CN112812165B

CN112812165B - Hermetia illucens antibacterial peptide Hidefensein 1 and application thereof

Info

Publication number: CN112812165B
Application number: CN202110190190.8A
Authority: CN
Inventors: 张吉斌; 张晶晶; 李嘉晖; 张弘源; 蔡珉敏; 郑龙玉; 喻子牛
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-02-18
Filing date: 2021-02-18
Publication date: 2022-03-08
Anticipated expiration: 2041-02-18
Also published as: CN112812165A

Abstract

The invention relates to the technical field of genetic bioengineering, in particular to a soldier fly antimicrobial peptide Hidefensein 1 and application thereof, wherein the protein Hidefensein 1 of the antimicrobial peptide is shown in SEQ ID NO.3, the antimicrobial peptide has small molecular weight and broad-spectrum antimicrobial activity: can effectively inhibit gram-positive bacteria staphylococcus aureus and gram-negative bacteria escherichia coli, and has good application prospect in the aspects of preparing antibacterial agents, functional feed additives and the like.

Description

Hermetia illucens antibacterial peptide Hidefensein 1 and application thereof

Technical Field

The invention relates to the technical field of genetic bioengineering, and particularly relates to a soldier fly antimicrobial peptide Hidefensein 1 and application thereof.

Background

Insect antimicrobial peptides are a class of antimicrobial active substances produced by autoimmunity when insects are accidentally injured or infected with pathogenic bacteria, and are mainly produced by fat bodies which are synthesized and released into haemolymph (lyng2014, Rocha et al 2016). The antibacterial peptide has many potential advantages, such as small molecular weight (30-60 amino acids), broad-spectrum antibacterial action, good thermal stability, no drug resistance and the like (Wang G et al 2016). Therefore, the compound can be used for developing a novel high-efficiency low-toxicity peptide (Lakshmiaah et al 2015) and used as a substitute drug of antibiotic drugs or combined with antibiotics to achieve the bacteriostatic effect.

In recent years, drug-resistant bacteria caused by abuse of drugs such as antibiotics due to various factors have become a problem in the world public health field, and the use of drugs such as antibiotics (Yi Kunlun et al 2015) is strictly controlled in more and more countries and regions, and research on novel safe and stable antibacterial substances has become a hot spot.

Insect defensins were originally isolated from the flies Sarcophaga and the blowfly Phormiaterranaae by Matuyama and Natori and Lambert et al, respectively (Lambert J et al 1989). With the progress of research, experts and scholars at home and abroad have found more than 30 defensins in the class Insecta, which includes Diptera, Coleoptera, Hymenoptera, Termoptera, Hemiptera and Aeschna, etc., isolated in vivo to obtain antibacterial peptides. The antibacterial peptides have various good characteristics, such as good water solubility, good heat stability, broad-spectrum antibacterial activity, capability of inhibiting various bacteria, fungi, plasmodium, viruses and tumor cells, no (low) toxic or side effect on normal somatic cells and the like.

Hermetia illucens (hermetiillucens L., diptera) is exposed to the environment where various pathogenic bacteria grow, such as livestock and poultry manure, kitchen waste and other waste organic matters, so that various novel antibacterial peptide genes may exist. Chemical synthesis and genetic engineering techniques are still the main methods for producing antibacterial peptides at present. By analyzing the Hermetia illucens genome, novel antibacterial peptide genes can be screened out and antibacterial peptide can be prepared.

Therefore, the research on the discovery of new antibacterial peptide from the hermetia illucens is of great significance to a plurality of fields such as medicine, food preservative, feed additive, biological control of animal and plant diseases and the like. Provides a molecular basis for the research of the function and action mechanism of the Hermetia illucens antibacterial peptide, and provides a theoretical basis for the application of the antibacterial peptide in biological prevention and treatment and medicine.

Disclosure of Invention

The invention aims to provide an antibacterial peptide derived from hermetia illucens, and the sequence of the antibacterial peptide is shown as SEQ ID No. 3.

The invention also aims to provide the bacteriostatic action of the antibacterial peptide.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant obtains a brand new antibacterial peptide Hidefensen 1 by analyzing the genome of the Hermetia illucens strain, wherein the sequence of the antibacterial peptide is shown in SEQ ID NO. 3; the antimicrobial peptides can be prepared using protocols conventional in the art, such as synthesis, prokaryotic or eukaryotic expression; the propeptide of the antibacterial peptide is shown in SEQ ID NO.2 and comprises a signal peptide.

The gene which codes the protein shown in SEQ ID NO.3 or the protein shown in SEQ ID NO.2 also belongs to the protection scope of the invention.

The application of the antibacterial peptide Hidefensen 1 in preparing a bacterial inhibitor, wherein the bacteria comprise staphylococcus aureus or escherichia coli.

Compared with the prior art, the invention has the following advantages:

1. the novel Hidefensen 1 antibacterial peptide is obtained by analyzing the genome of the soldier fly of the Wuhan Liangbang Libang.

2. The Hidefensen 1 has broad-spectrum antibacterial activity and good application prospect in the aspects of medicine, disease control, feed additives and the like.

3. The preparation method of the novel antibacterial peptide Hidefensen 1 is simple, convenient, rapid and low in cost.

Drawings

FIG. 1 is a schematic diagram of PCR amplification electrophoresis of an antibacterial peptide gene Hidefensein 1;

lane M: DL2000 DNA Marker; 1: band amplified by antibacterial peptide gene Hidefensen 1.

FIG. 2 is a schematic diagram of the construction of pET32 a-Hidefensen 1;

DEF1 is antibacterial peptide gene Hidefensen 1.

FIG. 3 is a schematic diagram of the induced expression and protein purification of recombinant E.coli BL21(DE3) -pET32 a-Hidefensein 1;

in FIG. 3, A is the expressed recombinant protein analyzed by 15% SDS-PAGE: lanes 1-2 are empty vector induced pre and post expression samples, respectively; lanes 3-4 are recombinant protein-induced pre-and post-expression samples, respectively; m1 is the Protein marker-Broad range Protein Ladder;

protein purification results from 15% SDS-PAGE analysis in FIG. 3B: lanes 1-3 are the 100mM, 200mM, 500mM imidazole solution purification elution phases, respectively; lane 4 is 0.05mg/mL standard Bovine Serum Albumin (BSA); m2 is the Protein Marker-Unstanated Protein Molecular Weight Marker.

FIG. 4 is a schematic diagram of a bacteriostasis experiment of TRX-Hidefensein 1 fusion protein;

FIG. 4A shows the result of TRX-Hidefensein 1 inhibiting E.coli, 1: purified TRX-Hidefensin1 fusion protein, 2: purified tag protein TRX (negative control), 3: 50 μ g/mL of the antibiotic ampicillin (positive control);

in FIG. 4, B is the result of TRX-Hidefensein 1 inhibiting Staphylococcus aureus, 1: purified TRX-Hidefensin1 fusion protein, 2: purified tag protein TRX (negative control), 3: 50 μ g/mL of the antibiotic ampicillin (positive control).

Detailed Description

The invention will be further elucidated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not specified in the following examples, are generally carried out according to conventional experimental conditions, in a molecular cloning laboratory Manual (third edition) [ J. SammBruk et al, 2003, Beijing: scientific press, or according to the conditions recommended by the manufacturer.

In the embodiment of the invention, the antibacterial peptide Hidefensen 1 is prepared by prokaryotic expression of Escherichia coli, and those skilled in the art can also prepare the antibacterial peptide containing the amino acid shown in SEQ ID No.3 by other conventional methods in the art, such as artificial synthesis, and other prokaryotic expression or eukaryotic expression methods;

example 1:

expression of antibacterial peptide Hidefensin 1:

amplification of antibacterial peptide gene Hidefensein 1

Primers required in this example

A Hidefensen 1 gene sequence synthesized by a company (shown in SEQ ID NO.1, for the expression of matched escherichia coli, and the sequence shown in SEQ ID NO.1 is a sequence subjected to codon optimization according to an expression mode of escherichia coli) is used as a template, PCR is carried out by using a PCR primer, and an amplification system and conditions are as follows:

the PCR amplification system is as follows:

the PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles; extending for 7-10min at 72 ℃. Taking 3-5 μ L of the reacted product, and verifying the product by 1% agarose gel electrophoresis under 135V electrophoresis conditions for 40 min. As a result, a band of about 250bp was obtained (FIG. 1), and the target product contained the sequence shown in SEQ ID NO.1 and the translated protein was shown in SEQ ID NO. 2.

(3) Recovery and purification of PCR products

After PCR amplification, 1% agarose electrophoresis is used for recovering gel, the gel is cut under an ultraviolet lamp, and DNA gel recovery is carried out by using a gel recovery kit (Beijing Solambio company), and the detailed steps are described in the specification of the gel recovery kit. Meanwhile, the recovery condition is detected by 1 percent agarose gel electrophoresis.

Secondly, construction of recombinant plasmid pET32 a-Hidefensein 1

(1) Double digestion of target fragment and plasmid pET32a

The target gene Hidefensen 1 and the expression vector pET32a are subjected to double enzyme digestion reaction by using endonucleases XhoI and BamHI respectively, and the reaction system and conditions are as follows:

PCR product double digestion system (30. mu.L):

plasmid pET32a double restriction system (20. mu.L):

preparing 50 mu L of each reaction system according to the proportion of the reagents, uniformly mixing, centrifuging for a short time, placing the reaction system at 37 ℃, reacting for 30min to ensure complete enzyme digestion, and heating at 80 ℃ for 5min to inactivate the endonuclease so as to avoid influencing the subsequent ligation reaction. The double enzyme digestion product is verified by 1% agarose gel, and then the enzyme digestion product is purified by a gel recovery method. The concentration and OD of the product were measured by using NanoDrop2000_260/280. Storing at-20 deg.C for use.

(2) Ligation and transformation of the fragment of interest

Connecting the target gene Hidefensen 1 and the plasmid pET32a after double enzyme digestion by using T4 DNA ligase, reacting in a 0.2mL EP tube, preparing a 10 mu L reaction system, uniformly mixing, centrifuging for a short time, placing the reaction system at 22 ℃, connecting for 1.5h, then terminating the reaction at 65 ℃ for 10min, and thermally inactivating the T4 DNA ligase.

The system of the ligation reaction is:

(3) coli E.coli DH5 alpha competent preparation and transformation

A. Dipping a small amount of bacterial liquid from a DH5 alpha glycerin tube preserved at the temperature of-20 ℃, streaking on an LB solid culture medium, culturing for 12 hours at the temperature of 37 ℃, selecting a single colony to be cultured in an LB liquid culture medium containing 5mL at the temperature of 37 ℃ at 180r/min, and shaking the culture platform overnight;

B. inoculating 5mL of the cultured strain into a triangular flask containing 50mL of LB culture medium, culturing at 37 ℃ at 200r/min until OD600 is between 0.3 and 0.4, and taking about 1.5 to 2.5 hours;

C. placing the culture solution on ice for 10min, and cooling;

D. operating according to the aseptic requirement, subpackaging the culture solution by a sterilized 50mL centrifuge tube, and balancing. Then centrifuging for 3min at 4 ℃ and 5000 r/min;

E. pouring off the supernatant, collecting thallus, inverting 50mL centrifuge tube for a moment to facilitate the culture solution in the tube to flow out and be sucked dry, and pouring out clean culture solution to make competent cells or not (the components in the culture solution can weaken CaCl)₂Permeability to membranes, but prevents the bacterial cells from flowing out due to long-term storage);

F. each 50mL of the cells of the initial culture medium was incubated with 0.1mol/L CaCl in 10mL of ice bath₂Placing in a direct ice bath, resuspending thallus precipitate (sucking and blowing thallus at the bottom uniformly by using a gun head), then placing on ice for 10min, wherein the placing is not suitable for overlong and overlong, and the placing is ensured for 10 min;

G. centrifuging at 4 deg.C and 5000r/min for 3min to collect thallus, and placing the centrifuged centrifuge tube on ice;

H. resuspending the thalli by using 0.1mol/L CaCl2 in 10mL ice bath every 50mL of initial culture solution for the second time, and then placing on ice for 30min, wherein the placing is not suitable for overlong and overlong, and the placing is ensured for 30 min;

I. finally, the thalli is collected by centrifugation at 5000r/min for 3min at 4 ℃.

J. Adding 2mL of ice-bath CaCl for the third time₂Solution (2 mL CaCl added according to 50mL culture solution)₂Solution, 100mL of culture medium with 4mL of CaCl₂Sucking and blowing the bottom thallus with a gun head, packaging with 1.5mL sterile EB tube, and storing at-80 deg.C with 100 μ L (containing 10% glycerol) tube to obtain competent cells of Escherichia coli, wherein glycerol may not be added if directly used for transformation on the same day;

K. thawing prepared 100 μ L DH5 α competent cells on ice, adding 10 μ L ligation product, adding 10 μ L sterilized water as control, mixing gently, and ice-cooling for 30 min;

heating at L.42 deg.C for 90s, and rapidly ice-cooling for 3 min;

adding 500-800 μ L LB liquid culture medium, shaking-culturing at 37 deg.C and 120r/min, and recovering for 1 h;

centrifuging at N.5000r/min for 5min, and collecting thallus. After aspirating about 500. mu.L of LB liquid medium, the remaining cells were resuspended, plated on LB-containing plates (containing 1 ‰ Amp), and cultured overnight at 37 ℃. Control 100 u L coated LB plate (containing 1 ‰ Amp), another suction 100 u L coated LB plate, at 37 degrees C overnight culture.

(4) Extraction and characterization of recombinant plasmids

Transformed DH5 alpha was plated on LB plates containing antibiotic Amp for 12-16h, after which 10 positive colonies were picked in PA bottles containing 5mL of liquid LB medium (1 ‰ Amp) and cultured overnight at 37 ℃ at 180 r/min. Collecting thallus, extracting plasmid with plasmid extraction kit, detecting with 1% agarose gel electrophoresis, and measuring its concentration and OD with NanoDrop2000_260/280. Then, PCR detection and identification are carried out, wherein the PCR system is the same as the embodiment 2 except that the plasmid is provided with a primer S · tag and a primer T7T, and the product is sent to the department of Onychidae to detect whether the target gene exists on the plasmid or not by sequencing. The construction of the recombinant plasmid can be seen in FIG. 2.

Thirdly, construction and induced expression of genetic engineering strains

(1) Coli BL21(DE3) competent preparation and transformation

Coli BL21(DE3) was made competent (or purchased) as DH5 α. Likewise using CaCl₂The transformation method is carried out by conventional transformation.

(2) Screening of E.coli BL21(DE3) Positive clones

From the plates with more colonies, 5 single colonies were picked up and cultured overnight at 37 ℃ at 180 rpm in 5mL LB liquid medium (5. mu.L Amp) PA bottles. And (3) carrying out target gene PCR by using the recombinant plasmid as a template, wherein the PCR system is the same as the step I, but the primers are replaced by plasmid primers S · tag and T7T. The PCR product was verified by 0.8% agarose gel electrophoresis at 135V for 40 min. And (3) verifying whether the recombinant plasmid containing the target gene is successfully transferred into the host bacterium, wherein the target fragment amplified after identification is a positive clone.

(3) Induced expression of target protein and SDS-PAGE identification

After resistance screening and PCR identification, the bacterial liquid corresponding to the positive clone in (2) is induced and expressed by 0.4mM IPTG inducer, BL21(DE3) -pET32a for transforming the empty vector is used as a control, and the specific operation steps are as follows (note that inoculation, sample adding, sampling, suspension treatment, thallus collection and the like in the operation are all operated under aseptic conditions):

A. picking a single colony of the transformed strain to 5mL of LB liquid culture medium (containing 1 thousandth Amp), and culturing at 37 ℃ overnight at 180 r/min;

B. inoculating 0.5mL of overnight culture into 50mL of LB liquid culture medium, placing in a 250mL conical flask, and culturing at 37 ℃ overnight at 180 r/min;

C. inducing with 0.4mmol/L IPTG, respectively taking 1mL bacterial liquid before and after induction, centrifuging at 12000 Xg for 2min, separating thallus and fermentation supernatant. Storing at-20 deg.C;

D. the cells were resuspended in 100. mu.L of sterile water and left at room temperature for 5 min. Centrifuging at 12000 Xg for 3min, resuspending the thallus with 5 XSDS Loading Buffer, and boiling water bath for 5-10 min;

E. all samples were centrifuged at 3000 Xg for 3min after boiling. The denatured sample was subjected to SDS-PAGE electrophoresis.

F. After the electrophoresis was completed, the gel was detached, stained in a coomassie brilliant blue staining solution (1g of coomassie brilliant blue R-250, ethanol: acetic acid: water: 5:1:4) for 1 hour, and then, destained with a destaining solution (ethanol: acetic acid: water: 4:1:5) and the solution was changed every 30 minutes until the background was completely removed. By observing the gel, it can be seen that: the target gene is induced and expressed in Escherichia coli BL21(DE3), and the size is about 28kDa (figure 3).

(4) Separation, purification and concentration of target protein

In order to obtain a large amount of target protein, it is necessary to express a large amount of positive clones, then collect the cells by centrifugation, crush the cells under high pressure to release intracellular target protein, then take the crushed cells and purify the supernatant to obtain protein, and since the vector pET32a contains a 6 XHis tag, the protein purification is performed by using a nickel column. The purified fractions were concentrated by freeze-drying and the purification results were checked by SDS-PAGE. By observing the gel, it can be seen that: the target protein has pure components under the elution of 200mM imidazole solution and has the size of about 28kDa (figure 3), the target protein comprises amino acid shown as SEQ ID NO.3, and the target protein is named as TRX-Hidefensen 1; the tag protein TRX is thioredoxin.

Example 2:

determination of TRX-Hidefensein 1 in vitro antibacterial activity

The strains to be detected are as follows: staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), and Salmonella enterica (Salmonella enterica).

The antibacterial activity of the strain is detected by adopting a perforated plate confronting culture method. Inoculating the strain to be detected which grows to the logarithmic phase to an LB culture medium according to the inoculation amount of 1%, and mixing and pouring the strain to be detected into a flat plate. Then, the culture medium was perforated symmetrically and uniformly with an Oxford cup (size: inner diameter-outer diameter-height: 6mm-8mm-10 mm). The expressed TRX-Hidefensen 1 protein was pipetted precisely at 200. mu.L (15. mu.g/mL) and added to an Oxford cup of medium in triplicate for each indicator. The pathogenic bacteria were cultured at 37 ℃ for 12h and the inhibitory effect was observed.

The bacteriostatic effects are shown in the following table and fig. 4:

diameter of zone of inhibition

Note: because the external diameter of the oxford cup is 8.00mm, the minimum detection limit of the diameter of the inhibition zone is 8.00 mm.

As can be seen from FIG. 4, the expression sample TRX-Hidefensen 1 has inhibitory effects on both gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli. Has no bacteriostatic effect on salmonella enteritidis, namely, pictures are not displayed.

Sequence listing

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atgcgttttc tcctcttcgc tacacttctt gccgctgttt tcttgttcgt gtatgcggat 60

cccatcctgc gtcttcccga aaatggagag gatttaggag acatcccagt acatcatcgt 120

gccaggcgtg ccacctgtga cctgttgagt gctacaaaag tcaaaagtac cgcctgcgct 180

gctcattgcc tcttgaaggg acacaaggga ggttattgca attccaaact tgtctgtgtc 240

tgccga 246

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Gly Asp Ile Pro Val His His Arg Ala Arg Arg Ala Thr Cys Asp Leu

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attagcatag aaggggtaac actcg 25

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tgaatacatc gcaagttcgt agtct 25

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Claims

1. An isolated protein shown as SEQ ID NO. 3.

2. A gene encoding the protein of claim 1.

3. The propeptide of the protein of claim 1, wherein the propeptide has a sequence shown in SEQ ID No. 2.

4. A gene encoding the propeptide of claim 3.

5. The gene of claim 4, which is represented by SEQ ID NO. 1.

6. Use of the protein of claim 1 in the preparation of a bacterial inhibitor of staphylococcus aureus (m:)Staphylococcus aureus) Or Escherichia coli (Escherichia coli）。

7. The use of the gene of claim 2 in the preparation of an antimicrobial peptide, wherein the antimicrobial peptide is Staphylococcus aureus or Escherichia coli.