阅读说明：本技术 一种鹦鹉螺血蓝蛋白衍生抗菌肽tyw4及其应用 (Nautilus nautilus hemocyanin derived antibacterial peptide TYW4 and application thereof ) 是由 包永波 袁春 张扬 于 2020-11-10 设计创作，主要内容包括：本发明公开了一种鹦鹉螺血蓝蛋白衍生抗菌肽TYW4及其应用。氨基酸序列如下所示：Arg Val Phe Ala Gly Phe Leu Arg His Gly lle Lys Arg Ser Arg。本发明通过分子改造已有鹦鹉螺血蓝蛋白天然衍生抗菌肽S4,创造出衍生抗菌肽TYW4,对得到的抗菌肽进行抗菌检测,发现其杀菌活力明显提高。衍生抗菌肽TYW4对溶藻弧菌A056及副溶血性弧菌2013V-1174的MIC分别为60μM、100μM,而且具有较好的α螺旋结构。本发明不仅为进一步了解鹦鹉螺免疫防御机制,提高其应用价值奠定了基础,还为研制新型抗菌药物提供了一种新的途径。(The invention discloses a nautilus hemocyanin derived antibacterial peptide TYW4 and application thereof. The amino acid sequence is shown as follows: arg Val Phe Ala Gly Phe Leu Arg His Gly lle Lys Arg Ser Arg. According to the invention, the existing nautilus snail hemocyanin naturally-derived antibacterial peptide S4 is molecularly modified to create derived antibacterial peptide TYW4, and the antibacterial activity of the obtained antibacterial peptide is obviously improved by carrying out antibacterial detection on the obtained antibacterial peptide. The MICs of the derivative antibacterial peptide TYW4 to Vibrio alginolyticus A056 and Vibrio parahaemolyticus 2013V-1174 are respectively 60 mu M and 100 mu M, and the derivative antibacterial peptide has a better alpha helical structure. The invention not only lays a foundation for further understanding the immune defense mechanism of the nautilus and improving the application value of the nautilus, but also provides a new way for developing novel antibacterial drugs.)

1. A peptide TYW4 derived from a nautilus hemocyanin antibacterial peptide, which is characterized in that the amino acid sequence is as follows:

Arg Val PheAla Gly Phe LeuArg His Gly lle Lys Arg SerArg。

2. use of the peptide TYW4 derived from the antimicrobial peptide derived from the nautilus snail hemocyanin according to claim 1 in the preparation of antimicrobial drugs.

3. The use of claim 2, wherein the antibacterial agent is a marine bacterial infectious disease treatment agent.

4. The use of claim 2, wherein the antibacterial agent is an agent against vibrio parahaemolyticus 2013V-1174 or vibrio alginolyticus a 056.

5. An antibacterial agent comprising the peptide TYW4 derived from the nautilus snail hemocyanin antibacterial peptide according to claim 1 as an active ingredient.

6. The antibacterial agent according to claim 5, wherein the antibacterial agent is a marine bacterial infectious disease treatment agent.

7. The antibacterial agent according to claim 5, wherein the antibacterial agent is an agent against Vibrio parahaemolyticus 2013V-1174 or Vibrio alginolyticus A056.

The technical field is as follows:

the invention belongs to the technical field of applied marine organisms, and particularly relates to a nautilus hemocyanin derived antibacterial peptide and application thereof.

Background art:

psittacosis is a cephalopod mollusk living in the deepwater western pacific, whose hemocyanin is a cyclic decamer of 350kDa polypeptide subunits, consisting of 7 different functional units (FU-a to FU-g). Psittacosis represent the oldest existing branches in the cephalopoda class, and their survival in the marine environment with a wide variety of microorganisms has so far shown that their innate immune system is efficient and powerful.

Antibacterial peptides (AMPs) are widely distributed small molecular polypeptides and are important components of the innate immune system of marine invertebrates. The number of microorganisms can reach 10 at most⁶Bacteria/ml and 10⁹In the seawater of each virus/ml, the antibacterial peptide can help marine invertebrates to quickly identify and kill pathogenic microorganisms such as invasive bacteria, viruses and the like by utilizing the broad-spectrum antibacterial activity of the antibacterial peptide. In recent years, it has been reported that hemocyanin of mollusk not only has oxygen delivery function, but also can participate in immune defense of organism through various ways.

The emergence of bacterial resistance, indicating that the efficacy of traditional antibiotics is decreasing, has increasingly highlighted the necessity of studying new antibacterials. The antibacterial peptide has the characteristics of broad-spectrum activity, multiple types, difficult generation of resistance mutation and the like, and is considered to have wide application prospect in the fields of medicines, foods and the like. However, the antibacterial activity of natural antibacterial peptides is not ideal enough compared with conventional antibiotics. Therefore, designing a completely new antibacterial peptide or molecular modification of an existing natural antibacterial peptide is an effective way to create a high-activity antibacterial peptide.

The invention content is as follows:

the first purpose of the invention is to provide a derivative peptide TYW4 of the nautilus hemocyanin antibacterial peptide with very good antibacterial effect.

The amino acid sequence of the derivative peptide TYW4 of the nautilus snail hemocyanin antibacterial peptide is shown as follows:

arg Val Phe Ala Gly Phe Leu Arg His Gly lle Lys Arg Ser Arg, which is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide the application of the peptide TYW4 derived from the nautilus snail hemocyanin antibacterial peptide in preparing antibacterial drugs.

The third object of the present invention is to provide an antibacterial agent comprising the peptide TYW4 derived from the nautilus snail hemocyanin antibacterial peptide described above as an active ingredient.

The antibacterial drug is a drug for treating marine bacterial infectious diseases.

The antibacterial drug is a drug for resisting vibrio parahaemolyticus 2013V-1174 or vibrio alginolyticus A056.

According to the invention, the existing nautilus snail hemocyanin naturally-derived antibacterial peptide S4 is molecularly modified to create derived antibacterial peptide TYW4, and the antibacterial activity of the obtained antibacterial peptide is obviously improved by carrying out antibacterial detection on the obtained antibacterial peptide. The MICs of the derivative antibacterial peptide TYW4 to Vibrio alginolyticus A056 and Vibrio parahaemolyticus 2013V-1174 are respectively 60 mu M and 100 mu M, and the derivative antibacterial peptide has a better alpha helical structure. The invention not only lays a foundation for further understanding the immune defense mechanism of the nautilus and improving the application value of the nautilus, but also provides a new way for developing novel antibacterial drugs.

Description of the drawings:

FIG. 1. inhibitory effect of antibacterial peptide S4 on Vibrio alginolyticus;

FIG. 2. inhibitory effect of antimicrobial peptide S4 on Vibrio parahaemolyticus;

FIG. 3. inhibitory effect of antibacterial peptide TYW4 on Vibrio alginolyticus;

FIG. 4. inhibitory effect of antimicrobial peptide TYW4 on Vibrio parahaemolyticus;

FIG. 5. three-dimensional structure of antimicrobial peptide TYW 4;

FIG. 6 3D model of antimicrobial peptide S4.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The strain related by the invention: escherichia coli DH5 alpha, Vibrio alginolyticus A056, and Vibrio parahaemolyticus 2013V-1174 are all from south sea research institute of Chinese academy of sciences.

The antimicrobial peptides TYW4 and S4 of the invention are diluted into mother liquor with the concentration of 0.01mol/L by sterile water, and are diluted into antimicrobial peptide solution with required concentration gradient (diluted by 1 × PBS) before MIC experiment.

Example 1:

three pieces of online software, namely AntiBP Server, CAMP and APD3 are combined to predict the psittacosis snail hemocyanin derived antibacterial peptide. The first of the three online software used was an antimicrobial peptide Server (AntiBP) (https:// webs. iiitd. edu. in/raghava/anti// index. html), which could be combined with the Nautilus snail hemocyanin sequence to look for potential AMPs. AMPs were then further screened using an antimicrobial peptide Collection Server (CAMP) (http:// www.camp.bicnirrh.res.in /). Finally, AMPs were verified using an antimicrobial peptide calculator and predictor (APD3) (http:// APs. unmac. edu/AP /), since APD3 allows the amphiphilicity and charge of AMPs to be obtained.

As shown in Table 1 below, 43 AMPs of 1.5-2.0 kDa were predicted in total. Considering that a large number of known AMPs have secondary conformational structures or β -rotations approximating α -helices, 6 potential AMPs were selected from which α -helix structures are likely to be present, predicted by APD3 software (S1, S2, S3, S4, S5 and S6).

TABLE 1 prediction of Nautilus Agitatus hemocyanin-derived antimicrobial peptides

TABLE 1

The antibacterial activity verification is carried out in the 6 potential AMPs (S1, S2, S3, S4, S5 and S6), and the specific steps are as follows:

a. the pathogenic bacteria (Escherichia coli DH5 alpha and Vibrio alginolyticus A056) stored at-80 ℃ are respectively activated in a small amount (a small amount of bacteria liquid is dipped by an inoculating loop and streaked on an LB culture medium), after overnight culture at 37 ℃, a single colony on a plate is picked up and placed in 50ml of LB liquid culture medium, and shake culture is carried out for 6-8h at 37 ℃ until logarithmic phase. Finally, the mixture is stored in a refrigerator at 4 ℃.

b. 1 μ L of the activated bacterial suspension was taken, and then diluted with 1000 μ L of 1 XPBS (10 μ L of the original bacterial suspension)^5-6CFU/mL)。

c. The same volume of the bacterial solution was mixed with diluted peptides (PBS dilution) of different concentration gradients (0. mu.M, 100. mu.M, 200. mu.M, 250. mu.M, 300. mu.M, 350. mu.M, 400. mu.M, 450. mu.M) (3. mu.L of bacterial cells + 3. mu.L of diluted peptides), and incubated at room temperature for 2.5 h. After completion of incubation, the mixture (3. mu.L of cell and 3. mu.L of diluted peptide) was placed on LB agar (12-well plate) and incubated overnight at 37 ℃. Colonies were counted after incubation was complete (PBS as negative control). Experiments were performed in triplicate, at least three replicates. The results are shown in Table 2.

TABLE 2 bacteriostatic Effect of six potential antibacterial peptides

Note: -represents no antibacterial activity

As shown in Table 2, the naturally derived antimicrobial peptide S4 from the nautilus hemocyanin has inhibitory effects on Escherichia coli and Vibrio alginolyticus, but the inhibitory effects are not very ideal.

Determination of Minimum Inhibitory Concentration (MIC) of antibacterial peptide S4 to Vibrio alginolyticus A056

(1) Respectively taking 1 μ L of activated Vibrio alginolyticus A056, adding 1000 μ L of 1 XPBS, and diluting (original bacterial liquid is 10%^5-6CFU/mL)。

(2) The diluted bacterial solution was mixed with antimicrobial peptide S4 (cell 3. mu.L + diluted peptide 3. mu.L) in different concentration gradients (0. mu.M, 100. mu.M, 200. mu.M, 250. mu.M, 300. mu.M) in equal volume, and incubated at room temperature for 2.5 h. After the incubation, the mixture (3. mu.L of the bacterial cells + 3. mu.L of the diluted peptide) was placed on LB agar (12-well plate) and incubated overnight (10-11 h) at 37 ℃. Colonies were counted after incubation was complete (PBS as negative control). Experiments were performed in triplicate, at least three replicates. The experimental results are shown in FIG. 1. As can be seen from FIG. 1, the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide S4 for Vibrio alginolyticus A056 was 250. mu.M.

Secondly, determination of Minimum Inhibitory Concentration (MIC) of antibacterial peptide S4 on Vibrio parahaemolyticus 2013V-1174

(1) Respectively taking 1 mu L of the activated vibrio parahaemolyticus 2013V-1174, and adding 1000 mu L of 1 XPBS for dilution (the original bacterial liquid is 10)^5-6CFU/mL)。

(2) The diluted bacterial solution was mixed with antimicrobial peptide S4 (cell 3. mu.L + diluted peptide 3. mu.L) in different concentration gradients (0. mu.M, 100. mu.M, 125. mu.M, 200. mu.M, 225. mu.M) in equal volume, and incubated at room temperature for 2.5 h. After the incubation, the mixture (3. mu.L of the bacterial cells + 3. mu.L of the diluted peptide) was placed on LB agar (12-well plate) and incubated overnight (10-11 h) at 37 ℃. Colonies were counted after incubation was complete (PBS as negative control). Experiments were performed in triplicate, at least three replicates. The experimental results are shown in FIG. 2. As can be seen from FIG. 2, the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide S4 against Vibrio parahaemolyticus 2013V-1174 was 200. mu.M.

Through SWISSMODEL presence server (https://swissmodel.expasy.org/interactive) The three-dimensional structure prediction of the amino acid sequence of antimicrobial peptide S4 was followed by opening in PyMol software for editing, and the 3D model of the position of antimicrobial peptide S4 in the nautilus hemocyanin was obtained as shown in fig. 6.

Example 2:

based on the beta folding part amino acid sequence RVFAGF in the sequence of the psittacine hemocyanin derived antibacterial peptide S4, arginine with positive charge is selected to replace two hydrophobic amino acids L and A in the beta corner part of S4. The psittacosis snail hemocyanin derived antibacterial peptide TYW4 is designed, and the amino acid sequence is as follows:

specifically shown as SEQ ID NO. 1.

The molecular weight was 1800.13Da, the hydrophobic ratio was 40%, and the charged amount was + 5.25. The three-dimensional structure of the antimicrobial peptide TYW4 predicted by an I-TASSER online server (https:// zhangglab. ccmb. med. umich. edu/I-TASSER /) is shown in FIG. 5.

The nautilus hemocyanin derived antibacterial peptide TYW4 is synthesized by Hefei peptide biotechnology limited, and the purity is more than 95%.

1. Determination of Minimum Inhibitory Concentration (MIC) of Vibrio alginolyticus A056 by Nautilus hemocyanin-derived antimicrobial peptide TYW4

(1) Respectively taking 1 μ L of activated Vibrio alginolyticus A056, adding 1000 μ L of 1 XPBS, and diluting (original bacterial liquid is 10%^5-6CFU/mL)。

(2) The diluted bacterial solution was mixed with antimicrobial peptide TYW4 (cell 3. mu.L + diluted peptide 3. mu.L) in different concentration gradients (0. mu.M, 10. mu.M, 25. mu.M, 30. mu.M, 50. mu.M, 60. mu.M, 70. mu.M) in equal volume, and incubated at room temperature for 2.5 h. After the incubation, the mixture (3. mu.L of the bacterial cells + 3. mu.L of the diluted peptide) was placed on LB agar (12-well plate) and incubated overnight (10-11 h) at 37 ℃. Colonies were counted after incubation was complete (PBS as negative control). Experiments were performed in six replicates, at least six replicates. The results of the experiment are shown in FIG. 3.

As can be seen from FIG. 3, the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide TYW4 for Vibrio alginolyticus A056 was 60. mu.M.

2. Determination of Minimum Inhibitory Concentration (MIC) of Naciotuo hemocyanin-derived antibacterial peptide TYW4 to Vibrio parahaemolyticus 2013V-1174

(1) Respectively taking 1 mu L of the activated vibrio parahaemolyticus 2013V-1174, and adding 1000 mu L of 1 XPBS for dilution (the original bacterial liquid is 10)^5-6CFU/mL)。

(2) The diluted bacterial solution was mixed with antimicrobial peptide TYW4 (cell 3. mu.L + diluted peptide 3. mu.L) in different concentration gradients (0. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 90. mu.M, 100. mu.M, 110. mu.M) in the same volume, and incubated at room temperature for 2.5 hours. After the incubation, the mixture (3. mu.L of the bacterial cells + 3. mu.L of the diluted peptide) was placed on LB agar (12-well plate) and incubated overnight (10-11 h) at 37 ℃. Colonies were counted after incubation was complete (PBS as negative control). Experiments were performed in six replicates, at least six replicates. The results of the experiment are shown in FIG. 4.

As can be seen from FIG. 4, the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide TYW4 against Vibrio parahaemolyticus 2013V-1174 was 100. mu.M.

Sequence listing

<110> Zhejiang Wanli college

South China Sea Institute of Oceanography, Chinese Academy of Sciences

<120> spirochete hemocyanin-derived antibacterial peptide TYW4 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Val Phe Ala Gly Phe Leu Arg His Gly Ile Lys Arg Ser Arg

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