Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to a Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide and a preparation method and application thereof. The present invention studies a horseshoe crab antimicrobial peptide: TaTrs_ALF_α1 for Chinese horseshoe crabs. It has been verified that the antimicrobial peptide has good inhibitory effects on common pathogenic bacteria in aquaculture: Staphylococcus aureus, Micrococcus luteus, Aeromonas hydrophila and / or Pseudomonas stutzeri. At the same time, it also has a good growth inhibition effect on gastric cancer cells SGC-7901 and / or esophageal cancer cells Eca-109. When used as a drug for treating esophageal cancer, the inhibitory effect on esophageal cancer cells Eca-109 can reach more than 90% when the concentration reaches 80 μg / mL, and the anticancer effect is good. Therefore, the antimicrobial peptide of the present application can be widely used in the fields of drug research and development and animal breeding immunoenhancers, and is a rare antimicrobial peptide.

Description

[Technical field]

[0001] The invention relates to the field of biotechnology, and in particular to Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide and a preparation method and application thereof. [Background technology]

[0002] As an ancient marine invertebrate, Chinese horseshoe crab lacks acquired immunity, and its resistance to infection by pathogens such as bacteria, fungi and viruses is mediated by innate immunity, which mainly includes cellular immunity and humoral immunity. The circulating blood cells of Chinese horseshoe crab are involved in the storage and release of various defense molecules, among which antimicrobial peptides (AMPs) are the main components of the innate immune defense system of marine invertebrates, which have the function of directly killing microbial pathogens and can also participate in regulating the host's immune response mechanism, indirectly achieving antibacterial function. Marine arthropods are a rich resource of natural antimicrobial peptides. Currently, several family members of antimicrobial peptides such as penaeidin, chitin, and anti-lipopolysaccharide factor have been isolated from crustaceans. However, the extent of exploration of antimicrobial peptides from the chelicerate subphylum, including horseshoe crabs, is still relatively low. The most outstanding research results are the horseshoe crab antimicrobial peptides (Tachyplesins, TPs), including TP-Ⅰ, TP-Ⅱ, TP-Ⅲ isolated from Chinese horseshoe crab and polyphemusin Ⅰ and polyphemusin Ⅱ from American horseshoe crab. They have biological activity in inhibiting and killing bacteria, fungi, viruses and parasites, and can also inhibit the proliferation of tumor cells.

[0003] Anti-lipopolysaccharide factors (ALFs) can bind and neutralize lipopolysaccharide (LPS), and their functional activity comes from the highly conserved LPS binding domain (LBD), hence the name. ALF was first isolated from the hemolymph of Limulus polyphemus and Tachypleus tridentatus, and can inhibit the activation of bacterial endotoxins and thus inhibit the hemolymph coagulation system of horseshoe crabs. Only one variant of ALFs has been described in horseshoe crabs, but it is the most widely studied antimicrobial peptide in crustaceans. To date, more than 300 proteins of this type have been isolated and identified, forming a genetically diverse family of antimicrobial peptides. Although there are a large number of research reports on crustacean ALFs, as of 2021, only the crystal structure of ALF-Pm3 from Penaeus monodon has been resolved, which was then used as the structural model of 7 representative ALFs (AG), mainly composed of 3 α helices and 4 antiparallel β folds. Compared with other crustacean antimicrobial peptides, the ALFs family has the broadest spectrum of antimicrobial activity, showing inhibitory effects on bacteria, fungi and viruses. Antimicrobial peptides are a class of small molecule polypeptides with broad-spectrum antimicrobial properties, usually composed of less than 100 amino acid residues, most of which are cationic in nature, with a cysteine-stabilized structure and amphipathic properties. Antimicrobial peptides are a potential alternative to antibiotics because of their special mechanism of action and pattern diversity, making it difficult for microorganisms to develop drug resistance. They have broad prospects for the development of new peptide antimicrobial drugs and can also be used as immune enhancers for farmed animals. However, in the current study, we found that there is currently no research and development of Chinese horseshoe crab antimicrobial peptide products based on the ALFs family.

[0004] In the process of functional verification of antimicrobial peptide products, we found that a class of antimicrobial peptides cannot inhibit the growth of all bacteria. Therefore, antimicrobial peptides need to be continuously verified to determine their antibacterial range. At the same time, although antimicrobial peptides have been reported to have certain tumor cell inhibitory activity, functional verification has shown that they do not have inhibitory effects on all tumor cells. Therefore, in order to enhance the application field of antimicrobial peptides, it is necessary to study antimicrobial peptides, continuously verify their functional effects, and enrich the application scope of antimicrobial peptides. [Summary of the invention]

[0005] In view of the above, it is necessary to conduct research and development on the ALFs family of Chinese horseshoe crabs, produce antimicrobial peptide products, and conduct certain functional verifications to verify its scope of antibacterial effects and the scope of inhibiting the growth of tumor cells, so as to improve the application scope of Chinese horseshoe crab antimicrobial peptides.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention comprises Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide, and the amino acid sequence of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide is shown as SEQ ID NO.3.

[0008] The present invention also includes the use of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide in the preparation of antimicrobial drugs.

[0009] Furthermore, the bacteria inhibited by the antibacterial drug are Staphylococcus aureus, Micrococcus luteus, Aeromonas hydrophila and / or Pseudomonas stutzeri.

[0010] Furthermore, the minimum inhibitory concentrations of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide against Micrococcus luteus, Pseudomonas stutzeri and Aeromonas hydrophila are <1.5 μM, 3-6 μM and 24-48 μM, respectively, and the minimum bactericidal concentrations are 1.5-3 μM, 6-12 μM and 24-48 μM, respectively; the minimum inhibitory concentration against Staphylococcus aureus is 12-24 μM, and the minimum bactericidal concentration is 12-24 μM.

[0011] The present invention also includes the use of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide in the preparation of anticancer drugs.

[0012] Furthermore, the cancer cells inhibited by the anticancer drug are gastric cancer cells and / or esophageal cancer cells.

[0013] Furthermore, the cancer cells inhibited by the anticancer drug are gastric cancer cells SGC-7901, and the esophageal cancer cells are Eca-109.

[0014] Furthermore, the concentration of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide as a drug for inhibiting esophageal cancer cells Eca-109 is greater than or equal to 80 μg / mL.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention has developed a horseshoe crab antimicrobial peptide: TaTrs_ALF_α1 for Chinese horseshoe crab. It has been verified that the antimicrobial peptide has a good inhibitory effect on common pathogenic bacteria in aquaculture: Staphylococcus aureus, Micrococcus luteus, Aeromonas hydrophila and / or Pseudomonas stutzeri. At the same time, it also has a good inhibitory effect on the growth of gastric cancer cells SGC-7901 and / or esophageal cancer cells Eca-109. When used as a drug for the treatment of esophageal cancer, its concentration reaches 80 μg / mL, and the inhibitory effect on esophageal cancer cells Eca-109 can reach more than 90%, and the anti-cancer effect is good. This shows that the antimicrobial peptide of the present application can be widely used in the field of drug research and development and animal breeding immunoenhancers, and is a rare peptide product.

Brief Description of the Drawings

[0017] Figure 1 Diagram of the tertiary structure of a protein;

[0018] Figure 2 Antimicrobial peptides can form an amphipathic α-helical structure;

[0019] Figure 3 Photograph of a petri dish of Aeromonas hydrophila. [Specific implementation method]

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0021] Embodiment 1:

[0022] 1. The gene sequence of TaTrs_ALF antimicrobial peptide from Chinese horseshoe crab was obtained by the following method:

[0023] Chinese horseshoe crab embryos and larvae were collected, total RNA was extracted, and transcriptome library sequencing was carried out after passing quality inspection. The trinity software was used to assemble the Chinese horseshoe crab reference transcripts. Finally, the blastp software was used to screen out a full-length cDNA sequence of the TaTrs_ALF antimicrobial peptide gene of Chinese horseshoe crab by homology alignment. The full-length cDNA sequence of the gene is shown in SEQ ID NO.1.

[0024] ATGATGAAAGTGACATTAGTCGCACTGTTGCTTATGTCAACCTACGTCTACAGAACAGCAGCACAGGGCGGAATTTGGACTCAACTTGCTCTCGCATTAGTAAAGAATTTAGCCACACTTTGGCAGAGCGGAGATTTTCAGTTTCTGGGCCATGAATGTCATTATCGAGTTAAT CCCACTATAAAACGTCTAAGGTGGAAATATAAAGGGAAGTTCTGGTGTCCCTCGTGGACTTCCATCACTGGAAGAGCTACCAAAAGCAGTAGGTCCGGCGCTGTAGAACATTCTGTTAGAGACTTTGTTAGCCAGGCCAAATCGTCAGGTTTAATCACAGAAAAAGAAGCACCA.

[0025] The full-length cDNA of TaTrs_ALF antimicrobial peptide gene was translated into an amino acid sequence. The full-length amino acid sequence of TaTrs_ALF antimicrobial peptide is SEQ ID NO.2, which is as follows:

[0026] MMKVTLVALLLMSTYVYRTAAQGGIWTQLALALVKNLATLWQSGDFQFLGHECHYRVNP

[0027] TIKRLRWKYKGKFWCPSWTSITGRATKSSRSGAVEHSVRDFVSQAKSSGLITEKEAP

[0028] 2. Obtaining the tertiary protein structure of the mature peptide and the TaTrs_ALF_α1 polypeptide fragment with antibacterial activity:

[0029] SignalP-6.0 software was used to predict the signal peptide and mature peptide of TaTrs_ALF antimicrobial peptide, and the physicochemical properties and tertiary structure of the mature peptide were predicted using the websites https: / / web.expasy.org / protparam / and https: / / swissmodel.expasy.org / , respectively.

[0030] The results showed that the mature peptide consisted of 95 amino acids and its molecular formula was C 593 H 932 N 164 O 161 S 5 , with a molecular weight of 10737.27 Daltons. The mature peptide carries 14 positive charges and 6 negative charges, and is a cationic antimicrobial peptide. Figure 1 As shown, it contains 2 β sheets and 2 α helices.

[0031] The website http: / / www.camp.bicnirrh.res.in / predict_c / was used to predict the polypeptide fragment with antibacterial activity of the mature peptide, and a polypeptide fragment with a length of 20 amino acids was obtained and named TaTrs_ALF_α1, and its amino acid sequence was SEQ ID NO.3; specifically: CHYRVNPTIKRLRWKYKGKF.

[0032] The physicochemical properties and structural characteristics of TaTrs_ALF_α1 were predicted using the website https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py.

[0033] The results showed that the antimicrobial peptide can form an amphipathic α-helical structure, such as Figure 2 As shown, it has obvious hydrophobic and hydrophilic surfaces and carries 7 positive charges. It is an α-helical cationic antimicrobial peptide.

[0034] 3. Preparation of TaTrs_ALF_α1 antimicrobial peptide from Chinese horseshoe crab:

[0035] The amino acid sequence of TaTrs_ALF_α1 antimicrobial peptide was submitted to Hubei Qiangyao Biotechnology Co., Ltd., and the company was commissioned to use solid phase chemical synthesis to synthesize the crude TaTrs_ALF_α1 antimicrobial peptide, which was then purified by high performance liquid chromatography and concentrated by a freeze dryer. Finally, a mass spectrometer was used for mass spectrometry identification. After qualified identification, high-purity TaTrs_ALF_α1 antimicrobial peptide was prepared.

[0036] 4. Determination of antimicrobial activity of TaTrs_ALF_α1 antimicrobial peptide from Chinese horseshoe crab:

[0037] In this example, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Corynebacterium glutamicum, Vibrio alginolyticus, Vibrio fluvialis, Micrococcus luteus, Shigella flexneri

[0038] Shigellaflexneri, Bacillus cereus, Vibrioharveyi, Aeromonas hydrophila and Pseudomonas stutzeri were used as test strains for antibacterial activity experiments. Among them, Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Pseudomonas stutzeri and Aeromonas hydrophila were cultured in MHA medium at 28°C, and Corynebacterium glutamicum, Bacillus subtilis, Vibrio alginolyticus, Vibrio fluvii, Shigella flexneri, Bacillus cereus and Vibrio harveyi were cultured in 2216E medium at 37°C. All the strains used were purchased from the Marine Culture Collection of China (MCCC).

[0039] The method for determining the antimicrobial activity is as follows:

[0040] (1) Activation of refrigerated strains: Streak the bacterial strains (i.e., the above-mentioned test strains) stored at -80°C on MHA or 2216E plates, culture them at 28°C (marine bacteria) or 37°C (non-marine bacteria) for 24 h, pick a single colony and transfer it to 10 mL of sterile MHA or 2216E liquid culture medium, and culture it at 28°C or 37°C, shaking at 180 rpm / min for 12 h;

[0041] (2) Preparation of bacterial suspension: Take a small amount of activated bacterial liquid and streak it on MHA or 2216E plate, invert and culture for 12 h, pick a single colony from the plate and streak it on MHA or 2216E slant culture medium, culture it at 28°C or 37°C for 12 h, elute the slant culture with NaPB (10 mM), and measure the OD value of the eluate with a microplate reader. Add a certain amount of the eluate to NaPB or working culture medium so that the OD value of the final bacterial suspension is 0.0018;

[0042] (3) Preparation of antimicrobial peptide working solution: The antimicrobial peptide dry powder was dissolved in ultrapure water to prepare a working solution. After filtering through a 0.22 μm filter membrane, the protein concentration was determined by the Bradford method. The protein concentrations of the working solutions were diluted to 1.5, 3.0, 6.0, 12, 24, 48, and 96 μM and stored at 4°C for later use.

[0043] (4) Determination of minimum inhibitory concentration (MIC): Design an experimental group, a blank control group, and a negative control group for the experiment, with three parallels for each antimicrobial peptide concentration; culture the cells in a 96-well cell culture plate for 24 hours and observe the experimental results.

[0044] The reagents added to each group are as follows:

[0045] Experimental group: 50 μL antimicrobial peptide working solution + 50 μL bacterial suspension;

[0046] Blank control: 50 μL sterile water + 50 μL bacterial suspension;

[0047] Negative control: 50 μL working culture medium + 50 μL antimicrobial peptide working solution with the lowest concentration.

[0048] (5) Determination of minimum bactericidal concentration (MBC): Pipette 1 μL of the mixed culture from each well of the incubated cell culture plate and drop it onto an MHA or 2216E plate. Incubate for 24 h (the culture temperature for non-marine bacteria is 37°C and the culture temperature for marine bacteria is 28°C). Observe the colony formation.

[0049] The specific test results are shown in Table 1:

[0050] Table 1 Results of antibacterial activity determination of TaTrs_ALF_α1 antimicrobial peptide

[0051]

[0052] As can be seen from Table 1, the results show that the TaTrs_ALF_α1 antimicrobial peptide has a strong inhibitory and killing effect on common pathogenic bacteria in aquaculture. Among them, the minimum inhibitory concentrations of Micrococcus luteus, Pseudomonas stutzeri and Aeromonas hydrophila are <1.5μM, 3-6μM and 24-48μM, respectively, and the minimum bactericidal concentrations are 1.5-3μM, 6-12μM and 24-48μM, respectively; it also has a strong inhibitory and killing effect on Staphylococcus aureus, a common foodborne pathogen in aquatic products, with a minimum inhibitory concentration of 12-24μM and a minimum bactericidal concentration of 12-24μM.

[0053] The plate culture of the killing test bacteria species takes Micrococcus luteus as an example. The photo of the culture dish of Micrococcus luteus is as follows Figure 3As shown in the figure, the plate is divided into 8 fan-shaped areas of the same size, and the concentrations of the antimicrobial peptide working solution in each area are: 1.5μM, 3.0μM, 6.0μM, 12μM, 24μM, 48μM, 96μM and 0.0μM (negative control group), among which the negative control group is not inoculated with Micrococcus luteus; the concentrations of the antimicrobial peptide working solution in different areas are marked with black text. It can be seen from the figure that at a concentration of 1.5μM, the plate can grow Micrococcus luteus, while at a concentration of 3μM, Micrococcus luteus cannot grow, which shows that the highest concentration of the antimicrobial peptide working solution that can grow Micrococcus luteus is 1.5μM, and the lowest concentration of the antimicrobial peptide working solution that kills Micrococcus luteus and cannot grow is 3μM. It is concluded that the minimum bactericidal concentration of TaTrs_ALF_α1 antimicrobial peptide against Micrococcus luteus is 1.5-3μM.

[0054] Embodiment 2:

[0055] The applicant discovered that TaTrs_ALF_α1 antimicrobial peptide (hereinafter referred to as α1 antimicrobial peptide) not only has a wide range of bactericidal effects, but also has an inhibitory effect on the growth of cancer cells, as follows:

[0056] 1. Materials: SGC-7901 cells are derived from human gastric adenocarcinoma cell line, human esophageal carcinoma cell line Eca-109, and TaTrs_ALF_α1 antimicrobial peptide.

[0057] 2. Experimental method: The cell proliferation ability was detected by MTT assay. Under sterile conditions, 5×10 3 Tumor cells were cultured for 24 hours, and 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 160 μg / mL of α1 antimicrobial peptide were added to the wells respectively. The experimental group with only tumor cells was used as the blank group. After culturing for 1d, 2d, 3d, 4d, 5d, 6d and 7d, 20 μL / well of freshly prepared MTT phosphate buffer was added respectively. After culturing for another 4 hours, the culture medium in the plate was poured out, DMSO150 μL was added to each well, and the culture was shaken for 10 minutes. The absorbance (A value) of each well was measured at a wavelength of 570nm using an enzyme marker. The average value of A of 4 wells in each group was taken as the average A value of each group. The cell growth inhibition rate on the 6th day was calculated according to the following formula: IF (%) = (1-A of the drug group / A of the control group) × 100%. The results are shown in Table 2:

[0058] Table 2 Tumor cell growth inhibition rate after adding α1 antimicrobial peptide on the 6th day

[0059]

[0060] Note: Data in the same column with the same lowercase letters or no letters on shoulders indicate no significant difference (P>0.05), while data with different lowercase letters on shoulders indicate significant difference (P<0.05).

[0061] As can be seen from Table 2, α1 antimicrobial peptide has a significant inhibitory effect on both SGC-7901 cells and Eca-109 cells, and as the concentration increases, the inhibitory effect becomes better. The inhibition rate of SGC-7901 cells on the 6th day can reach up to 58.85%, and that of Eca-109 cells can reach up to 95.51%. From the inhibitory effect of the two cells: the inhibitory effect of α1 antimicrobial peptide on Eca-109 cells is significantly better than that on SGC-7901 cells, which shows that α1 antimicrobial peptide can inhibit the growth of tumor cells, but due to the difference in cells, the inhibitory effect on esophageal cancer cells is better than that on gastric cancer cells. From the inhibitory effect, when the amount of antimicrobial peptide added is 10-20μ When the addition amount was 40 μg / mL, there was no significant difference in the inhibitory effect on SGC-7901 cells, but when the addition amount was 40 μg / mL, the inhibitory effect was significantly improved; when the addition amount of the antimicrobial peptide was 80-160 μg / mL, there was no significant difference in the inhibitory effect on Eca-109 cells, indicating that a good effect of inhibiting the growth of Eca-109 cells can be achieved when the addition amount reaches 80 μg / mL; from the cost consideration, a good effect can be achieved (the inhibition rate reaches more than 90%) when the addition amount is 80 μg / mL for the inhibitory effect on Eca-109 cells, indicating that if used as a drug to inhibit the growth of Eca-109 cells, the concentration can be selected to be above 80 μg / mL.

[0062] The TaTrs_ALF_α1 antimicrobial peptide of the present invention has been verified to have good antibacterial function and is effective against most common pathogenic bacteria in aquaculture. Cell experiments have found that the antimicrobial peptide also has a certain inhibitory effect on gastric cancer cells SGC-7901 and esophageal cancer cells Eca-109. When its concentration reaches 80 μg / mL, the inhibitory effect on esophageal cancer cells reaches more than 90%, indicating that the TaTrs_ALF_α1 antimicrobial peptide of the present application is a product with good antibacterial and anti-cancer functions.

[0063] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. TaTrs_ALF_α1 antimicrobial peptide from Chinese horseshoe crab, characterized in that: The amino acid sequence of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide is shown in SEQ ID NO.

3.

2. The use of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide in the preparation of antimicrobial drugs as claimed in claim 1, wherein the bacterium is Staphylococcus aureus ( Staphylococcus aureus ), Micrococcus luteus ( Micrococcus luteus ), Aeromonas hydrophila ( Aeromonas hydrophila ) and / or Pseudomonas stutzeri ( Pseudomonas stutzeri ).

3. The use according to claim 2, characterized in that: The Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide is effective against Micrococcus luteus ( Micrococcus luteus ), Pseudomonas stutzeri ( Pseudomonas stutzeri ) and Aeromonas hydrophila ( Aeromonas hydrophila ) were <1.5μM, 3~6μM and 24~48μM, and the minimum bactericidal concentrations were 1.5~3μM, 6~12μM and 24~48μM, respectively; for Staphylococcus aureus ( Staphylococcus aureus ) had a minimum inhibitory concentration of 12~24 μM, and a minimum bactericidal concentration of 12~24 μM.

4. Use of the Chinese horseshoe crab TaTrs_ALF_α1 antimicrobial peptide as claimed in claim 1 in the preparation of anticancer drugs; the cancer cells inhibited by the anticancer drugs are gastric cancer cells and / or esophageal cancer cells.

5. The use according to claim 4, characterized in that: The gastric cancer cells are SGC-7901, and the esophageal cancer cells are Eca-109.

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