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Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof

An antiviral and hybrid technology, applied in the fields of genetic engineering and biopharmaceuticals, can solve the problems of severe hemolysis, achieve the effects of reducing toxicity and hemolysis, antiviral biological characteristics, and resistance to drugs

Inactive Publication Date: 2014-09-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to severe hemolysis, it cannot be used alone as a drug, so it needs to be combined with other antibacterial peptides to form a new hybrid peptide.

Method used

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  • Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof
  • Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof
  • Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of hybrid antibacterial and antiviral polypeptide M-L

[0038] Escherichia coli Mach1 TM -T1 R , E. coli BL21 and expression vector pETSUMO (Cat. No.: inv-k300-01) were purchased from Invitrogen.

[0039] Escherichia coli Mach1 TM -T1 R A single colony of BL21 and BL21 was inoculated in 3-5ml LB (1% tryptone, 0.5% yeast powder, 1% sodium chloride) liquid medium, cultured with shaking at 37°C for about 12 hours, according to 1:100-1:50 Proportionally inoculate in 100ml LB liquid medium, culture with shaking at 37℃ for 2-3 hours to OD 600 = About 0.5. The bacterial suspension was inoculated into 100ml LB liquid medium at a ratio of 1:100-1:50, cultured with shaking at 37°C for 2-3 hours to OD 600 = About 0.5, stop the culture. The culture solution was transferred to a centrifuge tube, placed on ice for 10 minutes, and then centrifuged at 3000g for 10 minutes at 4°C to collect the bacteria. Add pre-cooled 0.05mol / L CaCl to the bacteria 2 The cells were g...

Embodiment 2

[0048] Example 2 Thermal stability test of polypeptide M-L

[0049] Take 1ml each of the purified recombinant protein and place them in a water bath at 40℃, 60℃, 80℃ and 100℃ for 10, 20 and 30 minutes respectively. Use the untreated recombinant protein as a control and use standard Staphylococcus aureus as an indication Bacteria, the bacteriostatic effect of the samples before and after treatment was measured by the diameter of inhibition zone method. The experiment was repeated 3 times, and the average value was taken as the measurement result. (Table 1)

[0050] Table 1 Diameter of inhibition zone of recombinant target protein M-L on Staphylococcus aureus after heat treatment

[0051]

[0052] Note: The control is the diameter of the inhibition zone of the recombinant target protein without heat treatment.

[0053] The results in Table 1 show that after different heating temperatures and time treatments, the antibacterial effect of the recombinant target protein is different. When...

Embodiment 3

[0054] Example 3 Inhibition of polypeptide M-L on common bacteria

[0055] Put the filter paper (diameter 6mm) sterilized by dry heat (160℃, 2h) into the recombinant target protein solution (32μg / ml) and soak it for 24h. Use a sterile spreader to dip the test bacteria solution evenly. Spread the ground on the surface of the culture medium to make a plate containing bacteria, take out the fully soaked filter paper and stick it on the plate containing bacteria, stick one piece per petri dish, and place it in a constant temperature incubator at 37°C for 24 hours, observe and measure The diameter of the zone of inhibition. (Table 2)

[0056] Table 2 Diameter of inhibition zone of recombinant target protein against common bacteria

[0057] Bacteria name

[0058] Escherichia coli (CICC1.907)

[0059] It can be seen from Table 2 that the antibacterial peptides have different degrees of antibacterial effect on each strain.

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Abstract

The invention provides a heterozygous antibacterial and antiviral polypeptide, the amino acid sequence and the nucleotide sequence of which are respectively shown as SEQ ID No.1 and SEQ ID No.2. The preparation method comprises the following steps: cloning a gene which encodes the polypeptide to an expression vector pETSUMO to obtain a recombinant expression vector; then, converting the vector into escherichia coli BL21 though a hot stress method; inducing expression by IPTG (Isopropyl-beta-d-Thiogalactoside); carrying out enzyme digestion on an expression product through SUMO enzyme digestion and purifying through a Ni-NTA Sepharose chromatographic column; separating to obtain the targeted polypeptide. The polypeptide has inhibiting and killing effects to various bacteria and viruses in vitro and is less in possibility of generating resistance, so that the polypeptide can be used as an ideal antibiotic substituent, a human and animal biological medicine, a feed additive, a preservative or a killing agent and the like.

Description

Technical field [0001] The invention relates to the fields of genetic engineering and biopharmaceuticals, in particular to a novel hybrid antibacterial and antiviral polypeptide, its preparation method and application. 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, more and more resistant strains. Monitoring found that among the most representative resistant strains, resistant staphylococci have risen to 40%; resistant coagulase-negative staphylococci have exceeded 77%. Nowadays, resistant bacteria that can fight all antibiotics have emerged, and the problem of antibiotic resistance has seriously threatened human health. Eliminating the growing threat of drug-resistant bacteria to human health has become an urgent problem to be s...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/17A61P31/04A61P31/10A61P31/12A23K1/165A01N47/44A01P1/00A01P3/00A23K20/147
Inventor 张日俊黄燕武如娟卫旭彪赵龙妹郑昭君商婷婷刘旭辉马广
Owner CHINA AGRI UNIV
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