 12000rpm at 4°C for 10min, the supernatant was taken, and then filtered with a 0.45μm filter membrane. Loaded in 10mM Na 2 HPO 4 -NaH 2 PO 4 The Q Sepharose Fast Flow anion exchange column equilibrated with pH7.0 buffer, the length of the exchange column is 100mm, and the diameter is 10mm, washed by 2.5 column volumes of the same buffer until the flow-through peak is completely eluted. with 50 mM NaCl containing 1.5 M 2 HPO 4 -NaH 2 PO 4 Gradient elution with pH8.5 buffer to collect peaks II and III.

[0051] The second step, hydrophobic interaction chromatography: the active peaks II and III obtained by ion exchange ch...

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Abstract

The invention discloses the method for purifying and preparing recombinant antibacterial peptide of Xinjiang silkworm, recombinant antibacterial peptide of Xinjiang silkworm, application of recombinant antibacterial peptide of Xinjiang silkworm in bacteriostasis and antitumor. recombinant antibacterial peptide of Xinjiang silkworm is obtained from Xinjiang silkworm through cloning, expressing in eukaryotic expression system. The preparing method is: centrifuging the fermentation broth to remove the precipitant, freeze-drying, separating and purifying through an ion exchange chromatography, a hydrophobic interaction chro-matography, and a gel filtration chromatography to obtain the final product. The recombinant antibacterial peptide of Xinjiang silkworm has high inhibition and killing activity on bacteria and tumor cells, no hemolysis activity, and no toxicity to normal tissue cells, and can be used for preparing drugs for treating infectious diseases caused by pathogenic microorganismand tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, it relates to a method for purifying and preparing the recombinant Xinjiang silkworm antibacterial peptide, the recombinant Xinjiang silkworm antibacterial peptide and its application in antibacterial and antitumor aspects. Background technique [0002] The discovery of penicillin freed people from the fear of diseases caused by pathogenic microorganisms, which led to the emergence of a large number of antibiotics and laid a solid foundation for the protection of human health. However, the abuse of traditional antibiotics and the continuous emergence of drug-resistant strains have seriously affected the clinical treatment of infectious diseases. Therefore, the development of a new generation of antibiotics has become the focus of new drug development in the 21st century. In the past 20 years, antimicrobial peptides from different sources have shown the characteristics of broad-spectru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12N15/09C07K1/34
Inventor 窦君张富春刘忠渊马正海马纪
Owner XINJIANG UNIVERSITY
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