CN110101838B

CN110101838B - Method for improving large enterobacteriaceae resistance of histone

Info

Publication number: CN110101838B
Application number: CN201910355139.0A
Authority: CN
Inventors: 刘梅; 马亚云; 郭荣
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2022-07-01
Anticipated expiration: 2039-04-29
Also published as: CN110101838A

Abstract

The invention discloses a method for improving the resistance of histone to enterobacter coli, which is to combine DNA and histone to form a histone-DNA compound, wherein the DNA is the DNA consisting of 8-15 repeated AT base sequences from 5 'end to 3' end. The method has the advantages of simple operation, short reaction time, low cost and no need of excessive chemical modification and complex large-scale instruments. Compared with histone, the histone-DNA compound can obtain obvious bactericidal effect under the condition of lower concentration of histone and reduce the cytotoxicity of histone.

Description

Method for improving large enterobacteriaceae resistance of histone

Technical Field

The invention belongs to the technical field of antibiosis, and particularly relates to a method for improving the antibacterial activity of histone.

Background

Escherichia coli (Escherichia coli) is a common pathogenic bacterium in life, often causes diarrhea and septicemia, is a gram-negative short bacillus with blunt ends, circumferential flagella, motion capability and no spores, is widely distributed in nature, so that food has a high probability of being polluted by the Escherichia coli and is easy to cause food poisoning. For example, spinach in the us was contaminated with e.coli in 2006, with disease spreading in half of the us; the European "poisonous cucumber" event in 2011 is also an infection caused by the contamination of vegetables with E.coli. Coli has caused numerous large-scale infections worldwide, leading to many deaths. In order to ensure the food safety, consumers can purchase green and healthy food with ease. The research on a novel escherichia coli sterilization method has very important significance.

At present, the common escherichia coli sterilization method mainly adopts antibiotics for sterilization, and as a large amount of antibiotics are used, the escherichia coli generates drug resistance to a plurality of antibiotics, wherein 70% of bacteria have resistance to at least one drug, and the high-concentration antibiotics have certain harm to human bodies. Aiming at the problem of antibiotic resistance of bacteria, novel antibiotics become a research hotspot. The cationic polymer as a brand new antibacterial agent has an antibacterial mechanism different from that of conventional antibiotics, so that bacteria hardly generate drug resistance, and the histone is one of typical representatives in the cationic antibacterial peptide

Histone (Histones) are basic structural proteins of chromosomes, are basic due to the abundance of the basic amino acids arginine and lysine, and can be tightly bound to acidic DNA. The histone comprises five components, the molecular weight is 11-23ku, and the components are respectively called H, H3, H2A, H2B and H4 according to the molecular weight from large to small. Histones contain large amounts of lysine and arginine, where arginine is a basic amino acid containing a guanidino group, however, guanidino groups have a high acid dissociation constant and are effectively soluble in water, which makes them more basic and more suitable for stable electrostatic interactions with negatively charged groups in phospholipids. Due to the abundant presence of these positively charged guanidino groups, histones electrostatically interact with negatively charged bacterial cell membranes, and as a result, the permeability of the cell wall or cell membrane is disrupted, thereby inhibiting the growth of bacteria, even though it is reported that they can penetrate the plasma membrane. Histone is considered as one of antimicrobial peptides to be an effective antimicrobial peptide bactericide, and its bactericidal properties were found as early as 1958, and subsequently, bactericidal activities of histone and its components were found in various organisms such as soft body, amphibian, and the like. As the concentration of histone increases, its antibacterial activity is higher, but cytotoxicity is also increased. Therefore, how to reduce the cytotoxicity of histone and improve the antibacterial effect thereof needs to be solved.

Disclosure of Invention

The invention aims to provide a method for achieving better large enterobacteriaceae resistance of histone at low concentration.

In order to achieve the above object, the present invention provides a method for binding a DNA to a histone to form a histone-DNA complex, wherein the DNA is a DNA comprising 8 to 15 repeated AT base sequences from the 5 'end to the 3' end.

In the method for improving the antibacterial activity of histone, preferably, the histone-DNA complex is formed by mixing the histone aqueous solution and the DNA aqueous solution, reacting the mixture at room temperature for 1 to 3 hours so that the concentration of DNA in the mixture is 0.001 to 0.03 mu mol/L and the concentration of histone is 10 to 100 mu g/mL.

In the method for improving the antibacterial activity of histone, it is further preferable that the histone-DNA complex is formed by mixing the histone aqueous solution and the DNA aqueous solution so that the concentration of DNA in the obtained mixed solution is 0.005 to 0.01. mu. mol/L and the concentration of histone is 25 to 50. mu.g/mL, and reacting at room temperature for 2 hours.

The DNA is preferably a DNA comprising 8 to 10 repeated AT base sequences from the 5 'end to the 3' end.

The invention has the following beneficial effects:

the method can quickly and effectively combine DNA and histone to form a histone-DNA compound, has simple operation, short reaction time and low cost, does not need excessive chemical modification and complex large-scale instruments, ensures that the histone can have excellent antibacterial performance under lower concentration, and reduces the cytotoxicity.

Drawings

FIG. 1 is a diagram showing UV-VIS absorption spectra of histone, DNA, and histone-DNA complex in example 1.

FIG. 2 is a circular dichroism spectrum of histone, DNA, histone-DNA complex in example 1.

FIG. 3 is a partial enlarged view of circular dichroism spectra of histone, DNA, and histone-DNA complex in example 1.

FIG. 4 shows the bactericidal effect of histones and the histone-DNA complexes obtained in examples 1 to 8 on Escherichia coli.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having base sequence 5'-ATATATATATATATATATAT-3' and 50. mu.L of a 100. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

As can be seen from the ultraviolet spectrum of FIG. 1, the histone has an absorption peak at 275nm, which is consistent with the peak position of ultraviolet absorption of calf thymus histone reported in the literature. DNA has a significant UV absorption at 265 nm. Compared with the ultraviolet absorption of a histone single component, the absorption peak of a complex formed after the interaction of DNA and histone is enhanced in intensity and slightly blue-shifted at the peak position of the histone, and the conformation of the histone is changed. This also confirms that the binding of histone to DNA does occur. As can be seen from the circular dichroism spectrum of FIG. 2, histone has an absorption peak at 210nm, and the absorption intensity of the histone-DNA complex at the position of the peak is increased and slightly blue-shifted, indicating that the molecular conformation of histone is changed and hydrophobicity is enhanced, in accordance with the ultraviolet absorption spectrum thereof. The wavelengths from 230nm to 300nm were individually plotted because of the small peak-to-peak absorbance of DNA. As shown in FIG. 3, it can be seen that the DNA absorbs at 245nm and 270nm, and the absorbance of the histone-DNA complex is decreased at 275nm as compared with the UV absorbance peak of the DNA.

Example 2

50. mu.L of a 0.05. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATATATAT-3' and 50. mu.L of a 50. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 3

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATATATAT-3' and 50. mu.L of a 50. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 4

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATAT-3' and 50. mu.L of a 50. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 5

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATAT-3' and 50. mu.L of a 50. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 6

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATATATATATATAT-3' and 50. mu.L of a 50. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 7

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATAT-3' and 50. mu.L of a 200. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

Example 8

50. mu.L of a 0.01. mu. mol/L aqueous DNA solution having an alkali sequence of 5'-ATATATATATATATATATAT-3' and 50. mu.L of a 25. mu.g/mL aqueous histone solution were mixed and reacted at room temperature for 2 hours to form a histone-DNA complex.

In order to prove the beneficial effects of the invention, the inventor carries out comparative tests on the antibacterial performance of histone and histone-DNA compound in examples 1-8, and the specific test conditions are as follows:

1. test for Escherichia coli resistance

The antimicrobial properties of an antimicrobial agent are generally characterized by a Minimum Bactericidal Concentration (MBC). MBC refers to the minimum concentration of antimicrobial agent required to completely kill a particular test bacterium, with smaller MBC values indicating greater ability of the material to kill the growth of that microorganism. The determination of MBC consists of the following steps:

(1) weighing 25.0g of LB broth culture medium, adding the LB broth culture medium into 1L of distilled water, boiling to fully dissolve the LB broth culture medium, adjusting the pH value to 7.2-7.3, adding 15g of agar powder, boiling to dissolve the agar powder, and then autoclaving at 121 ℃ for 20 min. After sterilization, the plates were poured on a sterile worktop for use. Taking out the Escherichia coli strain cryopreservation tube from a refrigerator at-80 deg.C, diluting the bacteria solution with sterile water by a proper time after the bacteria solution is dissolved, spreading 100 μ L of the bacteria solution on a plate, and culturing at 37 deg.C for 18 h.

(2) In a clean bench, 20mL of sterilized LB liquid medium was transferred to a 100mL conical flask, a 10. mu.L pipette was used to aspirate a complete colony from the plate, the pipette tip was pipetted into the conical flask, and the plate was incubated for 14h in a shaker (37 ℃ C., 260 r/min).

(3) Taking out the bacterial liquid incubated for 14h in the shaking table, sucking 5mL into a 10mL centrifuge tube, centrifuging (6000r/min, 2min), removing the supernatant, adding 5mL of physiological saline, mixing uniformly, centrifuging (6000r/min, 2min), repeating twice, adding 5mL of physiological saline into the thallus washed out of the culture medium, diluting with 1 × 10 PBS buffer solution with pH 7.4, and repeating⁴After doubling, 100 μ L of the mixture was taken, 100 μ L of a 1.25 μ g/mL aqueous histone solution or 100 μ L of the diluted histone-DNA complex (wherein the histone-DNA complexes of examples 1 to 5 were diluted 80-fold with water, the histone-DNA complex of example 6 was diluted 40-fold with water, the histone-DNA complex of example 7 was diluted 10-fold with water, and the histone-DNA complex of example 8 was diluted 20-fold with water) was added thereto, and then the mixture was diluted to 1000 μ L with a PBS buffer solution having a pH of 7.4, and incubated in a shaker (37 ℃, 260r/min) for 5h, 100 μ L was spread on a plate, and cultured in an inverted state at 37 ℃ for 18h, and the growth of colonies was observed; and a blank control experiment is carried out by using PBS buffer solution, and experiments are carried out in parallel for 3 times.

The calculation method of the sterilization rate comprises the following steps:

the bactericidal rate was (1-number of colonies in experimental group/number of colonies in blank group) × 100%

As can be seen from FIG. 4, under the condition that the concentration of histone is 0.125. mu.g/mL, compared with single histone, the antibacterial performance of the histone-DNA compound obtained in the embodiments 1-8 of the invention is obviously improved, wherein the antibacterial performance of the histone-DNA compound of the embodiments 1, 3, 4, 5 and 7 is improved by 1-3 times.

Claims

1. A method of increasing the resistance of a histone protein to enterobacteriaceae for non-therapeutic purposes, comprising: the method comprises the step of combining DNA and histone to form a histone-DNA compound, wherein the DNA is the DNA formed by 8-15 repeated AT base sequences from 5 'end to 3' end.

2. The method of claim 1 for non-therapeutic purposes of increasing the resistance of a histone protein to enterobacteriaceae, wherein: mixing a histone aqueous solution and a DNA aqueous solution to ensure that the concentration of DNA in the obtained mixed solution is 0.001-0.03 mu mol/L and the concentration of histone is 10-100 mu g/mL, and reacting at room temperature for 1-3 hours to form a histone-DNA compound.

3. The method of claim 2 for non-therapeutic purposes of increasing the resistance of a histone protein to enterobacteriaceae, wherein: mixing the histone aqueous solution and the DNA aqueous solution to ensure that the concentration of DNA in the obtained mixed solution is 0.005-0.01 mu mol/L and the concentration of histone is 25-50 mu g/mL, and reacting at room temperature for 2 hours to form a histone-DNA compound.

4. The method of claim 3 for the non-therapeutic purpose of increasing the resistance of a histone against large intestine bacilli, wherein: the DNA is composed of 8-10 repeated AT base sequences from 5 'end to 3' end.