阅读说明：本技术 一种对革兰氏阳性细菌具有广谱杀菌活性的多肽复合物 (Polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria ) 是由 刘丕龙 李龙 王利红 赵丽丽 严顺华 于 2021-10-13 设计创作，主要内容包括：本发明公开了一种对革兰氏阳性细菌具有广谱杀菌活性的多肽复合物,其由SEQ ID No.1和SEQ ID No.2所示序列的多肽组合构成。一种对革兰氏阳性细菌具有广谱杀菌活性的多肽复合物来源于标准敏感型金黄色葡萄球菌(ATCC 29213)。本发明所述的多肽复合物对革兰氏阳性细菌具有广谱杀菌活性,并对抗生素难以杀灭的耐甲氧西林细菌和耐万古霉素细菌具有杀灭作用。(The invention discloses a polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria, which is composed of polypeptide combinations of sequences shown in SEQ ID No.1 and SEQ ID No. 2. A polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria is derived from Staphylococcus aureus (ATCC 29213) which is a standard susceptible species. The polypeptide compound has broad-spectrum bactericidal activity on gram-positive bacteria and has a killing effect on methicillin-resistant bacteria and vancomycin-resistant bacteria which are difficult to kill by antibiotics.)

1. A polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, characterized in that said polypeptide complex comprises the amino acid sequences shown in SEQ ID No.1 and SEQ ID No. 2;

the SEQ ID No.1 is:

Lys-Lys-Leu-Lys-Ile-Glu-Glu-Leu-Lys-Lys-Phe-Asn-Gly-Gly-Asn-Lys-Trp-Gly-Ser-Ser-Ala-Ile-Gly-Ala-Met-Gly-Gly-Ala-Ala-Thr-Gly-Ile-Lys-Leu-Cys-Ser-Ala-Gly-Gly-Pro-Trp-Ala-Met-Ala-Gly-Cys-Gly-Val-Val-Gly-Gly-Ala-Ile-Gly-Gly-Phe-Thr-Gly-Tyr-His-Gly-Tyr；

the SEQ ID No.2 is:

Met-Lys-Lys-Leu-Glu-Thr-Lys-Glu-Leu-Val-Ser-Ile-Asn-Gly-Gly-Lys-Ly s-Asn-Thr-Trp-Gln-Gln-Asn-Val-Ser-Gly-Ala-Ile-Gly-Ser-Thr-Val-Ala-Gly-Ala-Gly-Leu-Gly-Gly-Ala-Ile-Cys-Gly-Pro-Ala-Cys-Ala-Val-Val-Gly-Ala-His-Tyr-Gly-Pro-Ile-Ile-Trp-Ala-Gly-Val-Ser-Gly-Ala-Thr-Gly-Ala-Phe。

2. the polypeptide complex of claim 1, wherein the polypeptide consisting of the amino acid sequences shown in SEQ ID nos. 1 and 2 is derived from staphylococcus aureus.

3. The polypeptide complex of claim 1, wherein the polypeptide consisting of the amino acid sequences shown in SEQ ID nos. 1 and 2 is derived from staphylococcus aureus (ATCC 29213) which is a standard susceptible type.

4. Use of a polypeptide complex according to any one of claims 1 to 3 for killing bacteria.

5. The use of claim 4, wherein the bacteria comprise gram positive bacteria.

6. The use of claim 4, wherein the bacteria comprise methicillin-resistant bacteria and vancomycin-resistant bacteria.

Technical Field

The invention belongs to the field of biomedicine, and relates to a polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria.

Background

The use of a large amount of antibiotics causes clinical pathogenic bacteria to evolve antibiotic resistance, and the appearance of drug-resistant pathogenic bacteria greatly reduces the sterilization effect and clinical expectation of the antibiotics. In recent years, relevant researches at home and abroad find that not only certain polypeptides extracted from organisms have biological activity, but also active peptides with special functions can be generated after protein is subjected to enzymolysis treatment.

Antibacterial peptides (AMPs) are small molecular protein substances with antibacterial activity, are widely distributed in various organisms, and are key factors of nonspecific immunity of organisms such as invertebrates, vertebrates and plants. The antibacterial peptide has the advantages of wide antibacterial spectrum, rapid target cell searching and killing, good thermal stability, difficult generation of drug resistance to target strains and the like, and is considered to be a good choice for replacing antibiotics in the future.

The present inventors have studied a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, which is an antimicrobial peptide capable of killing a variety of bacteria including drug-resistant pathogenic bacteria, and which is not shown in the known literature, and it is difficult for those skilled in the art to know the beneficial effects of the polypeptide complex for killing bacteria through the existing literature.

Disclosure of Invention

The invention aims to provide a polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria, which can effectively kill various bacteria including drug-resistant pathogenic bacteria.

Based on the above objects, the present invention provides a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, said polypeptide complex comprising the amino acid sequences shown in SEQ ID nos. 1 and 2.

Further, the polypeptide complex provided by the invention has the following sequence as shown in SEQ ID No. 1: Lys-Lys-Leu-Lys-Ile-Glu-Glu-Leu-Lys-Lys-Phe-Asn-Gly-Gly-Asn-Lys-Trp-Gly-Ser-Ser-Ala-Ile-Gly-Ala-Met-Gly-Ala-Ala-Thr-Gly-Ile-Lys-Leu-Cys-Ser-Ala-Gly-Pro-Trp-Ala-Met-Ala-Gly-Cys-Gly-Val-Val-Gly-Ala-Ile-Gly-Phe-Thr-Gly-Tyr-His-Gly-Tyr.

Further, the polypeptide complex provided by the invention has the following sequence shown in SEQ ID No. 2: Met-Lys-Lys-Leu-Glu-Thr-Lys-Glu-Leu-Val-Ser-Ile-Asn-Gly-Gly-Lys-Asn-Thr-Trp-Gln-Gln-Asn-Val-Ser-Gly-Ala-Ile-Gly-Ser-Thr-Val-Ala-Gly-Ala-Gly-Leu-Gly-Ala-Ile-Cys-Gly-Pro-Ala-Cys-Ala-Val-Val-Gly-Ala-His-Tyr-Gly-Pro-Ile-Ile-Trp-Ala-Gly-Val-Ser-Gly-Ala-Thr-Gly-Ala-Phe.

Further, the polypeptide complex provided by the invention is derived from standard sensitive staphylococcus aureus (ATCC 29213).

In addition, the invention also provides application of the polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria in the aspect of killing bacteria, wherein the bacteria are mainly used for killing bacteria, the bacteria comprise gram-positive bacteria, and the bacteria further comprise methicillin-resistant bacteria and vancomycin-resistant bacteria.

Compared with the prior art, the polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria has the beneficial effects or advantages that:

(1) compared with the prior art, the main contribution of the invention is to provide a polypeptide compound with broad-spectrum bactericidal activity for gram-positive bacteria;

(2) the inventor of the patent performs antibacterial property test on the polypeptide compound, and the polypeptide compound can kill gram-positive bacteria in a broad spectrum;

(3) the polypeptide compound disclosed by the patent can generate a killing effect on gram-positive bacteria with drug resistance to currently widely used antibiotics, particularly methicillin-resistant bacteria and vancomycin-resistant bacteria.

Drawings

FIG. 1 is a diagram showing the result of SephadexLH-20 gel column chromatography of the polypeptide complex of the present invention;

FIG. 2 is a graph showing the bactericidal effect of a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, wherein MRSA (Methicillin-resistant Staphylococcus aureus) is Methicillin-resistant Staphylococcus aureus; MRSH (Methicillin-resistant staphylococcus haemolyticus) is Methicillin-resistant staphylococcus haemolyticus; GISA (glucopeptide-intermediate Staphylococcus Aureus) is Glycopeptide-resistant antibiotic Staphylococcus Aureus;

FIG. 3 is a Western blot result chart of eluted peak 4.

Detailed Description

The following examples are given to illustrate the technical aspects of the present invention, but the present invention is not limited to the following examples.

Example 1

This example provides an extraction, isolation and identification assay for polypeptide complexes having broad-spectrum bactericidal activity against gram-positive bacteria.

(1) Extraction of polypeptide complexes

Centrifuging standard sensitive Staphylococcus aureus (ATCC 29213) fermentation broth at 4800r/min for 20min, collecting supernatant 50mL, placing in 100mL beaker, adding ammonium sulfate slowly in small amount until ammonium sulfate saturation is 80%, placing in 4 deg.C refrigerator overnight, centrifuging at 12000r/min at 4 deg.C for 20min with high speed centrifuge, dissolving precipitate with 20mmol/L Tris-HCl buffer (pH7.5), transferring into treated dialysis bag, placing in 20mmol/L Tris-HCl buffer, dialyzing with BaCl₂After detecting that no precipitate is generated, the liquid is taken out, freeze-dried and tested for activity.

(2) Isolation and purification of polypeptide complexes

Collecting the liquid, eluting 2-3 column volumes with 20mmol/LTris-HCl buffer solution at a flow rate of 1 second/drop, loading a sample with a volume of 3-4% of the column volume after the column is well balanced, controlling the flow rate at 15 s/drop and 2 mL/tube, detecting substances in each tube under ultraviolet light with a wavelength of 280nm, collecting absorption peaks, and detecting the activity of each peak by using S.aureus 3521 as an indicator bacterium, wherein an elution peak 4 (a specific elution curve is shown in figure 1) is found to have remarkable bacteriostatic activity. Collecting peak 4, ultrafiltering, concentrating, and performing reversed phase C₁₈And (5) performing column chromatography. After activity detection, the fraction eluted by acetonitrile with the volume fraction of 15% (containing TFA with the mass fraction of 0.5%) in the eluted fraction is the fraction where the target peptide is located.

(3) Identification of polypeptide complexes

And collecting the eluent corresponding to the elution peak 4, and identifying total protein in the fermentation broth, a crude product (protein not combined with the affinity chromatography column) and a polypeptide compound (eluent corresponding to the elution peak 4) with broad-spectrum bactericidal activity on gram-positive bacteria by using Western blot to obtain a graph 3. As can be seen from FIG. 3, the purified protein at peak 4 was confirmed to be the target protein, i.e., the polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, and the content of the target protein sample was 97. mu.g/g.

Example 2

This example provides a bactericidal assay for a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria.

The test uses a strain of Staphylococcus aureus (ATCC 29213) which is a standard susceptible strain and produces in its culture said polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria, the test strains comprising S.aurous 3521, S.aurous 1611, S.aurous Mu50, S.aurous Mu50 Δ recA, S.hemalyticus NW19, S.hemalyticus 5211, S.aurous 1511, S.aurous 3821, S.aurous E48, S.agnets 1932, S.epidermidis 2311, S.neri 3511, Bacillus licheniformis 1, Bacillus subtilis 1A747, E.coli K88, E.coli DH5 a. Test Standard susceptible Staphylococcus aureus (ATCC 29213) species and colonies of the species to be tested were inoculated in MH broth in sterilized test tubes and cultured overnight at 37 ℃ at 250 r/min. Transferring 30L of the cultured bacterial liquid into 3mL of fresh MH broth liquid culture medium, and carrying out shake culture at the constant temperature of 37 ℃ and 250r/min for 2-5 h to logarithmic phase. 100 mu L of ATCC 29213 culture solution containing the polypeptide compound with broad-spectrum bactericidal activity on gram-positive bacteria is dripped into the center of a solid (agar) culture dish, strains to be tested are respectively coated on the periphery of the culture dish, the bacteria are cultured overnight at 37 ℃, the bactericidal effect is observed, and the diameter of a bacteriostatic circle of the culture solution is measured and recorded, as shown in figure 2 and table 1.

FIG. 2 illustrates the bactericidal effect of a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria on each test species, and Table 1 shows the width of the zone of inhibition of a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria on each test species during the test.

TABLE 1 width of a broad-spectrum bactericidal activity of a polypeptide complex against gram-positive bacteria

Test strains	Width of antibacterial belt (mm)
		S.aureus 3521	5
S.aureus 1611	8
		S.aureus Mu50	2.4
S.aureus Mu50 ΔrecA	7.2
		S.heamolyticus NW19	4.8
S.heamolyticus 5211	11
		S.aureus 1511	14
S.aureus 3821	12
		S.aureus E48	4.5
S.agnetis 1932	9
		S.epidermidis 2311	8
S.warneri 3511	10
		Bacilus licheniformis CK1	4
Bacillus subtilis 1A747	15
		E.coli K88	5
E.coli DH5a	4

As can be seen from the results in Table 1 and FIG. 2, the polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria has broad-spectrum bactericidal activity against gram-positive bacteria, and particularly has good bactericidal effect against bacteria in which antibiotics such as methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus hemolyticus, and glycopeptide-resistant antibiotic Staphylococcus aureus are difficult to act.

Example 3

This example provides a stability test for a polypeptide complex having broad-spectrum bactericidal activity against gram-positive bacteria.

(1) Effect of concentration on the Fungicide Activity of the polypeptide complexes

1.0mL of polypeptide complex with the concentrations of 250, 125, 62.5 and 31.25mg/mL are respectively taken and subpackaged into 4 centrifuge tubes, and escherichia coli is taken as an indicator bacterium. The indicator bacteria are inoculated in LB liquid culture medium and cultured for 24h at the constant temperature of 37 ℃. Taking 100 μ L of the sample solution after filtration sterilization, adding 30 μ LL LB culture medium and 70 μ L bacterial suspension (the concentration of the bacterial suspension is 10)³～10⁴CFU/mL), mixing, at 37 deg.C, 150r/mAnd (3) incubating in a shaking table for 1h, pouring 100 mu L of the solution into a solid medium plate, culturing at 37 ℃ overnight, and calculating the colony number. And (4) calculating the bacteriostasis rate by taking deionized water as a control group. The test was repeated 3 times, and the results were averaged and are shown in table 2.

The bacteriostatic rate (%) was calculated as follows:

TABLE 2 Effect of concentration on the bactericidal Activity of the polypeptide complexes

As can be seen from Table 2, the antibacterial activity of the ultrafiltered polypeptide complex to Escherichia coli is enhanced with the increase of concentration, and the polypeptide complex still has an obvious antibacterial effect at a concentration of 31.25 mg/mL.

(2) Effect of pH on the Bactericidal Activity of the polypeptide complexes

Taking 1.0mL of polypeptide compound with the concentration of 125mg/mL, subpackaging the polypeptide compound into 6 centrifuge tubes, adding 1.0mL of prepared solution with the pH values of 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 into the centrifuge tubes, uniformly mixing, taking another 1 test tube, adding deionized water as a contrast, standing at room temperature for 1h, taking escherichia coli as an indicator bacterium, and carrying out a bacteriostatic test according to a method for influencing the bactericidal activity of the polypeptide compound by the concentration, wherein the test result is shown in Table 3.

TABLE 3 influence of pH on the bactericidal Activity of the polypeptide complexes

pH	Bacteriostatic ratio (%)
		2	40.63±1.76
4	54.50±1.15
		6	64.29±3.46
8	63.17±1.32
		10	54.83±3.38
12	40.47±1.16
		Control	68.82±1.84

As can be seen from Table 3, the polypeptide compound has high bacteriostatic activity under the condition that the pH value is 2.0-12.0, and the polypeptide compound has strong acid and alkali resistance.

(3) Effect of temperature on the Bactericidal Activity of the polypeptide complexes

Taking 1.0mL of the polypeptide compound with the concentration of 125mg/mL, subpackaging the polypeptide compound into 5 centrifuge tubes, respectively carrying out water bath for 10min under the conditions of 0, 20, 40, 60 and 80 ℃, taking 1 other centrifuge tube to contain an untreated sample as a reference, taking escherichia coli as an indicator bacterium, and carrying out a bacteriostasis test according to a method for influencing the bactericidal activity of the polypeptide compound by the concentration, wherein the test result is shown in table 4.

TABLE 4 Effect of temperature on the bactericidal Activity of the polypeptide complexes

Temperature (. degree.C.)	Bacteriostatic ratio (%)
		0	58.83±1.93
20	56.03±1.31
		40	60.52±3.04
60	60.60±4.03
		80	58.52±2.23
Control	60.25±1.88

As can be seen from Table 4, after the polypeptide compound is treated for 10min at the temperature of 0-80 ℃, the bacteriostasis rate of the polypeptide compound to escherichia coli tends to decrease after increasing with the increase of temperature, and the bacteriostasis rate is basically maintained between 54% and 64%. However, variance analysis shows that the antibacterial activity difference of the polypeptide compound treated at different temperatures is not significant (p is greater than 0.05); LSD showed no significant difference in the treated groups compared to the control, indicating that the functional polypeptide complex has excellent stability under both low and high temperature conditions.

As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.