CN108948160A - A kind of Lactococcus lactis antibacterial peptide, preparation method and application - Google Patents
A kind of Lactococcus lactis antibacterial peptide, preparation method and application Download PDFInfo
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Abstract
The present invention relates to gene engineering technology field more particularly to a kind of Lactococcus lactis antibacterial peptides, preparation method and application.Lactococcus lactis antibacterial peptide of the invention, amino acid sequence is as shown in SEQ ID NO.1, and nucleotide sequence is as shown in SEQ ID NO.2.Antibacterial peptide lactocin C1-8 of the invention has high-efficiency broad spectrum bacteriostasis, with the stronger ability for inhibiting drug-fast bacteria, significantly inhibits especially for staphylococcus aureus, bacillus cereus；Thermal stability is high, and acid-fast alkali-proof, toxicity is low, can be used as Substitutes For Antibiotic use.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of Lactococcus lactis antibacterial peptide, preparation method and
Using.
Background technique
Antibacterial peptide is the important effect molecule of biological congenital immunity, is widely present in animal, plant, in microorganism, belongs to
Fight the polypeptides matter of foreign pathogens.So far, have thousands of kinds of antibacterial peptides and separated to identify and, and have successively new
Natural antibacterial peptide be found, can substantially be divided into four classes: sterilization peptides, Defensins, bombesin and Antimicrobial Peptides From Plants.It is different
Molecular weight, antimicrobial spectrum, genetic origin and the biochemical characteristic of antibacterial peptide, the mode of action etc. are different from, and different antibacterial peptides generate
Bacterium all shows its unique immunologic mechanism.Most of antibacterial peptides are generated by eucaryote, are killing the same of pathogenic microorganism
When, probiotics inevitably can be also killed, to break microecological balance.Lactic acid bacteria as one kind be widely present in environment,
Flora in human and animal's enteron aisle, it is most healthy with important physiological action to sustaining life, the lactic acid generated,
The antibacterial substance such as antibacterial peptide plays extremely critical effect to the growth and field planting that inhibit harmful intestinal tract bacteria, to maintain enteron aisle
Normal microecological balance, and the growth of probiotics will not be jeopardized.
The use of antibiotic has caused a series of problems, such as since antibiotic is difficult to degrade, make environment be seriously damaged with
Pollution, and the generation of multiple resistance gene has been induced, the health of the mankind is by serious threat.Antibacterial peptide is as a kind of peace
The Substitutes For Antibiotic that loopful is protected, is all with a wide range of applications in fields such as pharmaceuticals industry, feed and food additives.
In antibacterial peptide reported at present, there are several antibacterial peptide matters from Lactococcus lactis to have been shown to have
Efficient bacteriostatic activity and good potentiality to be exploited, part of Lactococcus lactis are naturally present in the alimentary canal of animal.
The lactobacillus peptide being found earliest is a kind of peptide generated by Lactococcus lactis subspecies, nisin is named as, in 1969
Year is by FAO/WHO certification license in the anti-corrosion of food, being uniquely authorized in majority state in the world at present and allowing
Antibacterial peptide applied to field of food.The growth of most Gram-positive pathogenic bacteriums can be effectively suppressed in nisin, has stronger heat
Stability and pH stability show good bacteriostatic activity under acid and neutrallty condition, but basic mistake under alkaline condition
Deactivation cannot keep ideal bacteriostasis, thus obtain it is a kind of can acid-fast alkali-proof antibacterial peptide, its application range can be widened
And keep good application effect.Nisin can be generally generated in view of Lactococcus lactis, and minority can produce other wide spectrums or acidproof
Alkali antibacterial peptide is derived from the antibacterial peptide of lactic acid bacteria for further development and utilization, finds more antibacterial peptides and studied, thus after being
The practical application of phase lays the foundation.
Summary of the invention
In consideration of it, it is necessary to provide a kind of novel lactic acid bacteria antibacterial peptide, preparation method and application, institute regarding to the issue above
The activity that antibacterial peptide has stronger inhibition drug-resistant S. aureus, bacillus cereus is stated, and there is stronger heat
Stability and acid and alkali-resistance characteristic.
The present invention is achieved by the following technical solutions:
In a first aspect, the present invention provides a kind of antibacterial peptide, the antibacterial peptide is named as lactocin C1-8；The antibacterial
Shown in the following SEQ ID NO.1 of peptide amino acid sequence.
Preferably, shown in the following SEQ ID NO.2 of the antibacterial peptide nucleotide sequence.
Preferably, the molecular weight of the antibacterial peptide amino acid sequence is 3475Da.
The antibacterial peptide has stronger thermal stability, still keeps original active after handling 15min under the conditions of 121 DEG C
90% or more.
There is the antibacterial peptide acid-fast alkali-proof activity still to keep original work after being incubated for 2h under the conditions of pH2.0 or pH10.0
80% or more of property.
Preferably, the antibacterial peptide is from spot prelarva intestinal flora.
Second aspect, the present invention provide a kind of expression vector, institute's expression vector contain at least one copy such as first party
Nucleotide sequence described in face.
The third aspect, the present invention provide a kind of host cell of recombination, and the cell of the recombination contains such as second aspect institute
The expression vector stated.
Preferably, the host cell is appointing in Escherichia coli, bacillus subtilis, Pichia pastoris or Lactococcus lactis
It anticipates one kind, preferably Lactococcus lactis.
In the present invention, when the host cell is Escherichia coli, bacillus subtilis or Pichia pastoris, antibacterial peptide itself is removed
Outside sequence, it should be transferred in host and express together with its signal peptide sequence (SEQ ID NO.3) carried；And host selects
When Lactococcus lactis, the alternative former leader peptide of distinctive usp45 signal peptide.
Fourth aspect, the present invention provide a kind of preparation method of antibacterial peptide as described in relation to the first aspect, include the following steps:
(1) the macro genome of flora is extracted；
(2) according to antibacterial peptide nisin gene design primer, PCR amplification is carried out by template of the macro genome of the flora of extraction；
(3) PCR product is purified, and the purpose product and carrier is carried out to Nco I and Hind III double digestion respectively, tested
It is purified after card digestion success；
(4) digestion products are attached, construct the recombinant plasmid of secreting, expressing antibacterial peptide, imported host cell, obtain
Express the expression engineering bacteria of antibacterial peptide.
Preferably, step (2) primer is sequence shown in following SEQ ID NO.4-5；
Upstream primer (nisF) SEQ ID NO.4:
5’-CCGGAATTCATAAGGAGGCACTCAAAATG-3’
Downstream primer (nisR) SEQ ID NO.5:
5’-CCCAAGCTTTACTATCCTTTGATTTGGTT-3’
Preferably, step (4) the building secreting, expressing antibacterial peptide recombinant plasmid is by antibacterial peptide lactocin C1-8's
Gene order is connected to Nco I and Hind the III cloning site of pNZ8148 plasmid.
5th aspect, the present invention provides the antibacterial peptide of one kind as described in relation to the first aspect, the expression as described in second aspect carries
Body or the host cell as described in the third aspect are preparing the application in animal feed.
6th aspect, the present invention provides the antibacterial peptide of one kind as described in relation to the first aspect, the expression as described in second aspect carries
Body or the recombinant host cell as described in the third aspect are preparing the application in therapeutic agent.
7th aspect, the present invention provide a kind of antibacterial peptide as described in first invention, the expression load as described in second aspect
Body or application of the host cell in food processing and preservation as described in the third aspect.
In the present invention, it includes but is not limited to by staphylococcus aureus, waxy gemma that the drug, which can be used for treating,
Infection caused by bacillus or production treatment include but is not limited to infect as caused by staphylococcus aureus, bacillus cereus
Drug.
The invention has the advantages that:
(1) antibacterial peptide lactocin C1-8 prepared by the present invention has high-efficiency broad spectrum bacteriostasis, has stronger suppression
The ability of drug-fast bacteria processed is significantly inhibited especially for staphylococcus aureus, bacillus cereus；
(2) antibacterial peptide lactocin C1-8 prepared by the present invention, thermal stability is high, and acid-fast alkali-proof, toxicity is low, can be used as
Substitutes For Antibiotic uses；
(3) antibacterial peptide lactocin C1-8 prepared by the present invention is that further development and utilization antibacterial peptide is laid a good foundation.
Detailed description of the invention
Fig. 1 is the PCR product of antibacterial peptide gene of the present invention；
Fig. 2 is construction of recombinant plasmid schematic diagram of the present invention；
Fig. 3 is the plate single colonie figure of NZ9000 recon of the present invention；
Fig. 4 is inhibitory effect figure of the engineering bacterium fermentation liquid of the present invention to staphylococcus aureus；
Fig. 5 is inhibitory effect figure of the engineering bacterium fermentation liquid of the present invention to bacillus cereus；
Fig. 6 is antibacterial situation of change of the engineering bacterium fermentation liquid of the present invention after treatment of different temperature；
Fig. 7 is engineering bacterium fermentation liquid of the present invention through condition of different pH treated antibacterial situation of change.
Specific embodiment
The problem of solved in order to better illustrate the present invention, used technical solution and effect achieved, are now tied
It closes specific embodiment and related data is further described.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
Spot prelarva is purchased from Nansha Area, Guangzhou food market:
Lactococcus lactis subsp (Lactococcus lactics subsp.Cremoris) NZ9000, mating plasmid
PNZ8148 (chlorampenicol resistant) is purchased from Shanghai North Connaught biological technology CO., LTD.；
Escherichia coli MG1655 is bought from Novagen company；
Primer is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd；
Main agents are purchased from Shanghai Sheng Gong bioengineering Co., Ltd；
Plasmid extracts and DNA gel QIAquick Gel Extraction Kit is purchased from Tiangeng company；
Relevant enzyme is purchased from TaKaRa company.
The preparation of main agents:
(1) Lactococcus lactis GM17 culture solution/base
Soya peptone 5.0g, peptone 5.0g, yeast extract 2.5g, beef extract powder 5.0g, sodium ascorbate 0.5g, β-glycerol
Sodium phosphate 19.0g, magnesium sulfate 0.25g, adds ddH2O to 900mL, 121 DEG C of high pressure steam sterilization 15min.Divide after cooling subject to sterilization
It Jia Ru not 5% glucose, 5% lactose solution of the 50mL after 0.22 μm of membrane filtration.2% is added when preparing solid medium
Agar powder.
(2) LB culture solution/base
Tryptone (Tryptone) 10g, yeast extract (Yeast extract) 5g, NaCl 10g are gone with 950mL
Ionized water dissolution, with 5mol/L NaOH tune pH to 7.0, is settled to 1L with deionized water, in 121 DEG C of high pressure steam sterilizations
20min.2% agar powder is added when preparing solid medium.
(3) Lactococcus lactis bacterium competence cell culture G-SGM17 culture solution/base
Soya peptone 5.0g, peptone 5.0g, yeast extract 2.5g, beef extract powder 5.0g, sodium ascorbate 0.5g, β-glycerol
Sodium phosphate 19.0g, magnesium sulfate 0.25g, sucrose 171.1g, glycine 25g add deionized water to 900mL, steam in 121 DEG C of high pressures
Vapour sterilizing 20min.It is molten with 5% glucose, 5% lactose after 0.22 μm of membrane filtration that 50mL is separately added into after cooling subject to sterilization
Liquid.
(4) MRS culture medium
Peptone 10.0g, beef extract powder 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, lemon
Acid diamine 2.0g, Tween-80 1.0mL, dipotassium hydrogen phosphate 0.4g, magnesium sulfate 0.58g, manganese sulfate 0.29g, calcium carbonate 20.0g,
Agar 15.0g, deionized water dissolving are simultaneously settled to 1000mL, pH to 6.2-6.6 are adjusted, after mixing evenly in 121 DEG C of high steams
Sterilize 20min.
(5) chloromycetin solution
It weighs chloramphenicol to be dissolved in dehydrated alcohol, 0.22 μm of membrane filtration degerming, the packing of 1.5mL centrifuge tube, -20 DEG C of storages
It deposits spare.Storage concentration is 35mg/mL, and working concentration is 10 μ g/mL.
(6) Escherichia coli electricity turns incubation fluid SOC culture medium
2% (W/V) tryptone, 0.5% (W/V) yeast extract, 0.05% (W/V) sodium chloride, 2.5mM potassium chloride,
10mM magnesium chloride, 20mM glucose solution (0.22 μm of membrane filtration degerming, medium sterilization then be added).121 DEG C of high steams
Sterilize 20min, stores for future use for -20 DEG C after cooling.
(7) 50 × TAE (Tris- acetic acid) electrophoretic buffer
800mL deionized water is added in Tris alkali 242g, EDTA 18.622g, and 57.1mL vinegar is added after dissolution is sufficiently stirred
Acid mixes well, and adds deionized water and is settled to 1L, room temperature preservation.1 × TAE electrophoretic buffer is diluted to before use.
Embodiment 1 extracts spot prelarva intestinal flora genome
Spot prelarva enteron aisle genome is extracted, is included the following steps:
(1) by scissors, 75% alcohol disinfecting of tweezers；
(2) spot prelarva ileum is taken, is shredded, 5mL PBS is added, is transferred in 15mL sterile centrifugation tube, in oscillator whirlpool
It mixes；
(3) 4000 × g room temperatures are centrifuged 10min；
(4) supernatant is transferred in 2mL sterile EP tube, 1,2000rpm centrifugation 5min abandons supernatant, 200 μ L lysozymes are added
Solution (20mg/mL), 37 DEG C of water-bath 1h.
It (5) will be through the processed sample Ezup pillar soil DNA extraction agent box of lysozyme (raw work Sangon
Biotech genome) is extracted.
Embodiment 2 prepares antibacterial peptide recombinant expression carrier
(1) according to antibacterial peptide nisin gene order design primer:
Upstream primer: 5 '-CCGGAATTCATAAGGAGGCACTCAAAATG-3 ' (SEQ ID NO:4)
Downstream primer: 5 '-CCCAAGCTTTACTATCCTTTGATTTGGTT-3 ' (SEQ ID NO:5)
(2) PCR amplification: the spot prelarva intestinal flora genome extracted using embodiment 1 carries out PCR amplification, reaction as template
System is as follows:
Reaction system is prepared:
| Reagent component | rnx(μL) |
| ddH2O | 34.75 |
| 10×Ex Taq buffer(Mg2+plus) | 5 |
| Upstream primer | 2 |
| Downstream primer | 2 |
| Ex Taq Enzyme(5U/μL) | 0.25 |
| Template | 2 |
| 2.5mM dNTPs | 4 |
| Total | 50 |
It is arranged one group using water as the negative control of sample.
PCR amplification program is as follows:
PCR response procedures:
Obtained PCR product is verified, as a result as shown in Figure 1, bands visible is correct.
Gel extraction is carried out with the plastic recovery kit of Tiangeng company after verifying.
(3) fully synthetic:
PCR product is sequenced, sequence SEQ ID NO.6 is obtained, leader peptide sequences are SEQ ID NO.7.To sequence
SEQ ID NO.6 is redesigned, i.e., believes the distinctive secreting, expressing of leader peptide sequences SEQ ID NO.7 Lactococcus lactis
Number peptide Usp45 substitution, and Nco I restriction enzyme site is added at the end of sequence 5 ', Hind III digestion site is added at 3 ' ends, is named as
Lactocin C1-8, by Shanghai, Sheng Gong bioengineering Co., Ltd is synthesized:
(4) construction recombination plasmid:
Digestion connection, construction recombination plasmid, reaction system are as follows:
PCR product and plasmid pNZ8148 endonuclease reaction system are prepared:
| Reagent component | rnx(μL) |
| Plasmid pNZ8148/ antibacterial peptide gene lactocin C1-8 | 5 |
| QuickCut Nco I | 1 |
| QuickCut Hind III | 1 |
| 10×QuickCut Buffer | 2 |
| ddH2O | 11 |
| Total | 20 |
3h is reacted in 37 DEG C of thermostat water baths, digestion products are recycled through glue purification, and the carrier after digestion is carried out with gene
Connection, reaction system are as follows:
The preparation of coupled reaction system:
Component is mixed gently, 16 DEG C of water-bath reaction 2h is placed in, obtains recombinant vector pNZ8148-lactocin C1-8.
Embodiment 3 constructs antibacterial peptide host cell
(1) recombinant vector for preparing embodiment 2 converts MG1655 competent cell, applies chlorampenicol resistant LB plate, chooses
Positive colony is selected to carry out sequence verification.
The preparation of Escherichia coli MG1655 competence
1. choosing single bacterium to fall in 5mL LB culture solution, 37 DEG C, 200-250rpm cultivates 12h；
2. take 1mL bacterium solution to be seeded in 100mL LB culture solution, 37 DEG C, 200-250rpm culture, until OD600Reach
0.4-0.6；
After 3. 30min is pre-chilled on ice in bacterium solution, 4200rpm, 4 DEG C of centrifugation 10min；
4. abandoning supernatant, the ddH for sterilizing and being pre-chilled on a small quantity is added in centrifuge tube2O makes after precipitating suspension, then water is filled
Centrifuge tube, centrifugation, 4200rpm, 4 DEG C, 10min；
5. abandoning supernatant, add a small amount of pre-cooling aqua sterilisa, thallus is resuspended, then water is filled into centrifuge tube, 4200rpm, 4 DEG C of centrifugations
10min；
6. abandoning supernatant, a small amount of 10% glycerol (sterilizing, pre-cooling) is added into centrifuge tube, thallus is resuspended, then to fill it up with 10% sweet
Oil, 4200rpm, 4 DEG C of centrifugation 10min；
7. abandoning supernatant, 10% glycerol 2-3mL is added in each centrifuge tube, after so that precipitating is suspended, by bacterium solution with 100 μ L/
Pipe is sub-packed in the centrifuge tube of 1.5mL, is saved in use or -80 DEG C of refrigerators immediately, is ready to use in conversion.
(2) by recombinant plasmid transformed NZ9000 competent cell, positive colony is selected；
The preparation of lactococcus lactis subsp NZ9000 competent cell:
The preparation of Solution I: sucrose 171.1g, glycerol 100mL add ddH2O to 1000mL, 121 DEG C of high steams go out
Bacterium 20min.
The preparation of Solution II: sucrose 171.1g, glycerol 100mL, EDTA 18.612g add ddH2O to 1000mL,
121 DEG C of high pressure steam sterilization 20min.
The preparation of Solution III: G-SGM17 culture solution 100mL+1mL MgCl2(20mM)+400μL CaCl2
(0.5mM)。
1. choosing NZ9000 single colonie to be inoculated into 1.5mL G-SGM17 culture solution, 30 DEG C of standings, 14h culture；
2. 1mL bacterium solution is seeded in 15mL G-SGM17 culture solution (with ParafilmTM conical flask bottleneck), 30 DEG C of standings,
Cultivate 14h；
3. 10mL bacterium solution is seeded in 100mL G-SGM17 culture solution (with ParafilmTM conical flask bottleneck), 30 DEG C quiet
Culture is set, until OD600 reaches 0.3-0.5 (about 3-4h)；
4. 6000 × g, 4 DEG C of centrifugation 20min abandon supernatant；
5. cell is resuspended and washed with 100mL Solution I (pre-cooling), 6000 × g, 4 DEG C of centrifugation 20min abandon supernatant；
6. cell is resuspended with 100mL Solution II (pre-cooling), 15min, 6000 × g, 4 DEG C of centrifugation 20min of ice bath are abandoned
Supernatant；
7. washing cell with 100mL Solution III (pre-cooling), 6000 × g, 4 DEG C of centrifugation 20min abandon supernatant；
8. cell is resuspended with 3mL Solution I (pre-cooling)；
9. dispensing, every 40 μ L, mono- pipe is dispensed into 1.5mL centrifuge tube, and -80 DEG C of preservations are thawed on ice using preceding.
(3) expression of antibacterial peptide
1. the recombinant plasmid pNZ8148-lactocin C1-8 built is converted lactococcus lactis subsp NZ9000
Competent cell is cultivated on GM17 plate；
2. picking transformant is in 5mL GM17 fluid nutrient medium (the 10 μ g/mL containing chloramphenicol), 30 DEG C of culture 14h；
3. taking 0.8mL bacterium solution in the fresh GM17 culture solution of 10mL (the 10 μ g/mL containing chloramphenicol), until OD600Up to 0.4 or so；
4. the Nisin induction of final concentration of 10ng/mL is added, antibacterial peptide activity is detected after 12h.
Embodiment 4 constructs antibacterial peptide host cell
(1) recombinant plasmid can also pass through electrotransformation to Escherichia coli MG1655 competent cell, preparation method
It is as follows:
1. taking out 100 μ L of MG1655 competent cell from -80 DEG C of refrigerators, it is placed on and thaws on ice；It thaws to competent cell
Afterwards, it is mixed with 10 μ L connection products in the electric revolving cup for being added and being pre-chilled, ice bath 5min；
2. electricity is arranged on electric shock instrument to turn condition electric revolving cup is put into electric turn trough by: 2kV, press electric shock instrument Pulse button into
Row electrotransformation；
3. 1mL SOC culture medium (pre-cooling), ice bath 5min is added after electrotransformation immediately；
4. bacterium solution in electric revolving cup is transferred to 1.5mL centrifuge tube, 37 DEG C, 250rpm recovery 40min；
5. resuscitation fluid is centrifuged 2min in 12,000rpm, supernatant is abandoned, LB culture solution of the 200 μ L containing chlorampenicol resistant is added,
It mixes, draws 200 μ L bacterium solutions and add to LB screening flat board (the 10 μ g/mL containing chloramphenicol), with glass spreading rod bacterium solution even spread
In entire planar surface, 37 DEG C of inversions are incubated overnight (12-16h).
(2) recombinant plasmid can also by electrotransformation to lactococcus lactis subsp NZ9000 competent cell,
Preparation method is as follows:
The preparation of Solution I: sucrose 171.1g, glycerol 100mL add ddH2O to 1000mL, 121 DEG C of high steams go out
Bacterium 20min.
The preparation of Solution II: sucrose 171.1g, glycerol 100mL, EDTA 18.612g add ddH2O to 1000mL,
121 DEG C of high pressure steam sterilization 20min.
The preparation of Solution III: G-SGM17 culture solution 100mL+1mL MgCl2(20mM)+400μL CaCl2
(0.5mM)。
1. taking out 40 μ L of NZ9000 competent cell from -80 DEG C of refrigerators, it is placed on and thaws on ice；It thaws to competent cell
Afterwards, it is mixed with 10 μ L connection products in the electric revolving cup for being added and being pre-chilled, ice bath 5min (negative control group: 40 μ L competent cells+
4 μ L zero load pNZ8148)；
2. electricity is arranged on electric shock instrument to turn condition electric revolving cup is put into electric turn trough by: 2kV, press electric shock instrument Pulse button into
Row electrotransformation；
3. 1mL Solution III (pre-cooling), ice bath 5min is added after electrotransformation immediately；
4. bacterium solution in electric revolving cup is transferred to 1.5mL centrifuge tube, in 30 DEG C of incubation 1.5h；
5. bacterium solution is centrifuged 2min in 12,000rpm, supernatant is abandoned, GM17 culture solution of the 100 μ L containing chlorampenicol resistant is added,
It mixes, draws 100 μ L bacterium solutions and add to GM17 screening flat board (the 10 μ g/mL containing chloramphenicol), bacterium solution is uniformly applied with glass spreading rod
It is distributed in entire planar surface, with sealed membrane by plate seal, 30 DEG C of inversions are incubated overnight (12-16h).
Cultivation results are screened out from it positive colony as shown in figure 3, the single colonie figure occurred after 30 DEG C of cultures.
(3) expression of antibacterial peptide
1. the recombinant plasmid pNZ8148-lactocin C1-8 built is converted lactococcus lactis subsp NZ9000
Competent cell is cultivated on GM17 plate；
2. picking transformant is in 5mL GM17 fluid nutrient medium (the 10 μ g/mL containing chloramphenicol), 30 DEG C are incubated overnight；
3. taking 0.8mL bacterium solution in the fresh GM17 culture solution of 10mL (the 10 μ g/mL containing chloramphenicol), until OD600Reach 0.4 left side
It is right；
4. the nisin induction of final concentration of 10ng/mL is added, antibacterial peptide activity is detected after 12h.
The active detection of 5 antibacterial peptide of embodiment
(1) by two kinds of fermented liquids in 9000rpm, 4 DEG C of centrifugation 15min, the supernatant of acquisition is subjected to 0.22 μm of filtering
Sterile supernatant after being induced；
(2) using staphylococcus aureus and bacillus cereus as indicator bacteria (~106CFU/mL), expanded with Oxford cup agar
Arching pushing does bacteriostatic experiment, and each sample is added 100 μ L, does three repetitions；
(3) after 37 DEG C of culture 12h, the Recombinant Lactococcus lactis subsp. cremoris of lactocin C1-8 gene is imported
The visible apparent inhibition zone of NZ9000 (experimental group) supernatant samples, as a result as shown in Figure 4,5.
Embodiment 6 is heat-treated the influence to antibacterial peptide activity
1. taking experimental group supernatant samples to be divided into totally five groups of A, B, C, D, E, each 1mL；
2. by A-D group be respectively placed in 37 DEG C (controls), 60 DEG C, 80 DEG C, be incubated for 60min in 100 DEG C of water-baths, D group is placed in
15min is placed in 121 DEG C of high steams, reaches the cooling in ice water immediately after each processing time, every group is done three repetitions；
3. using staphylococcus aureus as indicator bacteria (~106CFU/mL), bacteriostatic experiment is done with Oxford cup agar diffusion method,
100 μ L are added in each sample；
4. after 37 DEG C of culture 12h, it is seen that compared with A group (control), the bacteriostatic activity of B, C, D group is substantially unchanged, E group
The reduction of activity only 9%, as a result as shown in Figure 6.
The detection of 7 antibacterial peptide soda acid field of activity of embodiment
1. taking experimental group supernatant samples to be divided into totally nine groups of a, b, c, d, e, f, g, h, i, each 1mL；
2. with 2M NaOH/HCl by a-i group sample pH value be adjusted to respectively 2.0,3.0,4.0,5.0,6.0 (controls), 7.0,
8.0,9.0,10.0, it is incubated for 2h in 37 DEG C of water-baths, every group is done three repetitions；
3. take 10 μ g/mL standard nisin samples, do it is same as experimental group handle, as control group.With golden yellow grape
Coccus is indicator bacteria (~106CFU/mL), bacteriostatic experiment is done with Oxford cup agar diffusion method, 100 μ L are added in each sample；
4. after 37 DEG C of culture 12h, it is seen that compared with nisin control, under pH2.0-pH7.0 treatment conditions, nisin is living
Property ratio lactocin C1-8 sample bacteriostatic activity is strong, the possible reason is the concentration of nisin is than lactocin in supernatant samples
The effective concentration of C1-8 is high；Under pH7.0-pH10.0 treatment conditions, experimental group is still able to maintain original active 80% or more, but
The fungistatic effect of nisin obviously dies down, as a result as shown in Figure 7.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Shenzhen Xianjin Technology Academe
Guangzhou Chinese Academy of Sciences Advanced Technology Laboratories
<120>a kind of Lactococcus lactis antibacterial peptide, preparation method and application
<130> CNP201810119
<141> 2018-07-26
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attacaagta tatcgctatg tacacccggt tgtaagacag gagctctgat gggttgtaac 60
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Claims (10)
1. a kind of Lactococcus lactis antibacterial peptide, which is characterized in that the amino acid sequence of the antibacterial peptide such as SEQ ID NO.1 institute
Show.
2. antibacterial peptide according to claim 1, which is characterized in that the nucleotide sequence of the antibacterial peptide such as SEQ ID
Shown in NO.2.
3. antibacterial peptide according to claim 1, which is characterized in that the molecular weight of the amino acid sequence of the antibacterial peptide is
3475Da。
4. antibacterial peptide according to claim 1 to 3, which is characterized in that the antibacterial peptide is returned from spot prelarva
Intestinal flora.
5. a kind of expression vector, which is characterized in that the expression vector contains the core as claimed in claim 2 of at least one copy
Nucleotide sequence.
6. a kind of host cell of recombination, which is characterized in that the host cell of the recombination contains expression described in claim 5
Carrier.
7. a kind of preparation method of antibacterial peptide as claimed in any one of claims 1-3, which is characterized in that including walking as follows
It is rapid:
(1) the macro genome of flora is extracted；
(2) according to antibacterial peptide nisin gene design primer, PCR amplification is carried out by template of the macro genome of the flora of extraction；
(3) PCR product is purified, and the purpose product and carrier is carried out to III double digestion of Nco I and Hind respectively, verify digestion
It is purified after success；
(4) digestion products and plasmid vector are attached, construct the recombinant plasmid of secreting, expressing antibacterial peptide, it is thin to import host
Born of the same parents obtain the expression engineering bacteria of expression antibacterial peptide；
(5) it with the engineering bacteria in (4), expresses and antibacterial peptide described in claim 1-3 any one is made.
8. a kind of such as antibacterial peptide of any of claims 1-4, expression vector as claimed in claim 5, such as right
It is required that recombinant host cell described in 6 is preparing the application in animal feed.
9. a kind of such as antibacterial peptide of any of claims 1-4, expression vector as claimed in claim 5, such as right
It is required that recombinant host cell described in 6 is preparing the application in therapeutic agent.
10. a kind of such as antibacterial peptide of any of claims 1-4, expression vector as claimed in claim 5, such as right
It is required that application of the recombinant host cell described in 6 in food processing and preservation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269296A (en) * | 2020-03-06 | 2020-06-12 | 山东大学 | nLsA protein, structural gene thereof and application thereof |
| CN112375712A (en) * | 2020-11-25 | 2021-02-19 | 昆明理工大学 | Lactococcus lactis and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5861376A (en) * | 1991-07-01 | 1999-01-19 | Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. | Synthetically derived peptide |
| CN105861522A (en) * | 2016-06-28 | 2016-08-17 | 江苏盐城源耀生物科技有限公司 | Antibacterial peptide Enterocin P and preparation method and application thereof |
| 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 |
| WO2018023003A1 (en) * | 2016-07-29 | 2018-02-01 | Isothrive Llc | Optimized individualized prebiotic compositions |
-
2018
- 2018-07-27 CN CN201810839942.7A patent/CN108948160A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5861376A (en) * | 1991-07-01 | 1999-01-19 | Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. | Synthetically derived peptide |
| CN105861522A (en) * | 2016-06-28 | 2016-08-17 | 江苏盐城源耀生物科技有限公司 | Antibacterial peptide Enterocin P and preparation method and application thereof |
| WO2018023003A1 (en) * | 2016-07-29 | 2018-02-01 | Isothrive Llc | Optimized individualized prebiotic compositions |
| 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 |
Non-Patent Citations (5)
| Title |
|---|
| CARLOS ARAÚJO,ET AL.: "Safety assessment, genetic relatedness and bacteriocin activity of potential probiotic Lactococcus lactis strains from rainbowtrout (Oncorhynchus mykiss, Walbaum) and rearing environment", 《EUROPEAN FOOD RESEARCH AND TECHNOLOGY》 * |
| JOHN W. M. MULDERS ET AL.: "Identification and characterization of the lantibiotic nisin 2, a natural nisin variant", 《EUR. J. BIOCHEM》 * |
| JUN-ICHI NAGAO ET AL.: "Methodologies and Strategies for the Bioengineering of Lantibiotics", 《CURRENT PHARMACEUTICAL BIOTECHNOLOGY》 * |
| SERGIO E PASTERIS ET AL.: "Characterization of a bacteriocin produced by Lactococcus lactis subsp.lactis CRL 1584 isolated from a lithobates catesbeianus hatchery", 《WORLD J MICROBIOL BIOTECHNOL》 * |
| 袁道强 等: "《生物化学实验和技术》", 28 February 2006, 中国轻工业出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269296A (en) * | 2020-03-06 | 2020-06-12 | 山东大学 | nLsA protein, structural gene thereof and application thereof |
| CN111269296B (en) * | 2020-03-06 | 2021-11-05 | 山东大学 | nLsA protein, structural gene thereof and application thereof |
| CN112375712A (en) * | 2020-11-25 | 2021-02-19 | 昆明理工大学 | Lactococcus lactis and application thereof |
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