CN109232727B

CN109232727B - Green pollution-free antibacterial peptide for library and preparation and application thereof

Info

Publication number: CN109232727B
Application number: CN201811032542.1A
Authority: CN
Inventors: 沈秀丽; 李改英; 赵团; 丁立刚; 杜志强
Original assignee: Inner Mongolia University of Science and Technology
Current assignee: Inner Mongolia University of Science and Technology
Priority date: 2018-09-05
Filing date: 2018-09-05
Publication date: 2020-09-08
Anticipated expiration: 2038-09-05
Also published as: CN109232727A

Abstract

The invention discloses a green pollution-free antibacterial peptide for a library and preparation and application thereof, and belongs to the technical field of biotechnology and antibiosis. The antibacterial peptides Pc-Crustin 4 and Pc-Crustin5 are derived from freshwater crayfish, the amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the preparation method comprises the following steps: extracting total RNA of the freshwater crayfish as a template, and carrying out reverse transcription to obtain cDNA; PCR amplification is carried out by taking cDNA as a template to obtain a nucleotide fragment containing an antibacterial peptide coding sequence, the amplification product and an expression vector are subjected to enzyme digestion, connection and conversion to construct escherichia coli engineering bacteria capable of expressing recombinant antibacterial peptide, and the escherichia coli engineering bacteria are subjected to induction culture to express the recombinant antibacterial peptide. The antibacterial peptide (especially the compound of the antibacterial peptide and the antibacterial peptide) has good antibacterial and degerming activities, and is particularly suitable for mildew prevention and degerming of libraries.

Description

Green pollution-free antibacterial peptide for library and preparation and application thereof

Technical Field

The invention relates to the technical field of biotechnology and antibiosis, in particular to an antibacterial peptide derived from freshwater crayfish (Procambarus clarkii) and a preparation method and application thereof.

Background

The library is a necessary functional unit for every college and university and mainly provides borrowing service of paper and electronic periodicals for teachers and students. Therefore, it is also a dense space for various books and periodicals. In a public place such as a library, the method has two obvious characteristics, firstly, the mobility of borrowers is very high, so that certain pressure is caused on the aspect of maintaining the environmental sanitation of the library; secondly, the borrowing object of the paper book has uncertainty, and the environment in which the borrowing object is located is not controllable, so that the sanitation and safety of the book have certain hidden dangers in the circulation link of book borrowing. In addition, the library is a relatively closed space, the mobility of air is poor, personnel exchange is very complicated, and the factors are superposed to cause certain influence on the environmental sanitation of the library. Especially, the control of various common pathogenic bacteria should be the key point of environmental sanitation management of libraries. The library has a clean environment, pathogens such as bacteria, fungi and the like are controlled within a safe range, on one hand, the library is beneficial to the health of a borrower who borrows books and periodicals and readers who read the books and periodicals in the library, and on the other hand, the clean library environment is also an important guarantee for collection and protection of the books.

At present, after bacteria and fungi bred in the book borrowing and storing processes are bred in the bookshelf, the ground, the corners and the like of a library, ultraviolet irradiation sterilization is mostly adopted, and the method has two defects, namely, the sterilization time is limited, and the library is required to be closed to use ultraviolet rays; secondly, ozone is generated during ultraviolet sterilization, air in a museum is polluted, and safety risk exists for human health. Therefore, the novel library bactericide which has no pollution to the environment and can efficiently sterilize is developed, and has a good application prospect.

Disclosure of Invention

The primary object of the present invention is to provide a novel antimicrobial peptide derived from freshwater crayfish (Procambarus clarkii). The invention also aims to provide a preparation method of the antibacterial peptide. Still another object of the present invention is to provide the use of the antibacterial peptide.

The purpose of the invention is realized by the following technical scheme:

the new antibacterial peptides derived from freshwater crayfish (Procambarus clarkii) are named as Pc-Crustin 4 and Pc-Crustin5 proteins respectively, the amino acid sequences of the new antibacterial peptides are shown as SEQ ID NO.1 and SEQ ID NO.2, and the new antibacterial peptides are 127 amino acid residues and 167 amino acid residues respectively.

Furthermore, in the freshwater crayfish body, the nucleotide sequences corresponding to the open reading frames of the antibacterial peptide Pc-Crustin 4 and Pc-Crustin5 protein genes are shown as SEQ ID NO.3 and SEQ ID NO.4, and are 384bp and 504bp in length respectively.

The preparation method of the antibacterial peptide comprises the following steps:

(1) and performing reverse transcription PCR by taking the extracted total RNA of the freshwater crayfish as a template to obtain cDNA.

(2) And (2) carrying out PCR amplification by taking the cDNA obtained in the step (1) as a template to obtain a nucleotide fragment containing the antibacterial peptide coding sequence (open reading frame sequence).

(3) And (3) carrying out enzyme digestion, connection and transformation on the amplification product obtained in the step (2) and a prokaryotic expression vector to construct an escherichia coli engineering bacterium capable of expressing the recombinant antibacterial peptide.

(4) And (4) performing induced culture on the escherichia coli engineering bacteria constructed in the step (3) to express the recombinant antibacterial peptide.

Further, when the antibacterial peptide is rPc-Crustin 4, the reverse transcription primer is: 5'-cccggtggaccgctagacgg-3', respectively; the PCR amplification primers are as follows: f1: 5' -N_ncactatggcttctacggt-3′，R1：5′-N_nctagacgggggccttgca-3′。

Further, when the antibacterial peptide is rPc-Crustin5, the reverse transcription primer is: 5'-caaagatcgggcacgatcaacaact-3', respectively; the PCR amplification primers are as follows: f2: 5' -N_ncatggctactacagtcac-3′，R2：5′-N_nctagacggtggctttgca-3′。

In the PCR amplification primers, N_nThe expression vector consists of a protective base and a restriction enzyme cutting site, wherein N represents any base, and N is a natural number and can be correspondingly adjusted according to the expression vector.

The invention has carried on antibacterial peptide rPc-Crustin 4, rPc-Crustin5 and their mixture and common pathogenic bacterium liquid bacteriostasis experiment, the sterilization experiment to the mixed bacterium on the surface of the general library book room bookshelf, the sterilization experiment to the mixed bacterium on the surface of the general library reading desk, the sterilization experiment to the mixed bacterium in the air of the special old book room of the library, the result shows that the antibacterial peptide rPc-Crustin 4, rPc-Crustin5 and their mixture (especially their mixture) of the invention have good bacteriostasis, degerming activity. Therefore, the antimicrobial peptide rPc-Crustin 4 and/or rPc-Crustin5 can be used for preparing bacteriostatic agents or degerming agents.

A bacteriostatic or degerming agent comprises antimicrobial peptide rPc-Crustin 4 and/or rPc-Crustin 5. Further, when the bacteriostatic agent or the degerming agent contains rPc-Crustin 4 and rPc-Crustin5, the mass ratio of the bacteriostatic agent to the degerming agent is 1: 1. The bacteriostatic agent or the degerming agent is applied to mildew prevention and degerming of libraries.

The invention has the advantages and beneficial effects that: the rPc-Crustin 4 and/or rPc-Crustin5 of the invention have good activities of inhibiting bacteria and fungi, and have good application prospects in the aspects of mildew prevention and sterilization in the process of collection of precious and old books in libraries, sterilization of the surfaces of objects in library rooms and sterilization of air in the libraries.

Drawings

FIG. 1 is a graph showing the results of induced expression and affinity purification of recombinant antibacterial peptide rPc-Crustin 4 of crayfish. Lane 1 shows the total protein of E.coli cells before induction, lane 2 shows the total protein of E.coli cells after IPTG induction, lane 3 shows the rPc-Crustin 4 recombinant protein after purification, and lane M shows the standard protein molecular weight.

FIG. 2 is a graph showing the results of induced expression and affinity purification of recombinant antibacterial peptide rPc-Crustin5 of crayfish. Lane 1 shows the total protein of E.coli cells before induction, lane 2 shows the total protein of E.coli cells after IPTG induction, lane 3 shows the rPc-Crustin5 recombinant protein after purification, and lane M shows the standard protein molecular weight.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 preparation of two recombinant antimicrobial peptides rPc-Crustin 4 and rPc-Crustin5 proteins of freshwater crayfish

(1) And (3) extracting the total RNA of the mixed tissue sample of the hepatopancreas, pancreas, gill and intestine of the freshwater crayfish with 3 kinds of equal-mass tissues by using an animal total RNA extraction kit to obtain the total RNA sample of the mixed tissue of the freshwater crayfish.

(2) And (2) taking the total RNA sample of the freshwater crayfish obtained in the step (1) as a template, taking specific primers 5'-cccggtggaccgctagacgg-3' (Pc-crustin 4) and 5'-caaagatcgggcacgatcaacaact-3' (Pc-crustin 5) as rear primers of a reverse transcription synthesis reaction, and performing reverse transcription synthesis on cDNA by using a one-step RT-PCR amplification kit to obtain two cDNA samples of the freshwater crayfish mixed tissue sample.

(3) Front and rear primers Pc-crustin 4F/R and Pc-crustin 5F/R for amplifying coding region expression fragments (mature peptides) of two antibacterial peptide genes Pc-crustin 4 and Pc-crustin5 of the freshwater crayfish are respectively designed:

Pc-crustin 4-F：5′-GCCGAATTCCACTATGGCTTCTACGGT-3′，

Pc-crustin 4-R：5′-GCCCTCGAGCTAGACGGGGGCCTTGCA-3′；

Pc-crustin 5-F：5′-GCCGAATTCCATGGCTACTACAGTCAC-3′，

Pc-crustin 5-R：5′-GCCCTCGAGCTAGACGGTGGCTTTGCA-3′。

respectively taking the cDNA samples of the mixed tissues of the freshwater crayfish in the step (2) as templates to carry out gradient PCR amplification reaction, wherein the program aiming at the Pc-crustin 4 amplification reaction is as follows: pre-denaturation at 98 ℃ for 3min, first amplification gradient: denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 45s, extension at 72 ℃ for 30s, and circulating for 15 circles; second amplification gradient: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 30s, and circulating for 15 circles; third amplification gradient: denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 30s, and circulating for 15 circles; post extension for 5 min. The procedure for the Pc-crustin5 amplification reaction was: pre-denaturation at 98 ℃ for 3min, first amplification gradient: denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 60s, and circulating for 15 circles; second amplification gradient: denaturation at 98 ℃ for 30s, annealing at 59 ℃ for 45s, extension at 72 ℃ for 60s, and circulating for 15 circles; third amplification gradient: denaturation at 98 ℃ for 30s, annealing at 61 ℃ for 45s, extension at 72 ℃ for 60s, and circulating for 15 circles; and then extending for 10 min. PCR amplification reaction products are respectively obtained.

(4) Respectively recovering and purifying PCR amplification reaction products obtained in the step (3), respectively carrying out double digestion treatment on EcoRI endonuclease and Xho I endonuclease, respectively connecting with escherichia coli pET-28-a plasmid vectors subjected to double digestion treatment on the EcoRI endonuclease and the Xho I endonuclease, respectively transforming competent cells of escherichia coli BL21(DE3) expression strains, respectively carrying out colony PCR reaction by using two pairs of primers Pc-crustin 4F/R and Pc-crustin 5F/R to screen positive expression strains of target genes, and respectively sending the screened positive clones to a sequencing company for sequence verification of the two target genes.

(5) And (3) carrying out IPTG concentration gradient composite temperature gradient induced expression on the target gene positive expression strain obtained in the step (4), wherein the method comprises the following steps: respectively inoculating two antibacterial peptide gene (Pc-crustin 4 and Pc-crustin5) positive expression strains of freshwater crayfish into a 1000mL triangular flask containing 200mL liquid LB culture medium, carrying out shake dark transfer culture for 3h at 37 ℃ and 180rpm, then adding IPTG with the final concentration of 0.1mM, continuing shake dark culture for 12h at 28 ℃ and 180rpm, then adjusting the final concentration of IPTG to 0.3mM, carrying out shake dark continuous culture for 12h at 28 ℃ and 180rpm, then adjusting the final concentration of IPTG to 0.5mM, carrying out shake dark continuous culture for 12h at 28 ℃ and 180rpm, and then carrying out centrifugation collection at 6000 rpm.

(6) And (3) respectively carrying out thallus crushing on the two thalli obtained in the step (5) in different low-temperature composite ultrasonic modes, firstly freezing the thalli for 60min at the temperature of minus 20 ℃, then freezing for 60min at the temperature of minus 40 ℃, then crushing bacterial thalli for 60min by using an ultrasonic crusher, then freezing the thalli for 60min at the temperature of minus 40 ℃, then freezing the thalli for 60min at the temperature of minus 20 ℃, and then centrifuging and collecting the crushed precipitates respectively at the speed of 10000 rpm.

(7) And (3) shaking the two crushed precipitates obtained in the step (6) by using 20mL of denatured liquid (0.15M Tris-HCl, 5mM DTT, 6M urea and pH 7.0) respectively under the conditions of 37 ℃ and 180rpm in a shaking way and avoiding light for 2h, then centrifuging and collecting supernate under the condition of 12000rpm respectively, and performing low-temperature dialysis treatment overnight for 12h and at 4 ℃ respectively by using dialysis bags with the retention capacity of 3.5kDa, wherein the formula of the dialysate is 0.15M Tris-HCl, 10mM Cysteins and pH 7.0.

(8) Centrifuging the dialyzed two liquids in the step (7) at 10000rpm respectively, collecting supernate, namely denatured-renaturated liquid of two target recombinant proteins, purifying by using a his-tag gel column pre-filled with 5mL, firstly washing the his-tag gel column for 3 times by using 10mL 1 × PBS buffer solution, then slowly circulating 10mL of the obtained denatured-renaturated liquid of the two target recombinant proteins for 3 times, eluting the column by using 15mL of eluent (500mM imidazole, 20mM Tris-HCl, pH 7.0), collecting eluent, dialyzing at 4 ℃ for 12 hours in 1 × PBS buffer solution by using a dialysis bag with the retention capacity of 3.5kDa, concentrating by using polyethylene glycol 12000 the next day, and respectively leading the final concentration of the two target recombinant proteins rPc-Crustin 4 and rPc-Crustin5 to be 200 mu g.mL^-1。

The induced expression and affinity purification results of the two recombinant freshwater crayfish antibacterial peptides rPc-Crustin 4 and rPc-Crustin5 are shown in figure 1 and figure 2. FIG. 1 shows that the recombinant protein has a significant expression level after IPTG induction compared with the total protein of E.coli before induction; furthermore, the target rPc-Crustin 4 recombinant protein with single component and higher concentration is obtained after affinity purification, and the position of the protein band in the lane 3 is close to the 17kDa band of the standard protein molecular weight, which is consistent with the theoretically expected 16.9kDa, indicating that the expression is correct. FIG. 2 shows that the recombinant protein has a significant expression level after IPTG induction compared with the total protein of E.coli before induction; furthermore, the target rPc-Crustin5 recombinant protein with single component and higher concentration is obtained after affinity purification, and the position of the protein band in lane 3 is between the 17kDa and 25kDa bands of the standard protein molecular weight, which is consistent with the theoretically expected 21.3kDa band, indicating that the expression is correct.

(9) Mixing the recombinant antibacterial peptide rPc-Crustin 4 and rPc-Crustin5 protein prepared in the step (8) according to equal proportion (mass volume concentration), and diluting with distilled water according to the volume ratio of 1:100 to obtain the antibacterial peptide complexing agent; meanwhile, the recombinant antibacterial peptides rPc-Crustin 4 and rPc-Crustin5 are respectively diluted by distilled water according to the volume ratio of 1:100 and are used for subsequent experiments.

Example 2 liquid bacteriostatic experiment of antimicrobial peptides and common pathogenic bacteria

The recombinant antimicrobial peptides rPc-Crustin 4, rPc-Crustin5 and the antimicrobial peptide complexing agent obtained in example 1 and common pathogenic bacteria are subjected to liquid bacteriostasis experiments, staphylococcus aureus, bacillus subtilis, bacillus megaterium, vibrio parahaemolyticus, vibrio alginolyticus, vibrio harveyi, pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, candida albicans and pichia pastoris are selected as pathogenic bacteria to be cultured over night conventionally, 100 microliter of different antimicrobial peptide solutions which are diluted by 2 times of gradient by using 1 XPBS buffer solution are mixed with 100 microliter of target pathogenic bacteria liquid respectively on the next day, the mixture is placed in sample application holes of a clean disposable 96-hole enzyme label plate to be incubated, meanwhile, Bovine Serum Albumin (BSA) is used as a control group, 100 microliter of liquid barren (PB) culture medium (1% tryptone is added into each sample application hole, NaCl 0.5% by mass/volume, pH 7.0). Then, standing and culturing for 24 hours at the temperature of 28 ℃; during the period, the growth condition of pathogenic bacteria in each bacteria mixed solution is measured by using the wavelength of 600nm of a spectrophotometer, and the minimum inhibitory concentration of different antibacterial peptides to various pathogenic bacteria is determined.

The experimental results show that: the recombinant antibacterial peptides rPc-Crustin 4, rPc-Crustin5 and the antibacterial peptide complexing agent can effectively inhibit the growth and the propagation of staphylococcus aureus, bacillus megaterium, bacillus subtilis, vibrio alginolyticus, candida albicans, escherichia coli and pseudomonas aeruginosa, and also have a certain degree of inhibiting effect on the growth and the propagation of vibrio parahaemolyticus, vibrio harveyi, klebsiella pneumoniae and pichia pastoris (table 1).

TABLE 1 minimum inhibitory concentrations of antimicrobial peptides against different pathogenic bacteria and comparative analysis

Example 3 antibacterial peptide experiments for sterilizing infectious microbes on the surface of the bookshelf in the general library

The recombinant antibacterial peptide rPc-Crustin 4, rPc-Crustin5 obtained in example 1 and the antibacterial peptide complexing agent are used for sterilization experiments against mixed bacteria on the surface of a bookshelf in a general library book room. Firstly, a small amount of sterile water is dipped in cotton balls subjected to high-temperature sterilization treatment, and 10cm of a bookshelf in a common book collection room of a library is wiped²The method comprises the following steps of putting 100 mu L of prepared mixed bacteria stock solution into a 1.5mL sterilized centrifuge tube, adding 100 mu L of antibacterial peptide diluted by distilled water according to the volume ratio of 1:100 prepared in example 1, then carrying out plate coating treatment on the mixed solution on a solid LB culture medium in a super clean bench, then putting the mixed solution into a 37 ℃ incubator for carrying out constant temperature culture for 12h for the next day, counting mixed bacteria colonies on the solid LB culture medium, simultaneously taking 100 mu L of sterilized water and 100 mu L of 1 × PBS buffer solution subjected to high temperature sterilization as a control group, repeating the experiment of each group for three times, and carrying out variance analysis.

The experimental results show that: compared with two control groups added with 100 mu L of sterilized water and 100 mu L of 1 XPBS buffer solution subjected to high-temperature sterilization treatment, the addition of 100 mu L of the antibacterial peptide can obviously reduce the number of the mixed bacteria in the mixed bacteria stock solution prepared from the mixed bacteria collected from a library, and the antibacterial peptide has stronger sterilization effect (Table 2). Moreover, the data show: the recombinant antibacterial peptide rPc-Crustin 4 and rPc-Crustin5 protein are mixed for use, can exert a synergistic effect on the inhibition effect of microorganisms, and the effect of mixing two antibacterial peptides is better than the sterilization effect of singly using one antibacterial peptide.

TABLE 2 antibacterial peptide results of sterilization experiment and comparative analysis of infectious microbes on the surface of bookshelf in general library

Note: table indicates that the difference was very significant compared to the treatment group to which sterilized water was added.

Example 4 Sterilization test of antimicrobial peptides against infectious microbes on the surface of a reading desk in a typical library reading room

The recombinant antibacterial peptides rPc-Crustin 4, rPc-Crustin5 obtained in example 1 and the antibacterial peptide complexing agent were used for sterilization experiments against bacteria on the surface of a reading desk in a library general reading room. Firstly, a small amount of sterile water is dipped in cotton balls subjected to high-temperature sterilization treatment, and 10cm of reading desk in a general reading room of a library is wiped²The method comprises the following steps of putting 100 mu L of prepared mixed bacteria stock solution into a 1.5mL sterilized centrifuge tube, adding 100 mu L of antibacterial peptide diluted by distilled water according to the volume ratio of 1:100 prepared in example 1, then carrying out plate coating treatment on the mixed solution on a solid LB culture medium in a super clean bench, then putting the mixed solution into a 37 ℃ incubator for carrying out constant temperature culture for 12h for the next day, counting mixed bacteria colonies on the solid LB culture medium, simultaneously using 100 mu L of sterilized water and 100 mu L of 1 × PBS buffer solution subjected to high temperature sterilization as a control group, repeating the experiment of each group for three times, and carrying out variance analysis.

The experimental results show that: compared with two control groups added with 100 mu L of sterilized water and 100 mu L of 1 XPBS buffer solution subjected to high-temperature sterilization treatment, the addition of 100 mu L of the antibacterial peptide can obviously reduce the number of the mixed bacteria in the stock solution prepared from the mixed bacteria collected from the surfaces of reading desks in general reading rooms of libraries, and the antibacterial peptide has stronger effect of removing the mixed bacteria (Table 3). Moreover, the data show: the recombinant antibacterial peptide rPc-Crustin 4 and rPc-Crustin5 protein are mixed for use, can exert a synergistic effect on the inhibition effect of microorganisms, and the effect of mixing two antibacterial peptides is better than the sterilization effect of singly using one antibacterial peptide.

TABLE 3 results of experiments for sterilizing infectious microbes on the surface of reading desk in general reading room of library and comparative analysis

Example 5 Sterilization experiment of antimicrobial peptides against infectious microbes in the air of a library's special old and old book collection

The recombinant antibacterial peptides rPc-Crustin 4, rPc-Crustin5 obtained in example 1 and the antibacterial peptide complexing agent are used for the sterilization experiment of the mixed bacteria in the air of special old and old books and special storehouses of libraries. Firstly, a sterilized glass culture dish with the diameter of 15cm is placed on a bookshelf in a special old book and old book collection room of a library, the cover of the culture dish and the cover of the culture dish are both opened and placed for 24 hours, and the openings are upward, so that sundry bacteria in the air are collected. Then, the petri dish and the cover of the petri dish are fully rinsed by using 10mL of sterilized water, and the petri dish and the cover of the petri dish are transferred to a new 50mL sterilized centrifuge tube, so that the mixed bacteria stock solution is obtained. Then, 100. mu.L of the prepared stock solution of infectious microbes was placed in a 1.5mL sterilized centrifuge tube, 100. mu.L of the antimicrobial peptide prepared in example 1 diluted with distilled water in a volume ratio of 1:100 was added, and the mixture was plated on a solid LB medium in a clean bench, and then placed in a 37 ℃ incubator for incubation for 12 hours. The following day, the colonies of the undesired bacteria on the solid LB medium were counted. Meanwhile, 100. mu.L of sterilized water and 100. mu.L of 1 XPBS buffer solution sterilized at high temperature were used as a control group. In addition, each set of experiments was repeated three times for analysis of variance.

The experimental results show that: compared with two control groups added with 100 mu L of sterilized water and 100 mu L of 1 XPBS buffer solution subjected to high-temperature sterilization treatment, the addition of 100 mu L of the antibacterial peptide can obviously reduce the number of the mixed bacteria in the mixed bacteria stock solution prepared from the mixed bacteria collected from the air of special old and old books in libraries, and the antibacterial peptide has stronger effect of removing the mixed bacteria (Table 4). Moreover, the data show: the recombinant antibacterial peptide rPc-Crustin 4 and rPc-Crustin5 protein are mixed for use, can exert a synergistic effect on the inhibition effect of microorganisms, and the effect of mixing two antibacterial peptides is better than the sterilization effect of singly using one antibacterial peptide.

TABLE 4 antibacterial peptide sterilization experimental results and comparative analysis of air mixed bacteria in special old book and old book storerooms of library

Note: table indicates that the difference was very significant compared to the treatment group to which sterilized water was added; indicates significant differences compared to the treatment group with added sterilized water.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of inner Mongolia science and technology

<120> green pollution-free antibacterial peptide for library and preparation and application thereof

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>127

<212>PRT

<213>Procambarus clarkii

<400>1

Met Ser Val Met Asn Tyr Gln His Leu Ala Leu Val Leu Thr Leu Gly

1 5 10 15

Val Leu Leu Ser Thr Glu Val Ala Gly His Tyr Gly Phe Tyr Gly His

20 25 30

Gln Gly Gln Tyr Arg Pro Phe Gly His Phe Gly Gly Gly Gly Gly Arg

35 40 45

Phe Gln Gly Gly Arg Gly Phe Pro Gly Asn Thr Phe Gly Gly Gly Arg

50 55 60

Ile Pro Gly Gly Gly Gly Gly Gly Thr Thr Val Asn Arg Pro Val Gly

65 70 75 80

Gly Thr Cys Pro Pro Val Arg Pro Ser Cys Thr Ala Ser Arg Ser Gln

85 90 95

Pro Ala Thr Cys Thr Ser Asp Arg Gln Cys Ser Gly Gly Phe Lys Cys

100 105 110

Cys Phe Asp Ala Cys Val Gln Thr Glu Val Cys Lys Ala Pro Val

115 120 125

<210>2

<211>167

<212>PRT

<213>Procambarus clarkii

<400>2

Met Cys Ala Thr Met Asn Cys Val Leu Leu Ala Val Val Leu Ser Leu

1 5 10 15

Gly Leu Leu Leu Gly Thr Asp Ala His Gly Tyr Tyr Ser His Arg Arg

20 25 30

Ser Tyr Arg Pro Tyr Gly Tyr Phe Gly Gly Gly Gly Ser Ile Phe Asn

35 40 45

Asp Asp Ile Phe Gly Gly Gly Arg Gly Pro Gly Ser Ile Phe Arg Gly

50 55 60

Asn Arg Phe Gln Gly Gly Gly Gly Leu Pro Gly Gly Phe Gln Gly Gly

65 70 75 80

Gly Gly Phe Pro Gly Gly Asn Gly Phe Pro Gly Gly Asn Ile Gly Gly

85 90 95

Gly Gly Ala Thr Gly Thr Asn Gly Ala Ser Gly Asn Thr Gly Ser Asn

100 105 110

Ala Ile Ile Thr Pro Gly Gly Gly Gly Ser Cys Pro Pro Ala Arg Leu

115 120 125

Ala Cys Pro Ser Thr Arg Ser Gln Pro Val Gln Cys Thr Ser Asp Ser

130 135 140

Glu Cys Leu Ala Gly Arg Lys Cys Cys Phe Asp Ala Cys Val Gln Thr

145 150 155 160

Glu Val Cys Lys Ala Thr Val

165

<210>3

<211>384

<212>DNA

<213>Procambarus clarkii

<400>3

atgtctgtga tgaattatca acatctcgcc ctcgtcttga ccctgggtgt cctcctgagc 60

acggaggtgg ctggacacta tggcttctac ggtcaccaag gccagtaccg accattcggt 120

cactttggag gaggaggagg cagattccaa ggaggtcgtg gctttcctgg gaataccttc 180

ggaggcggaa gaatcccagg tggtggtggt ggtggtacca cagttaacag gcccgtagga 240

gggacctgtc ccccagtgcg accttcctgc acagcctcca ggtcccagcc ggctacgtgt 300

acgtcggaca gacagtgttc agggggcttc aagtgctgct tcgacgcctg tgtccagacc 360

gaagtgtgca aggcccccgt ctag 384

<210>4

<211>504

<212>DNA

<213>Procambarus clarkii

<400>4

atgtgtgcga cgatgaactg tgtgcttctg gccgtggtct tgtccctggg tctcctcctg 60

ggcactgacg cccatggcta ctacagtcac cgtcgttcat accggcccta cggttacttt 120

gggggaggtg gctctatatt caatgacgac attttcggag gtggcagagg accgggaagt 180

atcttcagag gcaacagatt ccaaggtggc ggtgggttgc ctggtggatt ccaagggggc 240

ggtggattcc caggaggcaa tggattccct ggaggaaaca tcggaggcgg cggtgccaca 300

ggaaccaacg gagcgagcgg gaacaccgga tctaatgcta ttatcacacc tggaggagga 360

gggagctgcc ccccagcacg cttagcctgc ccgtctacga gatcgcagcc agtccagtgc 420

acgtcggaca gcgagtgtct ggcgggccgc aagtgctgct ttgatgcctg cgttcagacc 480

gaagtgtgca aagccaccgt ctag 504

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cccggtggac cgctagacgg 20

<210>6

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

caaagatcgg gcacgatcaa caact 25

<210>7

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gccgaattcc actatggctt ctacggt 27

<210>8

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gccctcgagc tagacggggg ccttgca 27

<210>9

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gccgaattcc atggctacta cagtcac 27

<210>10

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gccctcgagc tagacggtgg ctttgca 27

Claims

1. An antimicrobial peptide, characterized by: the antibacterial peptide is rPc-Crustin 4 or rPc-Crustin5, the amino acid sequence of rPc-Crustin 4 is 26-127 amino acids of the sequence shown in SEQ ID NO.1, and the amino acid sequence of rPc-Crustin5 is 25-167 amino acids of the sequence shown in SEQ ID NO. 2.

2. A gene encoding the antibacterial peptide of claim 1, characterized in that:

rPc-Crustin 4 antibacterial peptide gene open reading frame nucleotide sequence as SEQ ID NO.3 sequence of 76-384 nucleotides;

the nucleotide sequence of the open reading frame of the rPc-Crustin5 antibacterial peptide gene is 73-504 nucleotides of the sequence shown in SEQ ID NO. 4.

3. The process for producing the antibacterial peptide according to claim 1, wherein: the method comprises the following steps:

(1) performing reverse transcription PCR to obtain cDNA by using the extracted total RNA of the freshwater crayfish as a template;

(2) performing PCR amplification by using the cDNA obtained in the step (1) as a template to obtain a nucleotide fragment containing the antibacterial peptide coding sequence of claim 1;

(3) constructing an escherichia coli engineering bacterium capable of expressing recombinant antibacterial peptide by enzyme digestion, connection and transformation of the amplification product in the step (2) and a prokaryotic expression vector;

4. The production method according to claim 3, characterized in that: when the antibacterial peptide is rPc-Crustin 4, the reverse transcription primer is as follows: 5'-cccggtggaccgctagacgg-3', respectively; the PCR amplification primers are as follows: f1: 5' -N_ncactatggcttctacggt-3′，R1：5′-N_nctagacgggggccttgca-3′；

Wherein N is_nConsists of protective basic groups and restriction enzyme cutting sites.

5. The production method according to claim 3, characterized in that: when the antibacterial peptide is rPc-Crustin5, the reverse transcription primer is as follows: 5'-caaagatcgggcacgatcaacaact-3'; the PCR amplification primers are as follows: f2: 5' -N_ncatggctactacagtcac-3′，R2：5′-N_nctagacggtggctttgca-3′；

6. The application of the antibacterial peptide rPc-Crustin 4 and/or rPc-Crustin5 in the claim 1 in preparing bacteriostatic agent or degerming agent is characterized in that: the antibacterial or degerming species is Staphylococcus aureus, Bacillus megaterium, Bacillus subtilis, Vibrio alginolyticus, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Klebsiella pneumoniae, and Pichia pastoris.

7. A bacteriostatic or degerming agent, which is characterized in that: comprising the antibacterial peptide rPc-Crustin 4 and/or rPc-Crustin5 of claim 1.

8. The bacteriostatic or degerming agent as claimed in claim 7, wherein: when rPc-Crustin 4 and rPc-Crustin5 are contained, the mass ratio of the two is 1: 1.

9. The use of the bacteriostatic or degerming agent of claim 7 or 8 for mildew prevention and degerming in libraries, wherein: the mildew-proof and bacteria-removing types comprise staphylococcus aureus, bacillus megaterium, bacillus subtilis, vibrio alginolyticus, candida albicans, escherichia coli, pseudomonas aeruginosa, vibrio parahaemolyticus, vibrio harveyi, klebsiella pneumoniae and pichia pastoris.