A hermetia illucens defensin c-13326

Abstract

The present application provides a black soldier fly defensin C-13326, the nucleotide sequence of the black soldier fly defensin C-13326 is shown as SEQ ID NO.1, and the application of the black soldier fly defensin C-13326 in inhibiting multiple drug-resistant Aeromonas sobria is also provided.The black soldier fly defensin C-13326 provided by the present application can be applied to the preparation of anti-drug-resistant Aeromonas sobria products to cope with the serious harm caused by drug-resistant Aeromonas sobria.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological products, and particularly relates to a Hermetia illucens L. defensin C-13326. BACKGROUND

[0002] Hermetia illucens L., also known as bright spot flat angle water bug, belongs to the class of insects, diptera, brachyptera, and water bug family. The larvae of Hermetia illucens L. live a saprophytic life, and the living environment is similar to that of fly maggots. The worm body is often exposed to high concentrations of harmful microorganisms, so the larvae have a very powerful immune system and a large number of antibacterial peptides. Antibacterial peptides are a class of small molecule polypeptides with antibacterial activity, which can directly kill pathogens or activate the host immune defense system, and play an important role in the immune defense of the host against pathogen invasion. Among the antibacterial peptides of Hermetia illucens L., the number of defensins accounts for the largest proportion. Studies have shown that the defensins of Hermetia illucens L. have good bacteriostatic effect on gram-positive bacteria and negative bacteria. Studies have shown that among the defensin antibacterial peptides isolated from Hermetia illucens L., DLP3 can inhibit the growth of Escherichia coli, and DLP2 and DLP4 have been confirmed to inhibit the growth of methicillin-resistant Staphylococcus aureus. Due to the long-term abuse of antibiotics in some large-scale livestock and poultry breeding industries and aquaculture industries in China, on the one hand, it leads to excessive antibiotic residues in food, which seriously endangers food safety; on the other hand, it also leads to the continuous improvement of the drug resistance spectrum and drug resistance of pathogenic bacteria, and induces the breeding of "super bacteria". Aeromonas sobria, as a common pathogenic bacterium in aquaculture, is often reported to have different degrees of drug resistance, which seriously affects its prevention and treatment and brings huge economic losses to the aquaculture industry. SUMMARY

[0003] The purpose of the present application is to provide a Hermetia illucens L. defensin.

[0004] The purpose of the present application is also to provide an application of the Hermetia illucens L. defensin C-13326 in inhibiting multi-drug resistant Aeromonas sobria.

[0005] In order to achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0006] A Hermetia illucens L. defensin C-13326, the nucleotide sequence of the Hermetia illucens L. defensin C-13326 is shown in SEQ ID NO. 1.

[0007] Further, the amino acid sequence of the Hermetia illucens L. defensin C-13326 is shown in SEQ ID NO. 2.

[0008] Further, the expression method of the black soldier fly defensin C-13326 gene is first to construct the recombinant plasmid pPIC9K-C-13326 of the Pichia pastoris secretion vector and the C-13326 gene, and then to transform into the Pichia pastoris bacteria GS115 for expression.

[0009] Further, the application of the black soldier fly defensin C-13326 in inhibiting multiple drug-resistant Aeromonas sobria.

[0010] Compared with the prior art, the application has the following advantages:

[0011] The application discloses a black soldier fly defensin C-13326 amino acid sequence.

[0012] The application lays a foundation for exploring the immune defense mechanism of the black soldier fly defensin, and provides a theoretical basis for further developing the black soldier fly antibacterial peptide resource.

[0013] The application discloses that the black soldier fly defensin C-13326 has antibacterial activity on multiple drug-resistant Aeromonas sobria, the separated black soldier fly defensin C-13326 has an obvious antibacterial circle on the multiple drug-resistant Aeromonas sobria, and the diameter of the antibacterial circle is greater than 10 mm, belonging to a sensitive state. BRIEF DESCRIPTION OF DRAWINGS

[0014] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and serve as an aid in explaining the exemplary embodiments of the present application and their descriptions, and do not constitute an improper limitation on the present application. In the drawings:

[0015] Figure 1 For the defensin C-13326 gene to be transferred into the Pichia pastoris; wherein a is an agarose electrophoresis map of a PCR amplification defensin C-13326 gene product; b is a pPIC9K plasmid enzyme digestion spectrum; c is a recombinant plasmid yeast transformant agarose electrophoresis map;

[0016] Figure 2 For the induced expression and identification of the defensin C-13326 in the Pichia pastoris; wherein 1 is a result map of Tricine-SDS-PAGE electrophoresis and Coomassie brilliant blue staining after methanol induction of the defensin C-13326 expression, and N is a negative control;

[0017] Figure 3The drug sensitivity test of the multi-drug resistant Aeromonas shajii;

[0018] Figure 4 The antibacterial results of the defensin C-13326 on the multi-drug resistant Aeromonas shajii; wherein a is the antibacterial results of the defensin C-13326 on the multi-drug resistant Aeromonas shajii by the Oxford cup method; b is the antibacterial circle diameter measurement results of the defensin C-13326 on the multi-drug resistant Aeromonas shajii by the Oxford cup method;

[0019] Figure 5 The antibacterial pathway of the defensin C-13326; wherein (a) is the electron microscope image of the multi-drug resistant Aeromonas shajii without treatment; (b) is the electron microscope image of the multi-drug resistant Aeromonas shajii treated by the defensin C-13326. DETAILED DESCRIPTION

[0020] The following specific descriptions are exemplary and are intended to provide further explanation of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0021] The raw materials and reagents used in the present application are commercially available, and the multi-drug resistant Aeromonas shajii strain is preserved by Zhongkai Agricultural Engineering College Health Breeding Innovation Research Institute.

[0022] Example 1 Expression of the defensin C-13326 gene of black soldier fly

[0023] The black soldier fly larvae were pricked with a sterile needle, and after 24h of continuous feeding, the total RNA was extracted using TRIzol and the cDNA was obtained by reverse transcription. The primer was designed, the defensin C-13326 gene was amplified by PCR, the PCR product was subjected to agarose gel electrophoresis (see Figure 1 a), and was sent to Guangzhou Tianyihuiyuan Company for sequencing. The gene sequence is shown in SEQ ID NO. 1. The sequencing data was compared and bioinformatics analysis was performed to determine that the defensin c-13326 gene sequence length is 243bp.

[0024] The defensin C-13326 gene was purified and recovered by using a DNA gel recovery kit, and the secreted expression vector pPIC9K of Pichia pastoris was subjected to enzyme digestion using EcoR I and Not I, and was connected with the purified defensin C-13326 gene (see Figure 1b), the connection product is transformed into E. coli competent cells, then coated on LB agar plates containing ampicillin, and single colonies are picked for amplification culture and plasmid extraction to construct the Pichia pastoris expression vector pPIC9K-C-13326 containing the defensin C-13326 gene. The plasmid is verified by PCR using 5'AOX and 3'AOX primers, and the product is subjected to 0.8% agarose gel electrophoresis to obtain a band of about 750bp, which is a positive plasmid (see Figure 1 c Lane 1), lane N is pPIC9K plasmid as a negative control. The positive plasmid is sent to Guangzhou Tianyihuiyuan Gene Technology Co., Ltd. for Sanger sequencing, and the plasmid with correct sequence is selected.

[0025] We linearize the constructed pPIC9K-C-13326 recombinant plasmid and electrotransform it into Pichia pastoris GS115, and obtain Mut+ positive clone strains by screening. The target gene is verified by PCR using primers 5'AOX and 3'AOX to determine whether it is successfully integrated into the yeast chromosome. If the integration is successful, two bands can be seen in the agarose gel, one about 2000bp is carried by Pichia pastoris itself, and the other about 750bp is the pPIC9K-C-13326 recombinant plasmid (see Figure 1 c Lane 2). The successfully integrated strain is inoculated into BMGY liquid medium and cultured for 12h, then centrifuged to obtain a large number of positive clone strains, and then transferred to BMMY liquid medium for amplification culture. The target gene is induced by methanol, and defensin C-13326 should exist in the fermentation supernatant. After 4d of induction at 30°C, the fermentation supernatant is collected, concentrated by freeze-drying, and then subjected to Tricine-SDS-PAGE and Coomassie brilliant blue staining (see Figure 2 ), and the control group is the yeast transformant induced under the same conditions but without the defensin C-13326 gene but with the pPIC9K plasmid. Compared with the control group, the experimental group (lane 2) expresses a protein with a molecular weight of about 5.8kDa, which is consistent with the expected molecular weight of defensin C-13326 (4.2kDa). The above protein band is cut and sent to Guangzhou Huijun Biotechnology Co., Ltd. for mass spectrometry analysis, and the identified polypeptide amino acid sequence is consistent with the amino acid sequence of defensin C-13326, and the amino acid sequence is shown as SEQ ID NO. 2.

[0026] Example 2. Drug resistance identification of Shuberter aeromonas

[0027] The detection method is performed according to the standard of American Clinical Laboratory Standardization Institute (CLSI).

[0028] Disk diffusion method: Shuberter aeromonas liquid culture is adjusted to 10 8CFU / mL, 100 uL was taken and uniformly spread on BHI agar plates, then 16 antibiotic filter paper discs were placed on the plates, and the plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was measured (mm), and the drug resistance of the strain was determined according to the National Committee for Clinical Laboratory Standards (NCCLS). The results showed that the strain of A. schubertii was resistant to 16 kinds of antibiotics (see Table 1). Figure 3 and Table 1).

[0029] Table 1, results of drug sensitivity test of multi-drug resistant A. schubertii

[0030]

[0031]

[0032] R is resistant; S is sensitive; I is intermediate; - is no inhibition zone

[0033] Example 3 Inhibition of multi-drug resistant A. schubertii by defense protein C-13326 of black soldier fly

[0034] Oxford cup diffusion method: After the multi-drug resistant A. schubertii was cultured, the concentration was adjusted to 10 8 CFU / mL, 100 uL was taken and uniformly spread on BHI agar plates, then 16 antibiotic filter paper discs were placed on the plates, and the plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was measured (mm), and the drug resistance of the strain was determined according to the National Committee for Clinical Laboratory Standards (NCCLS). The results showed that the strain of A. schubertii was resistant to 16 kinds of antibiotics (see Table 1). Figure 4 a), indicating that the defense protein C-13326 can inhibit the growth of multi-drug resistant A. schubertii. At the same time, after the multi-drug resistant A. schubertii treated by the defense protein C-13326 was fixed, scanning electron microscopy was used to further observe the antibacterial effect, and the results showed that the cell membrane of the multi-drug resistant A. schubertii treated by the defense protein C-13326 was damaged (see Figure 5 ), indicating that the defense protein C-13326 kills the multi-drug resistant A. schubertii by damaging the cell membrane.

[0035] Genetic sequence of black soldier fly defense protein C-13326 SEQ ID NO. 1:

[0036] ATGAAGCTTACTTTGGTACTTGTCGTCTTTGCTGTTATCTGTTCAATGGCTTTAGCAAGACCTGAAAACTTGG

[0037] AGAACGTAGAAGATTCTGGAGTTGTGGAGCTGGTTCGCCATAAGCGTCTAAGTTGTCTCTTCGAGAACCAA

[0038] GCTGTCTCAGCAATAGCATGCGGAGCCAGCTGTATTACAAGGAAAGGAAAACGAGGTGGATGGTGTTCTAA

[0039] TGGAGTATGCAGATGTACACCTAATTAA

[0040] Amino acid sequence of Musca domestica defensin C-13326 SEQ ID NO. 2:

[0041] MKLTLVLVVFAVICSMALARPENLENVEDSGVVELVRHKRLSCLFENQAVSAIACGASCITRKGKRGGWCSNGV

[0042] CRCTPN

Claims

1. A Hermetia illucens defensin C-13326, characterized in that, The nucleotide sequence of the gene encoding the black soldier fly defensin C-13326 is shown as SEQ ID NO. 1, and the amino acid sequence of the black soldier fly defensin C-13326 is shown as SEQ ID NO.

2.

2. A method of expressing Hermetia illucens defensin C-13326 according to claim 1, characterized in that, The method is to first construct a Pichia pastoris secretion vector and a recombinant plasmid pPIC9K-C-13326 encoding the gene of the black soldier fly defensin C-13326 according to claim 1, and then transform it into Pichia pastoris strain GS115 for expression.

3. The use of the black soldier fly defensin C-13326 according to claim 1 in the preparation of a drug for inhibiting multi-drug resistant S. subputata.

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