CN106011153A - Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof - Google Patents
Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a probiotic antimicrobial peptide Enterocin P, and a preparation method and application thereof. The probiotic antimicrobial peptide Enterocin P gene has nucleotide sequence disclosed as SEQ ID No.1. The mature antimicrobial peptide Enterocin P encoded by the probiotic antimicrobial peptide Enterocin P gene has amino acid sequence disclosed as SEQ ID No.2. The preparation method of the antimicrobial peptide Enterocin P comprises the following steps: (1) synthesizing gene of which the nucleotide sequence is disclosed as SEQ ID NO:3; (2) establishing a recombinant plasmid capable of expressing the antimicrobial peptide; (3) transforming the host bacterium by using the recombinant plasmid to establish a gene engineering bacterium; (4) expressing the antimicrobial peptide by using the gene engineering bacterium; and (5) detecting the antibacterial activity of the antimicrobial peptide. The probiotic antimicrobial peptide Enterocin P has antibacterial activity on pathogenic bacteria, and can be used as an animal feed additive.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of prebiotic type antibacterial peptide Enterocin P and its preparation method and application.
Background technology
In recent years, the antibiotic a large amount of abuses in animal farming industry, cause antibiotic-resistant strains constantly to occur, drug residue, the problems such as environmental pollution and ecological disruption, serious threat to human health and the development of animal husbandry.Therefore, the novel antibacterial preparation finding a kind of energy substitute antibiotics is extremely urgent.Antibacterial peptide has a good anti-bacterial effect, and Heat stability is good is nontoxic, noresidue, has no side effect, safe and efficient, environmentally safe and be not likely to produce the advantages such as bacterial drug resistance, is expected to become the effective ways solving this difficult problem of abuse of antibiotics.
Antibacterial peptide is the peptide matters resisting exogenous pathogen produced in biological defensive system, is biological natural. the important component part of system of defense.Antibacterial peptide source is extremely extensive, and from plant, insecticide, birds, mammal, microorganism all has been observed that the generation of antibacterial peptide.Up to now, existing nearly thousand kinds of antibacterial peptides are by the most isolated and purified and qualification.
Bacteriogenic antibacterial peptide is the polypeptides matter with bacteriostatic activity produced by Ribosome biogenesis mechanism in its metabolic process.Antibacterial peptide is the most universal in probiotic bacteria.Almost all of probiotic bacteria can produce one or more antibacterial peptide materials.In recent years, Chinese scholars from Lactobacillus plantarum, bacillus acidophilus, bacillus bifidus, streptococcus thermophilus, isolated and purified in many probiotic bacterias such as lactococcus lactis obtain multiple antibacterial peptide material.
Antibacterial peptide Enterocin P is from the isolated and purified antibacterial peptide obtained of Enterococcus faecium P13, named Enterocin P.71 amino acid whose propetides of the structural gene coding of antibacterial peptide Enterocin P.Its propetide includes 27 amino acid whose signal peptides and 44 amino acid whose mature peptides.Antibacterial peptide Enterocin P has wider antimicrobial spectrum, especially to Listeria monoeytogenes. and the pathogen such as staphylococcus aureus have obvious inhibition, have preferable application prospect.
Antibacterial peptide Enterocin P content in its producing strains Enterococcus faecium P13 is extremely low, yield of antibacterial peptides is extracted low from producing strains, time-consuming length, complex process and somewhat expensive, large-scale production cannot be realized, thus restrict the actual application of antibacterial peptide Enterocin P.
Summary of the invention
It is an object of the invention to for the problems referred to above, it is provided that a kind of prebiotic type antibacterial peptide Enterocin P with significant anti-microbial pathogen effect and its preparation method and application.
For reaching above-mentioned purpose, present invention employs following technical proposal: the one prebiotic type antibacterial peptide Enterocin P gene of the present invention, there is the nucleotide sequence shown in SEQ ID No.1.
Prebiotic type antibacterial peptide Enterocin P gene encoding mature antibacterial peptide Enterocin P of the present invention has the aminoacid sequence shown in SEQ ID No.2.
The preparation method of prebiotic type antibacterial peptide Enterocin P of the present invention, comprises the steps:
(1) synthetic nucleotide sequence gene as shown in SEQ ID NO:3；
(2) the pNZ8148-Enterocin P recombiant plasmid of energy efficient expression antimicrobial peptides Enterocin P is built；
(3) with described pNZ8148-Enterocin P recombinant plasmid transformed Host Strains, the genetic engineering bacterium of lactococcus lactis NZ900 is built；
(4) described genetic engineering bacterium lactococcus lactis is utilized to express antibacterial peptide Enterocin P；
(5) bacteriostatic activity of antibacterial peptide Enterocin P is detected.
Further, in step (1), the Enterocin P gene of the synthetic both sides restriction enzyme site containing pst I and Hind III has the nucleotide sequence shown in SEQ ID No.3.
Further, in step (2), nucleotide sequence shown in SEQ ID No.3 is connected and is connected acquisition pNZ8148-Enterocin P connection product with linearizing pNZ8148 plasmid after restricted enzyme pst I and Hind III double digestion, product will be connected and import escherichia coli MC1061, it is thus achieved that containing pNZ8148-Enterocin P recombiant plasmid.
Further, in step (3), described Host Strains is escherichia coli MC1061；
PNZ8148-Enterocin P recombiant plasmid will be extracted, identify that correct plasmid uses electricity to proceed to carry out in Lactococcus lactis bacterium competence cell NZ9000 the abduction delivering of antibacterial peptide Enterocin P through PCR and enzyme action, electricity conversion processing parameter is 2.0kV, 25 μ F, 200 Ω, 5ms, through chlorampenicol resistant screening, PCR checking, enzyme action identifies the genetic engineering bacterium obtaining the correct lactococcus lactis NZ900 containing pNZ8148-Enterocin P recombiant plasmid.
Further, in step (4), the genetic engineering bacterium of the lactococcus lactis NZ900 containing pNZ8148-Enterocin P recombiant plasmid is inoculated in the GM17 fluid medium containing erythromycin, 30 DEG C cultivate to OD600nm be 0.5 time, adding the nisin abduction delivering of final concentration 10ng/ml, after cultivating 4h, 4000g is centrifuged 10min and takes supernatant, the supernatant of acquisition is carried out 0.22 μm and filters the sterile supernatant after obtaining induction, obtain prebiotic type antibacterial peptide Enterocin P.
Further, in step (5), using agar diffusion method to detect the bacteriostatic activity of antibacterial peptide Enterocin P, after 37 DEG C are cultivated, the bacteriostatic activity of staphylococcus aureus, Listeria monoeytogenes or Salmonella is detected by antibacterial peptide Enterocin P.
The prebiotic type antibacterial peptide Enterocin P of the present invention application in animal feed.
Further, described animal feed is feed additive.
Beneficial effect: the present invention has a stronger bacteriostatic activity to pathogen, safety is nontoxic, has no side effect, and the advantage such as antibiotic-free drug resistance can be used as animal feed additive.The constructed genetic engineering bacterium of the present invention not only has the prebiotic effect of lactococcus lactis itself, also has antibacterial peptide Enterocin P obvious anti-microbial pathogen effect, preferably can safely and effectively be applied to animal feed as prebiotic type feed additive.
Compared with prior art, present invention have the advantage that
(1) antibacterial peptide of the present invention is from animal health is given birth to the Enterococcus faecium P13 acted on, being intended in animal feed additive having animal preferably effect and probiotic effects, the mature peptide molecular weight about 4.5KDa of Enterocin P antibacterial peptide has significant anti-microbial pathogen effect.
(2) the antibacterial peptide Enterocin P expressed by the present invention comes from Enterococcus faecium P13, animal body do not had toxicity, therefore the antibacterial peptide prepared with the gene of the present invention, compared with traditional antibiotic medicine, will be the short peptide matters of a kind of safer anti-microbial pathogen.The prebiotic type antibacterial peptide Enterocin P gene of the present invention is probiotic bacteria Enterococcus faecium P13 prebiotic type antibacterial peptide Enterocin P gene Enterocin P, and coded antibacterial peptide Enterocin P is isolated from probiotic bacteria Enterococcus faecium P13 fermented supernatant fluid.
(3) genetic engineering bacterium that the present invention builds with conventional probiotic lactic acid Lactococcus for Host Strains, the probiotic bacteria that lactococcus lactis is inherently favourable to animal health, obtains effective secreting, expressing by antibacterial peptide Enterocin P in lactococcus lactis simultaneously.Enterocin P channel genes probiotic lactic acid Lactococcus will obtain genetic engineering bacterium and efficiently express, this genetic engineering bacterium is used for feed fermentation as fermentation strain, can be used as, in animal feed additive, improving nutritive value and the animal disease resistant ability of feedstuff.
Accompanying drawing explanation
Fig. 1 is extracting by the schematic diagram of the bacillus coli DH 5 alpha plasmid containing genes of interest, swimming lane 1: genes of interest plasmid according to plasmid extraction kit of the present invention；Swimming lane M:500bp ladder marker；
Fig. 2 is that the bacillus coli DH 5 alpha genes of interest plasmid enzyme restriction product of the present invention is through 1% agarose gel electrophoresis figure, swimming lane 1: genes of interest plasmid；Swimming lane M:500bp ladder marker；
Fig. 3 is the schematic diagram of the pNZ8148 plasmid map of the present invention；
Fig. 4 is the schematic diagram of employing TaKaRa Biotech restricted enzyme the Pst I and Hind III operating instruction double digestion pNZ8148 plasmid of the present invention, swimming lane 1:pNZ8148 plasmid；Swimming lane M:1kb ladder marker；
Fig. 5 is the schematic diagram of the recombinant plasmid transformed mono-bacterium colony of escherichia coli MC1061 of the present invention；
Fig. 6 be the mono-bacterium colony of escherichia coli MC1061 of the present invention be that the PCR primer of DNA profiling carries out 1% agarose gel electrophoresis figure, swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker；
Fig. 7 is to carry out 1% agarose gel electrophoresis figure, swimming lane 1: recombiant plasmid after sky of the present invention root plasmid extraction kit operating instruction extracts escherichia coli MC1061 recombiant plasmid；Swimming lane M:500bp ladder marker；
Fig. 8 is that the present invention extracts escherichia coli MC1061 recombiant plasmid employing TaKaRa Biotech restricted enzyme Pst I and Hind III operating instruction double digestion plasmid, and product is through 1% agarose gel electrophoresis figure, swimming lane 1: recombiant plasmid；Swimming lane M:500bp ladder marker；
Fig. 9 is the schematic diagram of the recombinant plasmid transformed mono-bacterium colony of lactococcus lactis NZ9000 of the present invention；
Figure 10 be the mono-bacterium colony of lactococcus lactis NZ9000 of the present invention be that the PCR primer of DNA profiling carries out 1% agarose gel electrophoresis figure, swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker；
Figure 11 is to carry out 1% agarose gel electrophoresis figure, swimming lane 1: recombiant plasmid after sky of the present invention root plasmid extraction kit operating instruction extracts lactococcus lactis NZ9000 recombiant plasmid；Swimming lane M:1kb ladder marker；
Figure 12 is that the present invention extracts lactococcus lactis NZ9000 recombiant plasmid employing TaKaRa Biotech restricted enzyme Pst I and Hind III operating instruction double digestion plasmid, and product is through 1% agarose gel electrophoresis figure, swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker；
Figure 13 be Enterocin P of the present invention to listerial fungistatic effect figure, wherein 1,2,3 is experimental group；
Figure 14 is the Enterocin P of the present invention fungistatic effect figure to staphylococcus aureus, and wherein 1,2,3 is experimental group；
Figure 15 is the Enterocin P of the present invention fungistatic effect figure to Salmonella, and wherein 1,2,3 is experimental group.
Detailed description of the invention
Further illustrate the present invention by the following examples.It should be understood that these embodiments are explaination and the citings of the present invention, limit the scope of the present invention the most in any form.
The one prebiotic type antibacterial peptide Enterocin P gene of the present invention, has the nucleotide sequence shown in SEQ ID No.1.
Prebiotic type antibacterial peptide Enterocin P gene encoding mature antibacterial peptide Enterocin P described in claim 1 has the aminoacid sequence shown in SEQ ID No.2.
The preparation method of prebiotic type antibacterial peptide Enterocin P of the present invention, comprises the steps:
(1) the Enterocin P gene of the synthetic both sides restriction enzyme site containing pst I and Hind III has the nucleotide sequence shown in SEQ ID No.3.Synthetic nucleotide sequence gene as shown in SEQ ID NO:3；
(2) the pNZ8148-Enterocin P recombiant plasmid of energy efficient expression antimicrobial peptides Enterocin P is built；Nucleotide sequence shown in SEQ ID No.3 is connected and is connected acquisition pNZ8148-Enterocin P connection product with linearizing pNZ8148 plasmid after restricted enzyme pst I and Hind III double digestion, product will be connected and import escherichia coli MC1061, it is thus achieved that containing pNZ8148-Enterocin P recombiant plasmid.
(3) with described pNZ8148-Enterocin P recombinant plasmid transformed Host Strains, the genetic engineering bacterium of lactococcus lactis NZ900 is built；Described Host Strains is escherichia coli MC1061；
PNZ8148-Enterocin P recombiant plasmid will be extracted, identify that correct plasmid uses electricity to proceed to carry out in Lactococcus lactis bacterium competence cell NZ9000 the abduction delivering of antibacterial peptide Enterocin P through PCR and enzyme action, electricity conversion processing parameter is 2.0kV, 25 μ F, 200 Ω, 5ms, through chlorampenicol resistant screening, PCR checking, enzyme action identifies the genetic engineering bacterium obtaining the correct lactococcus lactis NZ900 containing pNZ8148-Enterocin P recombiant plasmid.
(4) described genetic engineering bacterium lactococcus lactis is utilized to express antibacterial peptide Enterocin P；The genetic engineering bacterium of the lactococcus lactis NZ900 containing pNZ8148-Enterocin P recombiant plasmid is inoculated in the GM17 fluid medium containing erythromycin, 30 DEG C cultivate to OD600nm be 0.5 time, add the nisin abduction delivering of final concentration 10ng/ml, after cultivating 4h, 4000g is centrifuged 10min and takes supernatant, the supernatant of acquisition is carried out 0.22 μm and filters the sterile supernatant after obtaining induction, obtain prebiotic type antibacterial peptide Enterocin P.
(5) bacteriostatic activity of antibacterial peptide Enterocin P is detected.Using agar diffusion method to detect the bacteriostatic activity of antibacterial peptide Enterocin P, after 37 DEG C are cultivated, the bacteriostatic activity of staphylococcus aureus, Listeria monoeytogenes or Salmonella is detected by antibacterial peptide Enterocin P.
The prebiotic type antibacterial peptide Enterocin P of the present invention application in animal feed.
Described animal feed is feed additive.
Embodiment 1
Main material used in the present invention
Bacterial strain and carrier: escherichia coli MC1061, lactococcus lactis NZ9000 and pNZ8148 plasmid bacterial are purchased from Mo Bi Tec company of Germany；DNA Marker is purchased from Shanghai JaRa company limited；Listerella, staphylococcus aureus and Salmonella；Prebiotic type antibacterial peptide Enterocin P gene is synthesized by Sangon Biotech (Shanghai) Co., Ltd. and proceeds to bacillus coli DH 5 alpha；Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Main agents and test kit: M17 broth bouillon is purchased from Qingdao Hai Bo company；Restricted enzyme Pst I and Hind III .T4DNA Ligase.EX Taq is purchased from TaKaRa Biotech company；Chloromycetin plasmid extraction and DNA gel reclaim test kit purchased from Tian Gen company；Nisin is purchased from Sigma company.
The preparation of main agents:
121 DEG C of autoclaving 15min of GM17:M17 broth bouillon, add the glucose of filtration sterilization to final concentration of 0.5% (mass volume ratio) after cooling.
SGM17:M17 meat soup adds final concentration of 0.5M sucrose, 2.5% glycine .0.5% glucose.M17 meat soup adds 121 DEG C of autoclaving 15min after sucrose and glycine, adds the glucose of 0.22 μm filtration sterilization after cooling to final concentration of 0.5% (mass volume ratio).
0.5M sucrose/10% glycerol: 17.115g sucrose is dissolved in 80ml ultra-pure water, after adding 10ml glycerol, mixing is settled to 100ml, 0.22 μm filtration sterilization.
LB fluid medium: 10g peptone .5g yeast extract, 1g NaCl is dissolved in 900ml distilled water, after regulation pH value to 7.0, is settled to 121 DEG C of autoclaving 15min after 1L.
Key instrument: High speed refrigerated centrifuge, pipettor .PCR instrument is all purchased from Eppendorf company of Germany；Nucleic acid electrophoresis tank. electrophresis apparatus. electricity conversion instrument is purchased from Bio-Rad company；Gel imaging instrument is purchased from Shanghai Tian Neng company；Aseptic filtration film (0.22 μm) is purchased from What Man company.
Genes of interest obtains:
The most prebiotic type antibacterial peptide Enterocin P gene is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and both sides add the restriction enzyme site of Pst I and Hind III and import bacillus coli DH 5 alpha.As it is shown in figure 1, be that the present invention extracts by the schematic diagram of the bacillus coli DH 5 alpha plasmid containing genes of interest according to plasmid extraction kit；Swimming lane 1: genes of interest plasmid；Swimming lane M:500bp ladder marker.
The most as shown in Figure 2, for the present invention according in TaKaRa Biotech restricted enzyme Pst I and Hind III double digestion step 1 extract plasmid, digestion products is through 1% agarose gel electrophoresis, genes of interest fragment uses Tian Gen company DNA to reclaim reagent and operational approach carries out glue recovery, and-20 DEG C save backup；Swimming lane 1: genes of interest plasmid；Swimming lane M:500bp ladder marker.
Plasmid pNZ8148 linearisation:
As it is shown on figure 3, the schematic diagram of the pNZ8148 plasmid map used for the present invention；As shown in Figure 4, for employing TaKaRa Biotech restricted enzyme the Pst I and Hind III operating instruction double digestion pNZ8148 plasmid of the present invention through 1% agarose gel electrophoresis figure, genes of interest fragment uses Tian Gen company DNA to reclaim reagent and operational approach carries out glue recovery, and-20 DEG C save backup；Swimming lane 1:pNZ8148 plasmid；Swimming lane M:1kb ladder marker.
The genes of interest reclaimed and linearization plasmid connect
According to the T4DNA ligase test kit operating instruction of TaKaRa Biotech, recovery genes of interest and plasmid are attached.
Embodiment 2
Preparation escherichia coli MC1061 competent cell
1, the LB fluid medium of 1% inoculation MC1061, cultivates in 37 DEG C of shaken overnight；
2, take incubated overnight bacterium solution 1% and be inoculated in LB fluid medium, shaken cultivation 4h (now antibacterial is in exponential phase) at 37 DEG C, place 10min on ice；
3,4000g, 4 DEG C of centrifugal 10min collect logarithmic (log) phase cell, abandon supernatant, collect bacterium cell pellet；
4, with 1/10 in the 0.1M CaCl of the pre-cooling of fluid medium volume before2Thalline, places 30min on ice；
5,4000g, 4 DEG C of centrifugal 5min abandon supernatant, collect bacterium cell pellet；
6, with 1/100 in fluid medium volume before containing 0.1M CaCl2The resuspended thalline of solution；
7, draw 200 μ L and be sub-packed in centrifuge tube ,-80 DEG C of preservations.
Embodiment 3
Connect product and convert escherichia coli MC1061 competent cell
(1) take 100 μ l E.coli MCI061 competent cells, add 10-50 μ l and connect product, after soft mixing, ice bath 30min；
(2) EP pipe is put into it is preheated to 90S in the water-bath of 42 DEG C；
(3) quickly EP pipe is transferred in ice bath, make cell cool down 1-2min；
(4) often pipe adds 900 μ l LB cultivation liquid bases, and 37 DEG C of shaking table gentlenesses concussions are cultivated 1.5h, made bacteria resuscitation and give full expression to plasmid-encoded antibiotic-resistance marker's gene；
(5) draw 100 μ l recovery cultivation bacterium solution to be respectively coated on the LB culture medium flat plate containing 10 μ g/mL chloromycetin；
(6) flat board is placed in room temperature absorb to liquid, is inverted flat board, as it is shown in figure 5, be that 37 DEG C of cultivation 12-24h of the present invention are until there is single bacterium colony figure.
Embodiment 4
Escherichia coli MC1061 recombiant plasmid bacterium colony PCR and double digestion are identified
Single bacterium colony that on picking, step obtains carries out PCR qualification
Primer for PCR amplification
Forward primer: AAGTTGATGCAGCTACGC
Reverse primer: TGTCCCATACCTGCCAAA
Amplification purpose fragment length is 142bp.
As follows with single bacterium colony PCR reaction system as DNA profiling:
Amplification program: 94 DEG C, 5min, then 30 circulations (94 DEG C, 1min；58 DEG C, 50S；72 DEG C, 1min)；72 DEG C extend 10min.As shown in Figure 6, the PCR primer for the present invention carries out 1% agarose gel electrophoresis figure；Swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker.
Recombiant plasmid enzyme action is identified:
Picking through the PCR correct single bacterium colony of checking in LB liquid culture based on the good bacterium solution of 37 DEG C of shaken cultivation, as it is shown in fig. 7, carry out 1% agarose gel electrophoresis figure after extracting plasmid according to sky root plasmid extraction kit operating instruction；Swimming lane 1: recombiant plasmid；Swimming lane M:500bp ladder marker, as shown in Figure 8, the extraction plasmid for the present invention uses TaKaRa Biotech restricted enzyme Pst I and Hind III operating instruction double digestion plasmid, and product is through 1% agarose gel electrophoresis figure；Swimming lane 1: recombiant plasmid；Swimming lane M:500bp ladder marker.
Embodiment 5
Prepare lactococcus lactis NZ9000 competent cell
1. lactococcus lactis 1% is inoculated in SGM17 (GM17 sucrose Han 0.5M, 2.5% glycine), cultivates to OD600 ≈ 0.5-0.8；
2.5000g, 4 DEG C of centrifugal 10min collect thalline；
The solution washing thalline of the most ice-cold 0.5M sucrose containing 10% glycerol 2 times；
4. with the resuspended thalline of 0.5M sucrose solution containing 10% glycerol of 1/100 with culture volume before, in-80 DEG C of preservations.
Lactococcus lactis electricity converts:
After taking the correct recombiant plasmid of 2 μ L empirical tests and the mixing of 40 μ L competent cells, proceed to, in the electric revolving cup of pre-cooling, place 10min on ice.Electricity conversion condition: voltage 2000V, electric capacity 25 μ F, resistance 200 Ω.After electric shock, immediately toward after electricity revolving cup adds 1mL GM17 culture medium (containing 20mM MgCl2,2mM CaCl2), 30 DEG C of quiescent culture 2h, coat on the GM17 flat board containing 5 μ g/ml chloromycetin, as it is shown in figure 9, be that 30 DEG C of cultivations of the present invention are until there is single bacterium colony figure.
Lactococcus lactis NZ9000 recombiant plasmid bacterium colony PCR and double digestion checking
Single bacterium colony that on picking, step obtains carries out PCR checking
Primer for PCR amplification
Forward primer: AAGTTGATGCAGCTACGC
Reverse primer: TGTCCCATACCTGCCAAA
Amplification purpose fragment length is 142bp.
As follows with single bacterium colony PCR reaction system as DNA profiling:
Amplification program: 94 DEG C, 5min, then 30 circulations (94 DEG C, 1min；58 DEG C, 50S；72 DEG C, 1min)；72 DEG C extend 10min.As shown in Figure 10, the PCR primer for the present invention carries out 1% agarose gel electrophoresis figure；Swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker.
Picking through the PCR correct single bacterium colony of checking in GM17 liquid culture based on the good bacterium solution of 30 DEG C of shaken cultivation, as shown in figure 11, after extracting plasmid according to sky root plasmid extraction kit operating instruction, carry out 1% agarose gel electrophoresis figure；Swimming lane 1: recombiant plasmid；Swimming lane M:1kb ladder marker；Shown in Figure 12, the extraction plasmid for the present invention uses TaKaRa Biotech restricted enzyme Pst I and Hind III operating instruction double digestion plasmid, and product is through 1% agarose gel electrophoresis figure；Swimming lane 1: genes of interest；Swimming lane M:500bp ladder marker.
Embodiment 6
The abduction delivering of lactococcus lactis NZ9000 recombinant bacterium
Recombinant bacterium 4% is inoculated in that the GM17 fluid medium containing 5 μ g/ml chloromycetin is to be grown to add final concentration of 10ng/ml nisin inducing culture 4h when being 0.5 to OD600nm and obtain and cultivate bacterium solution, 4000g is centrifuged 10min and takes supernatant, the supernatant of acquisition is carried out 0.22 μm and filters the sterile supernatant after obtaining induction, for next step bacteriostatic experiment.
Embodiment 7
Recombinant antibacterial peptide bacteriostatic experiment
1. recombinant antibacterial peptide bacteriostatic experiment
This experiment uses agar diffusion method to detect the bacteriostatic activity of recombinant expressed antibacterial peptide Enterocin P.The staphylococcus aureus of exponential phase will be in. Listerella, Salmonella cultures respectively takes in 100 μ L coatings and 20ml solid medium, again with the card punch punching of 8mm, add the testing sample supernatant of 200 μ L, with the lactococcus lactis supernatant containing prebiotic type antibacterial peptide Enterocin P gene induced without nisin for comparison, cultivate for 37 DEG C and observe.
2. bacteriostatic experiment result
Use agar diffusion method that the bacteriostatic activity of recombinant antibacterial peptide is detected, after 37 DEG C are cultivated, result shows, as shown in figure 13, for Enterocin P of the present invention to listerial fungistatic effect figure, wherein 1, 2, 3 is experimental group, comparison is matched group, as shown in figure 14, for the Enterocin P of the present invention fungistatic effect figure to staphylococcus aureus, wherein 1, 2, 3 is experimental group, comparison is matched group, as shown in figure 15, for the Enterocin P of the present invention fungistatic effect figure to Salmonella, wherein 1, 2, 3 is experimental group, comparison is matched group, Enterocin P of the present invention is respectively provided with biocidal property to three kinds of pathogen, the strongest to Listerella inhibition.The genetic engineering bacterium of the prebiotic type antibacterial peptide Enterocin P of the present invention can be applied in feed additive.
The ultimate principle of the present invention has more than been shown and described. principal character and advantages of the present invention.Skilled person will appreciate that of the industry; the present invention is not restricted to the described embodiments; the principle that the present invention is simply described described in above-described embodiment and description; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and claimed scope is by appending claims. and description and equivalent thereof define.
Claims (10)
1. a prebiotic type antibacterial peptide Enterocin P gene, it is characterised in that there is the nucleotide sequence shown in SEQ ID No.1.
2. the prebiotic type antibacterial peptide Enterocin P gene encoding mature antibacterial peptide Enterocin P described in claim 1 has SEQ
Aminoacid sequence shown in ID No.2.
3. the preparation method of the prebiotic type antibacterial peptide Enterocin P described in claim 2, it is characterised in that comprise the steps:
(1) synthetic nucleotide sequence gene as shown in SEQ ID NO:3；
(2) the pNZ8148-Enterocin P recombiant plasmid of energy efficient expression antimicrobial peptides Enterocin P is built；
(3) with described pNZ8148-Enterocin P recombinant plasmid transformed Host Strains, the gene work of lactococcus lactis NZ900 is built
Journey bacterium；
(4) described genetic engineering bacterium lactococcus lactis is utilized to express antibacterial peptide Enterocin P；
(5) bacteriostatic activity of antibacterial peptide Enterocin P is detected.
The preparation method of prebiotic type antibacterial peptide Enterocin P the most according to claim 3, it is characterised in that: in step (1)
In, the Enterocin P gene of the synthetic both sides restriction enzyme site containing pst I and Hind III has shown in SEQ ID No.3
Nucleotide sequence.
The preparation method of prebiotic type antibacterial peptide Enterocin P the most according to claim 3, it is characterised in that: in step (2)
In, the nucleotide sequence shown in SEQ ID No.3 is connected and linearisation after restricted enzyme pst I and Hind III double digestion
PNZ8148 plasmid connect obtain pNZ8148-Enterocin P connect product, will connect product import escherichia coli MC1061,
Obtain the P recombiant plasmid Han pNZ8148-Enterocin.
The preparation method of prebiotic type antibacterial peptide Enterocin P the most according to claim 3, it is characterised in that: in step (3)
In, described Host Strains is escherichia coli MC1061；
PNZ8148-Enterocin P recombiant plasmid will be extracted, identify that correct plasmid uses electricity to proceed to breast through PCR and enzyme action
Yogurt coccus competent cell NZ9000 carries out the abduction delivering of antibacterial peptide Enterocin P, electricity conversion processing parameter be 2.0kV,
25 μ F, 200 Ω, 5ms, through chlorampenicol resistant screening, PCR checking, enzyme action is identified and is obtained correct containing
The genetic engineering bacterium of the lactococcus lactis NZ900 of pNZ8148-Enterocin P recombiant plasmid.
The preparation method of prebiotic type antibacterial peptide Enterocin P the most according to claim 3, it is characterised in that: in step (4)
In, the genetic engineering bacterium of the lactococcus lactis NZ900 containing pNZ8148-Enterocin P recombiant plasmid is inoculated in containing erythromycin
GM17 fluid medium, 30 DEG C cultivate to OD600nm be 0.5 time, add final concentration 10ng/ml nisin abduction delivering, training
Supporting after 4h, 4000g is centrifuged 10min and takes supernatant, and what the supernatant of acquisition carried out 0.22 μm filters after obtaining induction is aseptic
Supernatant, obtains prebiotic type antibacterial peptide Enterocin P.
The preparation method of prebiotic type antibacterial peptide Enterocin P the most according to claim 3, it is characterised in that: in step (5)
In, use agar diffusion method that the bacteriostatic activity of antibacterial peptide Enterocin P is detected, after 37 DEG C are cultivated, antibacterial peptide
The bacteriostatic activity of staphylococcus aureus, Listeria monoeytogenes or Salmonella is detected by Enterocin P.
9. the application in animal feed of the prebiotic type antibacterial peptide Enterocin P described in any one of claim 2 to 8.
Application the most according to claim 9, described animal feed is feed additive.
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| CN106432431A (en) * | 2016-10-19 | 2017-02-22 | 深圳市澳华农牧有限公司 | Antibacterial peptide as well as preparation method and application thereof |
| CN107916244A (en) * | 2017-06-20 | 2018-04-17 | 江西嘉博生物工程有限公司 | A kind of construction method of Recombinant Lactococcus lactis for expressing lysostaphin gene and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106432431A (en) * | 2016-10-19 | 2017-02-22 | 深圳市澳华农牧有限公司 | Antibacterial peptide as well as preparation method and application thereof |
| CN107916244A (en) * | 2017-06-20 | 2018-04-17 | 江西嘉博生物工程有限公司 | A kind of construction method of Recombinant Lactococcus lactis for expressing lysostaphin gene and application |
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