A kind of antibacterial polypeptide and its preparation method and application

A technology of antibacterial peptides and antibacterial drugs, which is applied in the field of preparation of antibacterial peptides and the antibacterial peptides, can solve the problems of low content of antibacterial peptides, difficulty in extraction, high cost, etc., and achieve the effect of less drug resistance

Active Publication Date: 2011-12-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the content of antimicrobial peptides in organisms is not high, and the extraction is difficult and the c

Method used

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  • A kind of antibacterial polypeptide and its preparation method and application
  • A kind of antibacterial polypeptide and its preparation method and application
  • A kind of antibacterial polypeptide and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0029] The preparation of embodiment 1 antibacterial polypeptide

[0030] Escherichia coli DH5α was purchased from Treasure Bioengineering (Dalian) Co., Ltd., methanol yeast (Pichia pastoris) GS115, and expression vector pPIC9K was purchased from Invitrogen.

[0031] Inoculate a single colony of GS115 in 5ml YPD (1% yeast extract, 2% tryptone, 2% D-glucose) liquid medium, culture overnight at 28-30°C, transfer 100μl to 50ml YPD medium to continue the cultivation, Stop culturing when the OD600 is between 1.1 and 1.3, centrifuge at 5000rpm for 5min, collect the bacteria, then wash twice with ice-cold deionized water and D-sorbitol, and finally add 1ml of pre-cooled 1M D-sorbitol, Aliquot 200 μl per tube into spare methanolic yeast GS115 competent cells, use immediately or store at -70°C.

[0032] According to the ABP-j1 nucleic acid sequence (SEQ ID No.1) and the restriction endonuclease site characteristics on the Pichia pastoris expression vector pPIC9K, by artificial synthes...

Embodiment 2

[0037] The thermostability test of embodiment 2 antibacterial polypeptide

[0038] Take 1ml of each yeast expression supernatant and place them in water baths at 40, 60, 80 and 100°C for 10, 20, and 30 min respectively. Use the untreated yeast expression supernatant as a control, and standard Staphylococcus aureus as an indicator bacterium. The antibacterial effect of the samples before and after treatment was determined by the diameter of the inhibition zone. The experiment was repeated 3 times, and the average value was taken as the measurement result.

[0039] Table 1 The diameter of inhibition zone of yeast expression supernatant against standard Staphylococcus aureus after heat treatment

[0040]

[0041]

[0042] Note: The control is the diameter of the inhibition zone of the yeast expression supernatant without heat treatment.

[0043] The results in Table 1 show that the effects of different heating temperatures and times on the antibacterial effect of the yeas...

Embodiment 3

[0044] Example 3 Antimicrobial polypeptide challenge protection test

[0045] The virus strain is Escherichia coli K88.

[0046] Determination of median lethal dose (LD50) Escherichia coli K88 was inoculated in nutrient broth containing 0.5% glucose, cultured at 37°C for 24 hours, and the OD650 value was 0.765. The mice were injected intraperitoneally with doses of 0.05, 0.10, 0.15, 0.20, and 0.25 mL, respectively, and 5 mice were inoculated with each dose, and the death conditions were observed and recorded.

[0047] Table 2 The death of mice inoculated with different doses of bacterial solution

[0048] Dosage (ml / piece)

[0049] With the increase of the inoculum amount, the number of death of mice increased linearly. Calculated by Reed and Meunch method, the median lethal dose (LD50) of the bacterial solution to mice is 0.142mL.

[0050] Divide 30 mice into 4 groups, namely blank control group, virus strain control group, empty vector yeast supernatant control ...

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Abstract

The invention relates to an antimicrobial polypeptide, and a preparation method and application thereof, belonging to the field of biomedicine. The amino acid sequence of the antimicrobial polypeptide provided by the invention is disclosed as SEQ ID NO.2. The preparation method comprises the following steps: after linearizing the coding sequence of the antimicrobial polypeptide with restriction endonuclease, transforming into Pichia pastoris GS115 by an electric shock method, recombining onto the Pichia pastoris chromosome by homology, screening high-expression strains with G418, and inducingthe expression with methanol, thereby obtaining the antimicrobial polypeptide. The test proves that the antimicrobial polypeptide has the functions of inhibiting and killing multiple bacteria, viruses, tumor cells, carcinomatous solid tumors, fungi and protozoa, can not easily generate resistance, and thus, can be used as an ideal antibiotic substitute, biological medicine for human and livestock, feed additive, preservative, bactericide or the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an antibacterial polypeptide, a preparation method and application of the antibacterial polypeptide. Background technique [0002] Antibiotics play an important role in the control, prevention and treatment of various infectious diseases. Due to the use of antibiotics, by the 1980s, humans could conquer almost all infectious diseases. However, with the passage of time and the abuse of antibiotics, there are more and more drug-resistant strains. Surveillance has found that among the most representative drug-resistant strains, drug-resistant staphylococcus has risen to 40%; drug-resistant coagulase-negative staphylococcus exceeds 77%. Now, drug-resistant bacteria that can resist all antibiotics have appeared. Obviously, the problem of antibiotic resistance has seriously threatened human health. [0003] Removing the increasingly serious threat of drug-resistant bacteria to human being...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N1/19A61K38/17A61P31/04A61P31/10A61P31/12A23K1/17A01N61/00A01P1/00A01P3/00A23K20/195
Inventor 张日俊胡晓年解军于保锋李翔王俊丽向前余占桥马青山赵龙妹
Owner CHINA AGRI UNIV
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