CN113214409B - Melittin-mortiferin hybrid peptide mutant MTM and application thereof - Google Patents

Abstract

The invention discloses a melittin-mortiferin hybrid peptide mutant MTM and application thereof, belonging to the technical field of genetic engineering. The invention discloses application of melittin-mortin hybrid peptide mutant MTM in inhibiting activities of escherichia coli, salmonella and staphylococcus aureus. The invention firstly utilizes the SamyX-6His-EDDIE-MTM fusion fragment modified by the bacillus subtilis gene to construct an efficient recombinant bacillus subtilis BSMW expression system for biosynthesis of MTM. MTM was obtained up to 254.38mg from 1L fermentation supernatant. The size of the purified MTM was consistent with the expected molecular weight of 3.2 kDa. MTM has antibacterial activity against Escherichia coli, Salmonella, and Staphylococcus aureus.

Description

Melittin-mortiferin hybrid peptide mutant MTM and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to melittin-thanatin hybrid peptide mutant MTM and application thereof.

Background

The problem of drug resistance due to antibiotic abuse is becoming more serious, and the search for new types of alternative drugs is the key to solving the problem of drug resistance. The antibacterial peptide is expected to be an ideal substitute of antibiotics due to unique advantages, but the source problem of the antibacterial peptide is always the bottleneck of development and application of the antibacterial peptide. The rapid development of genetic engineering technology has promoted the research and development of antibacterial peptides to a certain extent. However, compared with the conventional antibiotics, the antibacterial peptide has a short plate in the aspects of inhibitory activity, stability, cytotoxicity and the like, researchers need to further explore the antibacterial mechanism and structure-activity relationship of the antibacterial peptide, and structurally modify the existing antibacterial peptide or design a new antibacterial peptide molecule, so that the actual use value of the antibacterial peptide is improved to the maximum extent on the premise of ensuring the source of the antibacterial peptide.

Generally, there are three routes of obtaining antibacterial peptides: extracting from an organism; obtained by a chemical synthesis method; the target antibacterial peptide is obtained by adopting a gene engineering expression technology. However, the natural antibacterial peptide is an active substance generated by a biological organism under the induction of external factors, and has low content in the organism, so that the efficiency of directly extracting from the organism is low, the extraction difficulty is high, the steps are complicated, the standardized production is difficult, the cost is high, the purity of the extract is difficult to ensure, the large-scale production is difficult to realize, and the method is not practical for some higher animals and plants and human beings. With the continuous improvement of polypeptide synthesis technology, the artificial synthesis of peptide substances with biological activity is realized and rapidly developed. Generally, the natural antibacterial peptide has small relative molecular mass, so the natural antibacterial peptide with biological activity can be obtained by adopting a chemical synthesis method, but the chemical synthesis has strict technical requirements, long synthesis time, high price and high cost, and is difficult to realize large-scale production. The rapid development of modern genetic engineering technology provides an effective way for large-scale production of the antibacterial peptide, makes up the defects of the two acquisition methods, and is an economic and effective method for acquiring the antibacterial peptide.

Melittin, also called melittin, is the main active ingredient of bee venom, accounting for 50% of the dry weight of bee venom, and is the substance playing a major role in bee venom. Melittin has been reported to have antibacterial, antiviral, anti-inflammatory, and radioprotective effects, and recently has been studied on its anticancer effects. Melittin has strong inhibitory action on gram-positive bacteria and gram-negative bacteria, but it also has strong hemolytic activity, limiting its clinical application. Melittin has the property of destroying cell membranes and killing cells, so that to obtain active melittin from prokaryotic and eukaryotic expression systems, it is required to express it as a fusion protein.

The Thanatin (Thanatin) is an insect antimicrobial peptide having a broad spectrum of antimicrobial activity isolated from hemiptera insect lygus bug (podius maculiventris) hemolymph, and is also an antimicrobial peptide having inhibitory activity against gram-positive bacteria, gram-negative bacteria and fungi at the first physiological concentration. The peptides have bactericidal and fungicidal action, with one of the greatest antibacterial spectrum of insect defence peptides observed to date.

Therefore, the problem to be solved by those skilled in the art is to provide a melittin-mortiferin hybrid peptide mutant MTM and the application thereof.

Disclosure of Invention

In view of the above, the invention provides melittin-mortiferin hybrid peptide mutant MTM and application thereof.

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

a melittin-mortiferin hybrid peptide mutant MTM has the following amino acid sequence: GIWAVLKVLTTWKPVPIIYCNRRTWKCQRM, respectively; SEQ ID NO. 4.

Furthermore, the melittin-mortiferin hybrid peptide mutant MTM is applied to inhibiting the activities of escherichia coli, salmonella and staphylococcus aureus.

Further, the construction method of the recombinant bacillus subtilis BSMW containing the melittin-mortin hybrid peptide mutant MTM comprises the following specific steps:

(1) the SamyX-6His-EDDIE-MTM is connected with a pDM030 carrier through enzyme digestion to obtain a recombinant plasmid pDM 045; the nucleotide sequence of the SamyX-6His-EDDIE-MTM is shown in SEQ ID NO. 5;

(2) transforming the recombinant plasmid pDM045 into escherichia coli DH5 alpha, screening positive clones by using chloramphenicol, and verifying a transformant by sequencing;

(3) and (3) converting the positive clone verified to be correct in the step (2) into the bacillus subtilis WB700 by an electroporation method, and constructing to obtain the recombinant bacillus subtilis BSMW.

According to the technical scheme, compared with the prior art, the melittin-mortin hybrid peptide mutant MTM and the application thereof are disclosed, and a high-efficiency recombinant bacillus subtilis BSMW expression system is constructed by utilizing a bacillus subtilis gene modified SamyX-6His-EDDIE-MTM fusion fragment for the first time and is used for biosynthesis of MTM. MTM was obtained up to 254.38mg from 1L fermentation supernatant. The size of the purified MTM was consistent with the expected molecular weight of 3.2 kDa. MTM has antibacterial activity against Escherichia coli, Salmonella, and Staphylococcus aureus. Has potential application value in the fields of feed additives, food, cosmetics, preservation and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the secondary structure of the MTM antimicrobial peptide of the present invention;

FIG. 2 is a schematic diagram of the construction of a pDM045 plasmid according to the invention;

FIG. 3 is the bacterial load of the recombinant Bacillus subtilis BSMW strain induced by IPTG according to the invention;

FIG. 4 is a diagram showing the transcription amount of the SamyX-Eddie-MTM fusion gene of the present invention;

FIG. 5 is a graph showing the cumulative yield of MTM in accordance with the present invention;

FIG. 6 is a SDS-PAGE and Westernblot analysis of MTM in the recombinant BSMW strain fermentation broth of the present invention;

wherein A is Tricine-SDS-PAGE analysis of MTM in recombinant BSMW strain fermentation liquor; b is Westernblot analysis of MTM in recombinant BSMW strain fermentation liquor; in a and B, lane 1: protein Marker, # P7702, New englangdbiolabs, USA; lane 2: after the recombinant bacillus subtilis BSMW culture solution is induced by IPTG, continuously culturing for 8h to obtain a fermentation liquor treatment sample; lane 3, the fermentation broth treatment sample of the Bacillus subtilis WB700 culture solution after being treated with IPTG and continuously cultured for 8 h;

FIG. 7 is an electrospray ionization mass spectrometry analysis of the recombinant MTM of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 design, physicochemical Properties and Structure prediction of mutant MTM of hybrid peptide

(1) Design of hybrid peptide mutant MTM

The original polypeptide sequences of melittin and mortiferin were obtained from an antimicrobial peptide database (http:// APs. unmac. edu/AP/main. php). Melittin consists of 26 amino acids, and has a primary structure of GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO. 1. The death hormone consists of 21 amino acids, and the primary structure is GSKKPVPIIYCNRRTGKCQRM; SEQ ID NO. 2. The organism company synthesized the nucleotide sequence based on the sequence GIGAVLKVLTTGKPVPIIYCNRRTGKCQRM (SEQ ID NO.3) of the hybrid peptide (M-T), inserted into the pDM030 vector, and transformed the Bacillus subtilis WB700 strain to produce the hybrid peptide (M-T). Then, a melittin-mortin hybrid peptide mutant (MTM) is obtained through multiple experiments by using an antibacterial peptide molecule design method, wherein the hybrid peptide mutant comprises 30 amino acids, 3 sites (bold and underline) are subjected to site-directed mutation, and the amino acid sequence of the hybrid peptide mutant is as follows:

(2) physical and chemical property and structure prediction of MTM

By using an on-line tool (polypeptide parameter analysis: https:// web. expasy. org/protparam /), the secondary structure, molecular weight and isoelectric point of the original peptide as well as the designed hybrid peptide can be predicted. Secondary structure prediction is based on GOR (http:// npsa-Pbil. ibcp. fr/cgi-bin/npsa _ Automat. plpage ═ npsa _ gor4.html) and HNN method (https:// prabi. ibcp. fr/htm/site/web/home). The secondary structure of MTM is shown in FIG. 1.

The isoelectric points and molecular weights of the mutant MTMs of the hybrid peptides are shown in Table 1.

TABLE 1 physical and chemical Properties of MTM

Example 2 construction of recombinant strains

The pDM030 vector is a shuttle vector which is derived from pGJ222 plasmid (construction of a bacillus subtilis high-efficiency expression system; Pitaifei, huxiong and the like; journal of northwest university of agriculture and forestry, vol.39, No. 11) and can be replicated in escherichia coli and bacillus subtilis by replacing a Pglv promoter of the PGJ222 plasmid with a Pgrac promoter, and contains a promoter Pgrac, a chloramphenicol and spectinomycin resistance gene and a beta-galactosidase gene. The vector was digested with EcoR I and SacI. A702 bp fusion fragment of SamyX-6His-EDDIE-MTM containing SamyX signal peptide, EcoR I and Sac I sites is synthesized by Beijing Okko Biotechnology Limited, and the nucleotide sequence of the fusion fragment is as follows:

gaattcatggtttcaattagaagatcatttgaagcatatgttgatgatatgaatattattacagttcttattccggcacatcatcatcatcatcatatggaacttaatcattttgaacttctttataaaacaaataaacaaaaaccggttggcgttgaagaaccggtttatgatacaacaggcagaccgctttttggcgatccgtcagaagttcatccgcaatcaacacttaaacttccgcatgatagaggcgaagatgatattgaaacaacacttagagatcttccgagaaaaggcgattgcagatcaggcaatcatcttggcccggtttcaggcatttatattaaaccggggccggtttattatcaagattatatggggccggtttatcatagagcaccgcttgaattttttgatgaaacacaatttgaagaaacaacaaaaagaattggcagagttacaggctcagatggcaaactttatcatatttatgttgaagttgatggcgaaattcttcttaaacaagcaaaaagaggcacaccgagaacacttaaatggacaagaaatacaacaaattgcccgctttgggttacatcatgcggcatttgggcagttcttaaagttcttacaacatggaaaccggttccgattatttattgcaatagaagaacatggaaatgccaaagaatgtaataagagctc；SEQ ID NO.5。

the recombinant plasmid pDM045 was obtained by substituting the β -galactosidase gene in the pDM030 vector and cloning it into the pDM030 vector. Recombinant plasmid pDM045 is an inducible expression plasmid containing an operon comprising the IPTG-inducible promoter of Pgrac and a fusion fragment encoding SamyX-6 His-EDDIE-MTM. Wherein the amino acid sequence of the SamyX-6His-EDDIE-MTM is as follows:

MVSIRRSFEAYVDDMNIITVLIPAHHHHHHMELNHFELLYKTNKQKPVGVEEPVYDTTGRPLFGDPSEVHPQSTLKLPHDRGEDDIETTLRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYMGPVYHRAPLEFFDETQFEETTKRIGRVTGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWTRNTTNCPLWVTSCGIWAVLKVLTTWKPVPIIYCNRRTWKCQRM；SEQ ID NO.6。

the construction of the recombinant plasmid pDM045 is shown in FIG. 2.

The recombinant plasmid pDM045 was transformed into E.coli DH 5. alpha. and positive clones were selected with chloramphenicol at a concentration of 5. mu.g/ml. The transformants were verified by sequencing by Beijing Okkomy Biotechnology Ltd, and the verified pDM045 was transformed into Bacillus subtilis WB700 strain by electroporation, thereby constructing recombinant Bacillus subtilis BSMW.

Example 3 biosynthesis of MTM

Screening out a recombinant bacillus subtilis BSMW positive clone from LB agar containing 50 mu g/ml spectinomycin; the positive clones were cultured in 250ml shake flasks containing 50ml LB liquid medium at 37 ℃ with a stirring speed of 225 rpm. When the recombinant bacteria reach the late logarithmic growth stage 12h after inoculation, IPTG solution is added into the culture solution to the final concentration of 100 mu M, and then the culture is carried out for 24 h. The bacillus subtilis WB700 strain is used as a parallel control, and the treatment is carried out under the same condition.

(1) Extraction of total RNA and fluorescent quantitative PCR:

cultures were collected every 3h after fermentation was started until 36 h. Bacterial total RNA was extracted using SV Total RNA isolation kit (# Z3100; Promega, USA). The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription system kit (# A3500; Promega, USA). Real-time PCR was then performed using the real-time PCR kit (# DRR 041S; Takara, Japan). The SamyX-6His-EDDIE-MTM gene was amplified with primers AEM-up/AEM-down.

Wherein, the primer sequences of AEM-up/AEM-down are as follows:

AEM-up：5’-CCGGCACATCATCATCATCA-3’；SEQ ID NO.7；

AEM-down：5’-GGAACCGGTTTCCATGTTGTAA-3’；SEQ ID NO.8；

the PCR reaction procedure was as follows: the reaction was carried out at 50 ℃ for 2min, at 95 ℃ for 10min, then at 95 ℃ for 42s, at 49 ℃ for 60s and at 72 ℃ for 30s for 35 cycles. The reaction was performed in an IQ5 real-time PCR detection system (Bio-Rad, USA).

(2) IPTG induction and MTM biosynthesis

Simultaneously, 2 bottles of bacillus subtilis BSMW strains are subjected to shake culture, wherein 1 bottle is subjected to IPTG induction treatment after the beginning of culture for 12 hours, and the other bottle is used as a parallel control and is not subjected to IPTG induction treatment. Both 2 flasks reached late logarithmic growth 12h after the start of the culture. After 18h of initial culture, the amount of recombinant BSMW strain of bacillus subtilis induced with IPTG was significantly reduced compared to BSMW strain of bacillus subtilis not induced with IPTG (fig. 3).

The promoter Pgrac efficiently promoted transcription of the fusion gene encoding SamyX-Eddie-MTM after IPTG induction in the late logarithmic growth phase (fig. 4). The expression of MTM can be detected after IPTG induction, and the maximum expression level of 254.38mg MTM can be detected in 1L fermentation medium after the recombinant strain grows for 20 h; however, the recombinant bacteria began to decline 8h after IPTG induction and declined to about half of the maximum value after 36h of culture (fig. 5).

Example 4Western blot

And (2) centrifuging fermentation liquor which is obtained by inducing the bacillus subtilis BSMW culture solution by IPTG and then continuously culturing for 8 hours for 10 minutes, mixing the supernatant with 4 multiplied Laemmli loading buffer solution 3: 1 mixing, and heating in boiling water for 5 min. The treated samples were subjected to Tris Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) on a 10% gel, and then transferred to a PVDF membrane for Western blot analysis. Mouse anti-Eddie monoclonal antibody (MAb) and mouse anti-MTM monoclonal antibody (MAb) were prepared and purified by CwBiotech, Beijing, China. After incubation with the appropriate HRP-conjugated secondary antibodies, signals were detected using a ChemiDoc XRS imaging system and QuantityOne analysis software (Bio-Rad, usa).

Analysis of the total extracellular protein of the recombinant Bacillus subtilis BSMW strain by Tricine-SDS-PAGE showed that the molecular weight of MTM was around 3.2kDa (FIG. 6A). Western blot analysis confirmed the results of Tricine-SDS-PAGE analysis, demonstrating the presence of MTM in the culture supernatants (FIG. 6B).

EXAMPLE 5 purification of MTM

The fermentation broth after 8 hours of continued culture of the Bacillus subtilis BSMW broth after induction with IPTG was centrifuged at 7000 Xg for 10min and the supernatant was filtered through a 0.22- μm pore filter (Nucleopore, Costar). The filtered samples were processed with a reverse phase high performance liquid chromatography system (reverse phase high performance liquid chromatography, agilent, usa) using a semi-preparative Zorbax300SB-C8 column (250mm x 9.4mm, 5 μm particle size, 300A pore size) (agilent, usa). The column was equilibrated in 0.1% (v/v) trifluoroacetic acid and 10% acetonitrile and then developed linearly with a gradient of 0% to 60% acetonitrile at a flow rate of 1.0 ml/min. MTM samples were purified and collected using MTM standards (synthesized by Shanghai Chu peptide Biotech Co., Ltd.). The molecular weight of the purified MTM was determined by electrospray ionization mass spectrometry (Agilent, USA). Electrospray ionization mass spectrometry analysis showed that the molecular weight of MTM was 3273.996 (FIG. 7), consistent with 3274.01 of theoretical predictive value, and also consistent with Tricine-SDS-PAGE analysis.

Example 6 antibacterial Activity

Coli ATCC 25922, salmonella ATCC 14028, staphylococcus aureus ATCC 29213 and their corresponding drug-resistant strains (ampicillin-resistant strains, enrofloxacin-resistant strains, methicillin-resistant strains isolated in the laboratory) were obtained from american type culture banks (rockval, maryland, usa) as indicator strains. The antimicrobial activity of the biosynthetic MTM against the indicator strain was investigated using the microdilution method and the results are shown in Table 2. The results in Table 2 show that the biosynthetic MTM shows good bacteriostatic activity on Escherichia coli, salmonella and staphylococcus aureus.

TABLE 2

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims (2)

1. The application of melittin-mortiferin hybrid peptide mutant MTM in preparing an agent for inhibiting activities of escherichia coli, salmonella and staphylococcus aureus is characterized in that the amino acid sequence of the melittin-mortiferin hybrid peptide mutant MTM is as follows: GIWAVLKVLTTWKPVPIIYCNRRTWKCQRM are provided.

2. The construction method of the recombinant bacillus subtilis BSMW for expressing the melittin-mortin hybrid peptide mutant MTM as claimed in claim 1 is characterized by comprising the following specific steps:

(1) the SamyX-6His-EDDIE-MTM is connected with a pDM030 carrier through enzyme digestion to obtain a recombinant plasmid pDM 045; the nucleotide sequence of the SamyX-6His-EDDIE-MTM is shown in SEQ ID NO. 5;

(2) transforming the recombinant plasmid pDM045 into escherichia coli DH5 alpha, screening positive clones by using chloramphenicol, and verifying a transformant by sequencing;

(3) and (3) converting the positive clone verified to be correct in the step (2) into the bacillus subtilis WB700 by an electroporation method, and constructing to obtain the recombinant bacillus subtilis BSMW.

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