CN105968214A - Antibacterial and antiviral hybrid peptide as well as preparation method and application thereof - Google Patents
Antibacterial and antiviral hybrid peptide as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an antibacterial and antiviral hybrid peptide as well as a preparation method and application thereof. An amino acid sequence and a nucleotide sequence of the antibacterial and antiviral hybrid peptide are as shown in SEQ ID No. 1 and 2. A gene for coding the antibacterial and antiviral hybrid peptide is cloned to an expression vector pPICZ[alpha]A to obtain a recombinant vector, then Pichia pastoris is converted, methyl alcohol is used for inducing expression, and an expression product is purified to obtain the target antibacterial antiviral hybrid peptide. The antibacterial and antiviral hybrid peptide can prevent and kill various bacteria and viruses, has low probability of resistance, and therefore, can serve as an ideal antibiotic substituent, a people and livestock biological antibacterial and antiviral medicine, a feed additive, a preservative, a sanitizer or the like.
Description
Technical field
The present invention relates to genetic engineering and field of biological pharmacy, specifically, relate to a kind of novel anti-bacteria and anti-virus miscellaneous
Close polypeptide and preparation method and application.
Background technology
Antibiotic is for controlling, preventing and treat various infectious disease have important function.Due to the use of antibiotic,
To the eighties in 20th century, the mankind almost can conquer most infection class disease, but, As time goes on and antibiosis
The abuse of element, Resistant strain is the most.Monitoring is had to find, in the middle of the most representational Resistant strain, the staphylococcus of drug resistance
Have risen to 40%；The coagulase negative staphylococcus of drug resistance is then more than 77%.Now, occurred in that and can resist all antibiosis
The fastbacteria of element, the safety of antibiotics resistance problem serious threat to human and animal is with healthy.Release fastbacteria to the mankind
The most serious healthy threat becomes problem demanding prompt solution, and therefore the discovery of new class antimicrobial material has become with development
For the focus studied in the world, but to have antibacterial and antiviral bioactive peptide research concurrently less simultaneously.In poultry and aquatic animal
In cultivation, either pathogenic microorganism primary infection or secondary infection, the most often can develop into antibacterial and virus mixed infection,
And the clinic caused due to variation or atypia virus (such as bird flu virus) and pathogen more in recent years or subclinical sense
Dye is more and more, the existence of serious harm animal and health, China annual Died Of Disease poultry (wherein dead chicken more than 300,000,000) straight
Connect economic loss and be up to more than 40,000,000,000 yuan, simultaneously because of feedstuff, artificial, drug-induced indirect loss more than 100,000,000,000 yuan.
Antibacterial peptide (Anti-microbial and Virus Peptide, AMVP) is by 20～60 amino acid residue groups
Become, have antibacterial and antiviral bioactive peptide concurrently.From Boman at the end of the seventies in last century etc. first from cherishing discovery giant silkworm guppy giant silkworm
Since element, antibacterial peptide has attracted the concern of more and more experts and scholars because of the bactericidal mechanism of its uniqueness and excellent physicochemical property,
And become the ideal chose of safe green Replacer of Antibiotic Additive, before Animal husbandry production has wide application
Scape.
Cecropin A is from cherishing the antibacterial peptide with αhelix extracted guppy giant silkworm immunoblastic lymphoma, by 35
Amino acid residue forms, and has broad-spectrum antibacterial activity.Utilize fluorescence microscope, ultramicroscope and immune colloidal gold technique pair
Cecropin A carries out research and finds, after it acts on pathogenic microorganism, can form electromotive force and rely on microbial cell film
Type hole, causes Cytoplasm overflow, ultimately results in cell death, but its hemolytic also ratio is more serious, it is therefore desirable to by it and its
His antibacterial peptide is combined into new T1249 and uses.LL-37, relative molecular mass about 5000 dalton, is to find in human body so far
Antibacterial peptide (cathelicidin) family in unique member, be also uniquely there is in human body amphiphilic αhelix anti-
Bacterium peptide.In its blood cell being widely distributed in human body and epithelial cell, there is broad-spectrum antibacterial action.Antibacterial activity relies on its spiral shell
The formation of rotation conformation, kills antibacterial by " carpet sample " mechanism.Additionally, LL-37 also has the endotoxic effect of neutralization, can be with
LPS and CD14 combines, and neutralizes the biological activity of LPS；Utilize FPRL1 as receptor-mediated chemotaxis, recruit immunocyte and arrive
Reach infection site, remove pathogen；Promote the effects such as angiogenesis.
Along with going deep into antibacterial peptides structure, function with sterilization mechanism research, research worker begins attempt to utilize biology
Method design disinfection vitality is higher, the wider array of novel antimicrobial peptide of antimicrobial spectrum.A lot of research reports, by miscellaneous for difference antibacterial peptide molecule
Conjunction is the effective ways of the novel antibacterial peptide molecule obtaining high antibacterial activity low hemolytic.
Summary of the invention
It is an object of the invention to provide a kind of novel anti-bacteria and anti-virus hybrid polypeptide and preparation method and application.
In order to realize the object of the invention, the present inventor is to the sequence of Cecropin A and LL-37, the basis of structural analysis
On, carry out heterozygosis optimization, it is thus achieved that a kind of novel anti-bacteria and anti-virus hybrid polypeptide, named C-L.With source of parents PEPC ecropin
A, LL-37 compare, in addition to there is higher antibacterial action, and the most obvious antivirus action.The aminoacid sequence of described polypeptide
As shown in SEQ ID No.1, or this sequence amino acids formed has equal function through replacing, lack or add one or several
Aminoacid sequence.
Such as, the Leu of the 4th is replaced with Ile, or at its terminal deletion tri-aminoacid of Leu Arg Asn, the most not
The function of albumen can be caused too much influence.Therefore, the anti-bacteria and anti-virus hybrid polypeptide of the present invention also includes SEQ ID No.1
Shown in aminoacid sequence be substituted, replace and/or increase one or several aminoacid, formation have equal function by SEQ
The polypeptide that ID No.1 is derivative.
The present invention also provides for encoding the gene of described anti-bacteria and anti-virus hybrid polypeptide.The nucleotide sequence of described gene is such as
Shown in SEQ ID No.2.
Furthermore, it is to be understood that consider degeneracy and the Preference of different plant species codon, the art technology of codon
Personnel can select as required to use and be suitable for the codon that specific species are expressed.
The present invention also provides for the expression cassette containing coded polypeptide C-L gene or expression vector.
The present invention also provides for containing coded polypeptide C-L gene or above-mentioned expression cassette or the transgenic cell line of expression vector
And engineering bacteria.
The gene of coded polypeptide C-L can be operably connected with expression vector, build that obtain can express polypeptide C-L
Recombinant expression carrier, and then the such as transgenic method such as heat stress and electrotransformation can be passed through, described expression vector is imported
Host cell, obtains having imported the transformant of C-L gene, such as genetic engineering bacterium.
The present invention also provides for the method preparing described peptide C-L, the expression containing peptide C-L encoding gene built is carried
Body converts Host Strains, screening positive clone, abduction delivering target polypeptides, and isolated and purified expression product.
Preferably, the gene of coded polypeptide C-L is cloned into expression vector pPICZ α A, converts Pichia sp., the screening positive
Clone, then expresses target polypeptides, and isolated and purified expression product with methanol induction.
It is highly preferred that the gene of coded polypeptide C-L to be cloned into expression vector pPICZ α A, obtain recombinant expression carrier
PPICZ α A-C-L, is converted to Pichia pastoris GS115 by electrotransformation, expresses target polypeptides, expression product with methanol induction
Through Ni-NTA Sepharose chromatography, separate and obtain target polypeptides.
Using escherichia coli, staphylococcus aureus, Salmonella, sarcina as indicator bacteria, found that this polypeptide
These bacterium are respectively provided with killing action, additionally, show that peptide C-L all has significantly suppression to virus, common bacteria by mensuration
Effect, can develop new and effective Substitutes For Antibiotic.Peptide C-the L of the present invention can be used for preparing antibacterials, antiviral agents
Thing, feed additive, preservative or elimination agent etc..And then the present invention also provides for a kind of antibacterial containing effective dose peptide C-L
Medicine, antiviral drugs, feed additive, preservative or elimination agent etc..
Described peptide C-L is to including but not limited to staphylococcus aureus, Salmonella, sarcina, escherichia coli, shuttle
Shape bacillus cereus, yeast, ocean rhodotorula, aspergillus oryzae, Bacillus cercus, bacstearothermophilus, Gall's chain coccus, addicted to
Hot streptococcus, bargen's streptococcus, anthrax bacillus, Bacillus typhi, bacillus typhi murium, sonne bacillus, vibrio cholera, pneumonia bar
Bacterium has antibacterial effect in interior microorganism；To including but not limited to that influenza virus, reproductive and respiratory syndrome virus etc. have in interior virus
Antiviral effect.
The present invention is first by Cecropin A and LL-37 heterozygosis, and the peptide C-L obtained can improve fungistatic effect, and significantly
Reduce toxicity and hemolytic, become preferable Substitutes For Antibiotic, there is huge using value.
Use the inventive method can prepare described anti-bacteria and anti-virus hybrid polypeptide, obtained antibiosis in a large number, efficiently
Element succedaneum has the biological characteristics of broad-spectrum antiseptic, antiviral, and is not likely to produce drug resistance.
Accompanying drawing explanation
Fig. 1 is the structure flow chart of recombinant expression carrier pPICZ α A-C-L in the embodiment of the present invention 1.
Fig. 2 is the pcr amplification product of the corresponding genes of interest of peptide C-L in the embodiment of the present invention 1 (including histidine-tagged)
Gel electrophoresis results: wherein, M, DNA molecular amount standard；Fragment for the purpose of 1-5.
Fig. 3 be in the embodiment of the present invention 1 methanol induction express T1249 C-L purified after SDS-PAGE electrophoresis result；
Wherein, M: protein molecular weight standard；1-4 is respectively methanol induction 48h, 72h, 96h and 24h fermented supernatant fluid mesh after purification
PROTEIN C-L band；5 is matched group.
Fig. 4 is to observe peptide C-L in the embodiment of the present invention 4 under scanning electron microscope to escherichia coli CVCC245 and golden yellow Portugal
The bactericidal effect of grape coccus ATCC25923；Wherein, A1-A3 represent respectively T1249 C-L process escherichia coli CVCC245 0,2,
Thalli morphology structure chart after 4 hours, B1-B3 represent respectively T1249 C-L process staphylococcus aureus ATCC25923 0,
2, the thalli morphology structure chart after 4 hours.
Fig. 5 is the anti-bacteria and anti-virus hybrid polypeptide C-L stability to simulation gastro-intestinal Fluid in case study on implementation 6 of the present invention；Wherein A
It is staphylococcus aureus ATCC43300 for staphylococcus aureus CVCC1882, B；In figure, 1 is the inhibition zone of experimental group, and 2 are
The inhibition zone of matched group.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
All according to conventional laboratory conditions, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or the condition according to manufacturer's description suggestion.
Competent escherichia coli cell Top 10, Pichia pastoris GS115 and the expression vector used in following example
PPICZ α A is purchased from Invitrogen company.
The preparation of embodiment 1 anti-bacteria and anti-virus hybrid polypeptide C-L
By Pichia pastoris GS115 list colony inoculation in 3-5ml YPD (2% tryptone, 2% glucose, 1% yeast powder,
PH 7.0) in fluid medium, shaken cultivation about 12 hours at 30 DEG C, it is inoculated in 100ml by the volume ratio of 1:100-1:50
In YPD fluid medium, 30 DEG C of shaken cultivation to OD600About=1.3-1.5, stops cultivating.Culture fluid is proceeded to centrifuge tube
In, place on ice 10 minutes, then at 4 DEG C, 1500g is centrifuged 5 minutes and collects thalline.The sterilized water of pre-cooling is added in thalline
Suspension cell gently, after placing 15-30 minute on ice, is centrifuged 5 minutes in 4 DEG C of 1500g.Supernatant discarded, adds 10ml pre-cooling 1M
Aseptic sorbitol solution, resuspended thalline, i.e. make competent cell suspension.Often pipe 200 μ l is distributed into standby Pichia sp.
Competent cell, uses immediately or is stored in-70 DEG C.
According to peptide C-L aminoacid sequence (SEQ ID No.1) and Pichia sp. codon preference, design and synthesize polypeptide
The gene order (SEQ ID No.2) of C-L, by being connected (Fig. 1) with expression vector pPICZ α A, builds the large intestine bar of this polypeptide
Bacterium recombinant expression carrier pPICZ α A-C-L, then electricity converts escherichia coli Top 10 competent cell.
Extract recombiant plasmid according to the operational approach of the little extraction reagent kit of TIANGEN Biotech's plasmid, make
Carry out PCR for template and expand C-L gene, forward primer: 5 '-GCGAGAAAAGAAAGTGGAAGT-3 '；Downstream primer: 5 '-
GAATAATGGTGATGGTGATGGT-3′.Amplified production carries out agarose gel electrophoresis, observes under uviol lamp, identifies and converts knot
Really, show purpose band and there is (Fig. 2).Order-checking identifies that the insertion of purpose fragment is the most correct further, and checks insertion sheet
The fidelity of section.Successfully construct through verifying this antibacterial polypeptide Recombinant protein expression carrier pPICZ α A-C-L.
The Plastid transformation Pichiapastoris expression strain GS115 that will extract in positive colony, method is as follows:
Linearizing recombiant plasmid 5-10 μ g and 200 μ l has activated the mixing of Pichia pastoris GS115 competent cell, moves into electricity
Revolving cup, ice bath 5-30min, after ice bath, electricity turns some seconds (electricity never vibrates during turning), then rapidly mixed liquor is placed in ice
On, it is rapidly added the 1M sorbitol of 1ml ice bath, makes thalline suspension mixing go to, in 1.5ml EP pipe, be placed on ice, be subsequently adding
2ml YPD fluid medium, cultivates 3h, makes bacterial strain recover and form resistance for 30 DEG C.Bacterium to be reorganized takes 200 μ l trainings after forming resistance
The nutrient solution coating YPDS flat board containing 100 μ g/ml kanamycin, is then inverted flat board in 30 DEG C of incubated overnight.Select above-mentioned YPDS
The single colony inoculation grown on flat board is incubated overnight in the YPD fluid medium containing 100 μ g/ml kanamycin, and plasmid is little to be carried
Middle amount test kit extracts plasmid, then carries out PCR checking.
Select positive colony to be inoculated in the BMGY culture medium containing 100 μ g/ml kanamycin, 30 DEG C, 180-200rpm shakes
Bed is cultivated to OD600During for 2-6, low-speed centrifugal collects thalline, with the resuspended thalline of BMMY culture medium to OD600Be 1,30 DEG C,
200rpm shaken cultivation, adds methanol to final concentration of 5% abduction delivering every 24h.Respectively methanol induction 0h, 24h, 48h,
Taking 2ml bacterium solution when 72h, 96h, 12000rpm is centrifuged 5min, collects-20 DEG C of preservations after supernatant.Make with the supernatant without induction
For negative control.The supernatant of collection is carried out Tricine-SDS-PAGE electrophoresis, and electrophoresis result has demonstrated T1249 C-L table
Reaching, T1249 expression can reach about 190.65mg/L after measured.
During due to design C-L gene order, with the addition of histidine-tagged (6 × His), the purpose therefore expressed at its C end
Fermented supernatant fluid can be washed with variable concentrations imidazole elution by albumen with Ni ions binding in Ni-NTA Sepharose chromatographic column
Take off and eluting peak sample is collected, the isolated and purified of destination protein can be realized.The Ni-NTA Sepharose of destination protein
Chromatography method, with reference to product operation instruction, collects each eluting peak sample, by Tricine-SDS-PAGE electrophoresis detection
Purification effect (Fig. 3).
The heat stabilization test of embodiment 2 peptide C-L
In Example 1, each 200 μ l of the recombiant protein of purification are under 40 DEG C, 60 DEG C, 80 DEG C and 100 DEG C of water bath condition
Process 10 respectively, 20,30min, with untreated recombiant protein as comparison, with standard gold Staphylococcus aureus as indicator bacteria,
The fungistatic effect of sample before and after processing by antibacterial circle diameter method mensuration.Experiment is repeated 3 times, and takes its meansigma methods as measurement result
(table 1).
The restructuring destination protein C-L antibacterial circle diameter to staphylococcus aureus after table 1 heat treated
Result shows, through the process of different heating temperature and time, the impact on restructuring destination protein fungistatic effect is
Different, when heating-up temperature is less than 60 DEG C, along with the prolongation of heat time heating time, its antibacterial circle diameter is not changed in substantially；When
Heating-up temperature is increased to 80-100 DEG C, and its antibacterial circle diameter increases with temperature and time lengthening slightly reduces, but still shows good
Good fungistatic effect.
The embodiment 3 peptide C-L inhibitory action to common bacteria
Restructuring destination protein solution (50 μ g/ will be placed in through the filter paper (diameter 6mm) of dry heat sterilization (160 DEG C, 2h)
Ml) fully soaking 24h in, dip for examination bacterium solution with aseptic spreader, the surface being coated in culture medium equably is made containing bacterium flat board,
Taking out the filter paper through fully soaking to be attached to containing on bacterium flat board, each culture dish pastes 1, is placed in the constant incubator of 37 DEG C training
Support 24h, observe and measure the diameter (table 2) of inhibition zone.
Table 2 T1249 C-L antibacterial circle diameter to each common bacteria
Note: CVCC refers to veterinary microorganism culture presevation administrative center of China；This laboratory refers to that China Agricultural University raises
Material Biotechnology Experiment room.
From table 2 it can be seen that antibacterial hybrid polypeptide C-L is to escherichia coli, staphylococcus aureus, Bacillus cercus etc.
Pathogenic bacterium have stronger inhibitory action, and also have inhibition in various degree for fungus such as yeast, mycete etc., but right
The least in the inhibitory action of probiotic bacteria such as Bacillus subtillis, Bacillus coagulans, Lactobacillus plantarum, or the most do not suppress to make
With.
The bactericidal effect of peptide C-L is observed under embodiment 4 scanning electron microscope
By escherichia coli CVCC245 and staphylococcus aureus ATCC25923 incubated overnight to exponential phase, with cultivation
Base dilution bacterium solution is to 0.5 Maxwell colorimetric unit (about 108CFU/ml), then peptide C-L is joined in the bacterium solution after dilution
To its final concentration of 1 × MIC, 37 DEG C of 180rpm shaken cultivation, taking mixed liquor 4ml respectively at 0h, 2h, 4h, 12000rpm is centrifuged
10min, abandons supernatant, cleans bacterial sediment 3 times with PBS, adds the 500 resuspended fixing thalline of μ l 2.5% glutaraldehyde, 4 DEG C
Overnight.Having fixed rear PBS to rinse 3 times, each 10min, osmic acid secondary fixes 2h；Flush three times with PBS again, graded ethanol
(50%-100%) dehydration, each 15min, finally process 2 times with dehydrated alcohol, process 15min every time；Process by acetone treatment
30min；Sample is overnight processed with pure embedding medium；70 DEG C overnight add thermal polymerization, cut into slices with Lycra ultrathin section instrument；Use acetic acid dioxygen
Uranium 50% saturated solution dyeing 15min～1h, distilled water cleans to dry and is placed under Hitachi's H-7650 transmission electron microscope observation.
From fig. 4, it can be seen that the escherichia coli processed without antibacterial peptide and aureus cell smooth surface, present
Complete shaft-like and botryoidal structure；And after heterozygous antibacterial peptide C-L processes 2h, escherichia coli and aureus cell
Film surface all occurs in that a lot " hole ", and along with the prolongation of action time, after processing 4h, can see a lot of thin in the visual field
The form of born of the same parents changes, some cell indents, flat state occurs；There is filament in some cell surfaces；Even a lot of thalline are thin
Born of the same parents have been cracked into substantial amounts of fragment.These results disclose somatic cells surface " hole " and formed is to cause somatic cells to split
Solution, main causes of death, be also the major way of heterozygous antibacterial peptide C-L performance bacteriostasis.
The embodiment 5 peptide C-L inhibitory action to virus
1, drug cytotoxicity experiment (LD0 mensuration)
Peptide C-L is carried out the dilution of 2 times of Concentraton gradient, totally 8 dilution factors, i.e. 100,50,25,12.5,6.25,3.125,
1.563 and 0.782 μ g/ml.Take each dilution factor liquid 0.2ml and add in cell pipe (each dilution factor 3 is managed), cultivate 7 days for 37 DEG C, see
Examine Marc-145 cytopathy.Result shows that disease does not occurs in peptide C-L the 2nd pipe (50 μ g/ml) to the 8th pipe (0.782 μ g/ml)
Become, therefore LD0=100.
2, virus infective dose titration (TCID50 mensuration)
(1) influenza virus 10 times dilution, totally 3 dilution factors, i.e. 10-1、10-2、10-3.Each dilution factor virus liquid 0.2ml adds
Enter (every dilution factor 5 is managed) in cell pipe, cultivate 7 days for 37 DEG C, observe Marc-145 cytopathy.Calculate by Reed-Muench method
Result: TCID50=10-5.7。
(2) reproductive and respiratory syndrome virus 10 times dilution, totally 3 dilution factors, i.e. 10-1、10-2、10-3.Every dilution factor virus liquid 0.2ml
Add (each dilution factor 5 is managed) in cell pipe, 37 DEG C hatch 1h after suck virus liquid, hand ' s liquid is washed 3 times, adds and maintains liquid, 37
DEG C cultivate 7 days, observe Marc-145 cytopathy.By Reed-Muench method result of calculation: TCID50=10-4.6。
Embodiment 6 peptide C-L stability in simulating gastrointestinal digestion liquid
The supernatant containing anti-bacteria and anti-virus hybrid polypeptide taking 0.5mL embodiment 1 preparation adds to the people of 5mL pH value 2.5
In work simulated gastric fluid, it is placed in 37 DEG C of water-baths, lucifuge insulation digestion 3h, then adjusts pH value with 0.1mol/L sodium hydroxide solution
To 6.8, add 5mL artificial simulation intestinal juice, be placed in 37 DEG C of water-baths, lucifuge insulation digestion 4h, after digestion completes, rapidly
Cooling.Add with the isopyknic normal saline of Digestive system as a control group simultaneously.
Process with staphylococcus aureus ATCC43300 and staphylococcus aureus CVCC1882 for indicator bacteria observation respectively
The activity change of hybrid polypeptide C-L front and back.Draw 0.2mL staphylococcus aureus ATCC43300 and Staphylococcus aureus respectively
The bacteria suspension of bacterium CVCC1882 is spread evenly across media surface；4 Oxford cups equidistantly put into by each flat board, and each Oxford cup adds
Enter the hybrid polypeptide C-L 200 μ L of respective handling, make 3 flat boards and repeat.It is placed in constant incubator, cultivates 24h for 37 DEG C.Take out
Oxford cup, measures the antibacterial circle diameter with T1249 C-L adding normal saline process after gastro-intestinal Fluid digests respectively, compares it
Activity change.
Anti-bacteria and anti-virus hybrid polypeptide C-L after simulation gastro-intestinal Fluid digestion to the fungistatic effect of staphylococcus aureus such as
Shown in Fig. 5.Result shows, hybrid polypeptide C-L all can retain the Antibacterial Activity of about 90% (table 3) for two strain indicator bacterias, says
This polypeptide bright has higher stablizing in Imitative gastroenteric environments (such as pepsin, trypsin, highly acid pH value 2.5)
Property.
Table 3 anti-bacteria and anti-virus hybrid polypeptide C-L is antibacterial circle diameter after simulation gastro-intestinal Fluid processes
By tables such as high temperature resistant test, protest test, antibacterial experiment, antivirus test, the liquid of resistance to gastro-intestinal digestion tests
Bright, the anti-bacteria and anti-virus hybrid polypeptide C-L of the present invention is a kind of stable broad spectrum antimicrobial peptide, and has gram positive bacteria
Stronger lethality, this is probably what the kind by antibacterial peptide and characteristic determined.Meanwhile, although antibacterial polypeptide has broad-spectrum antiseptic
Activity, but it is for different microorganism bacterium the most of the same race not homophyletics, there may be difference in killing-efficiency.
Experiment shows, the derivant of peptide C-L also has and the anti-bacteria and anti-virus function of peptide C-L approximation, such as, will be many
The Leu that PEPC-L is the 4th replaces with Ile, or the polypeptide obtained at its terminal deletion tri-aminoacid of Leu Arg Asn derives
Thing.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. an anti-bacteria and anti-virus hybrid polypeptide, it is characterised in that the aminoacid sequence of described polypeptide such as SEQ ID No.1 institute
Show, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with equal function.
2. the gene of polypeptide described in coding claim 1.
3. gene as claimed in claim 2, it is characterised in that its nucleotide sequence is as shown in SEQ ID No.2.
4. contain expression cassette or the expression vector of gene described in Claims 2 or 3.
5. contain gene described in Claims 2 or 3, or expression cassette described in claim 4 or the engineering bacteria of expression vector.
6. the preparation method of polypeptide described in claim 1, it is characterised in that the expression vector described in claim 4 is converted place
Main bacterium, screening positive clone, abduction delivering target polypeptides, and isolated and purified expression product.
Method the most according to claim 6, it is characterised in that gene described in claim 3 is cloned into expression vector
PPICZ α A, converts Pichia sp., screening positive clone, then expresses target polypeptides with methanol induction, and isolated and purified expression is produced
Thing.
8. polypeptide described in claim 1 is in preparation antibacterials, antiviral drugs, feed additive, preservative or elimination agent
Application.
Application the most according to claim 8, it is characterised in that described polypeptide is to including staphylococcus aureus, Salmonella
Bacterium, sarcina, escherichia coli, clostridium, yeast, ocean rhodotorula, aspergillus oryzae, Bacillus cercus, thermophilic fat
Fat bacillus cereus, Gall's chain coccus, streptococcus thermophilus, bargen's streptococcus, anthrax bacillus, Bacillus typhi, bacillus typhi murium, Song Nei Shi
Dysentery bacterium, vibrio cholera, pneumobacillus have antibacterial effect in interior microorganism；To including influenza virus, reproductive and respiratory syndrome virus
In interior virus, there is antiviral effect.
10. contain the antibacterials of polypeptide described in claim 1, antiviral drugs, feed additive, preservative or elimination agent.
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| CN110066342A (en) * | 2019-04-03 | 2019-07-30 | 中国农业大学 | It is a kind of to clear up endotoxin and the hybrid peptide of anti-inflammatory properties and the preparation method and application thereof with immunological regulation, neutralization |
| CN110128548A (en) * | 2019-05-23 | 2019-08-16 | 中国农业大学 | One kind, which has both, adjusts immune, anti-oxidant, anti-inflammatory and multi-functional hybrid peptide of removing toxic substances and the preparation method and application thereof |
| CN110305222A (en) * | 2019-06-06 | 2019-10-08 | 中国农业大学 | It is a kind of to have both removing toxic substances, anti-inflammatory, anti-apoptotic, protection gut barrier and the hybrid peptide and its application that promote wound healing |
| CN110526960A (en) * | 2018-05-24 | 2019-12-03 | 中国农业大学 | One antiviral polypeptide and the preparation method and application thereof |
| CN111848817A (en) * | 2020-07-28 | 2020-10-30 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, antiviral, immunoregulatory and anti-inflammatory activities, and preparation method and application thereof |
| CN111944060A (en) * | 2020-07-29 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, anti-inflammatory and detoxifying activities and application thereof |
| CN112480230A (en) * | 2020-11-30 | 2021-03-12 | 青岛农业大学 | Antibacterial peptide with better heat resistance function and application thereof |
| CN113527432A (en) * | 2021-07-08 | 2021-10-22 | 山东省健牧生物药业有限公司 | Polypeptide for resisting H9N2 subtype avian influenza virus and application thereof |
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| CN110526960A (en) * | 2018-05-24 | 2019-12-03 | 中国农业大学 | One antiviral polypeptide and the preparation method and application thereof |
| CN110066342A (en) * | 2019-04-03 | 2019-07-30 | 中国农业大学 | It is a kind of to clear up endotoxin and the hybrid peptide of anti-inflammatory properties and the preparation method and application thereof with immunological regulation, neutralization |
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| CN110128548A (en) * | 2019-05-23 | 2019-08-16 | 中国农业大学 | One kind, which has both, adjusts immune, anti-oxidant, anti-inflammatory and multi-functional hybrid peptide of removing toxic substances and the preparation method and application thereof |
| CN110128548B (en) * | 2019-05-23 | 2020-12-01 | 中国农业大学 | Hybrid peptide with functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and preparation method and application thereof |
| CN110305222A (en) * | 2019-06-06 | 2019-10-08 | 中国农业大学 | It is a kind of to have both removing toxic substances, anti-inflammatory, anti-apoptotic, protection gut barrier and the hybrid peptide and its application that promote wound healing |
| CN111848817A (en) * | 2020-07-28 | 2020-10-30 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, antiviral, immunoregulatory and anti-inflammatory activities, and preparation method and application thereof |
| CN111944060A (en) * | 2020-07-29 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, anti-inflammatory and detoxifying activities and application thereof |
| CN112480230A (en) * | 2020-11-30 | 2021-03-12 | 青岛农业大学 | Antibacterial peptide with better heat resistance function and application thereof |
| CN113527432A (en) * | 2021-07-08 | 2021-10-22 | 山东省健牧生物药业有限公司 | Polypeptide for resisting H9N2 subtype avian influenza virus and application thereof |
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Application publication date: 20160928 |