CN108794635A - A kind of Bovine lactoferricin-human lysozyme fusion protein, gene and its application - Google Patents
A kind of Bovine lactoferricin-human lysozyme fusion protein, gene and its application Download PDFInfo
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Abstract
The invention discloses a kind of Bovine lactoferricin-human lysozyme fusion proteins, it is characterised in that the fusion protein is followed in series to form by pig fribrillin anti-oxidation peptide, Bovine lactoferricin, chicken egg white anti-oxidation peptide, pig myogen myosin anti-oxidation peptide, flexible peptide linker, human lysozyme.By the anion anti-oxidation peptide of amalgamation and expression animal origin, the positive charge of antibacterial peptide is offset, mitigates inhibition of the antibacterial peptide to host strain；Anti-oxidation peptide is conducive to increase the storage stability of antibacterial peptide, and 4 DEG C of the fusion protein is placed in 20d only declines 7.7% to Escherichia coli (ATCC 25922) bacteriostasis antibiosis vigor.It only loses 15.4% to the vigor of Escherichia coli (ATCC 25922) after 30d.
Description
(1) technical field
The present invention relates to a kind of Bovine lactoferricin-human lysozyme, gene and its applications.
(2) background technology
The abuse of medical treatment and animal-breeding field antibiotic causes the prevalence of drug-resistant bacteria, and serious prestige is caused to human health
The side of body.There is an urgent need to research and develop novel antibacterial drug or feed addictive to substitute antibiotics.Antibacterial peptide and lysozyme are a kind of
Natural polypeptides with bactericidal activity and albumen are widely used to clinical medicine and food work as Substitutes For Antibiotic
Industry.
The Antibacterial Mechanism of antibacterial peptide is that positively charged antibacterial peptide is combined with the bacterial cell membrane with negative electrical charge, by making
Film forming perforation inhibits the physiological metabolism process of bacterium to cause bacterial death.Bovine lactoferrin N-terminal is containing there are two by protease
Digest the antibacterial structure domain lactoferricin (LFC) 17-30 generated and lactoferrampin (LFA) 265-284.Bacteriolyze
Enzyme plays its bactericidal effect by decomposing β-(1,4) glycosidic bond of peptide glycan on cell wall.Lactoferrin antimicrobial peptide and lysozyme
With good thermal stability, especially in acid condition, heating even high temperature high pressure process does not have shadow to its antibacterial activity
It rings, is good conventional antibiotic substitute.
Chinese invention patent CN101649311B discloses a kind of preparation method of human lysozyme-antibacterial peptide fusion protein,
It builds human lysozyme-antibacterial peptide recombinant plasmid using the method for genetic engineering, then in eukaryotic expression human lysozyme-antibacterial peptide
Fusion protein.However since lysozyme and antibacterial peptide usually carry cationic positive charge, may occur with the DNA with anion
Interaction there is certain cytotoxicity, the form of generally use fusion protein to be expressed genetic engineering host strain, with
Weaken the cytotoxicity.It is flexible that Chinese invention patent CN105061603A discloses a kind of antibacterial peptide thanatin albumen, 3GSA
Peptide, T4 Lysozyme albumen hybrid protein preparation method, it melts hybrid protein in Escherichia coli with Sumo labels
Expression is closed, later purified fusion albumen, cuts off Sumo labels, obtain purpose antibacterial peptide-lysozyme fusion protein.Li Qing and Zhou Xiao
The DNA sequence dna of macro (Food Science 2013) artificial synthesized coding polycation antibacterial peptide and polyanion peptide fusion protein,
And it is expressed in saccharomycete.However fusion protein used in both methods itself does not help final product function, obtains
To product need to post-process, purifying cost it is too high.
(3) invention content
It is an object of the present invention to provide a kind of lactoferricin-human lysozyme hybrid protein and applications, and product is without cracking, i.e.,
Have antibacterial activity more stronger than lactoferricin and human lysozyme and storage stability.
The technical solution adopted by the present invention is：
The present invention provides a kind of lactoferricin-human lysozyme fusion protein, and the fusion protein is by pig muscle fibril
Protein antioxidant peptide (SEQ ID NO.1), Bovine lactoferricin (SEQ ID NO.4), chicken egg white anti-oxidation peptide (SEQ ID
NO.2), pig myogen myosin anti-oxidation peptide (SEQ ID NO.3), flexible peptide linker (SEQ ID NO.7), human lysozyme
(SEQ ID NO.5) is followed in series to form, wherein pig fribrillin anti-oxidation peptide (SEQ ID NO.1), bovine lactoferrin
DPNG catenation sequences, Bovine lactoferricin (SEQ ID NO.4), chicken egg white antioxygen are connected between peptide (SEQ ID NO.4)
NGDPE sequences, flexible peptide linker (SEQ ID NO.7) and human lysozyme (SEQ ID are connected between change peptide (SEQ ID NO.2)
NO.5 EDPNG sequences (a in Fig. 1) are connected between).NGDPE and EDPNG between above-mentioned sequence be be easy to by acid, azanol and
The amino acid sites of pepsin hydrolysis.
Further, the amino acid sequence of the fusion protein is shown in SEQ ID NO.6.
The present invention also provides a kind of Bovine lactoferricin-human lysozyme fusion protein encoding gene, the coding bases
The nucleotides sequence of cause is classified as shown in SEQ ID NO.8.
Moreover, it relates to which a kind of Bovine lactoferricin-human lysozyme fusion protein is preparing antiseptic feed
Application in additive.
Further, the antiseptic feed additive is additive for farm animal feed, the more preferably described antiseptic feed additive
For additive for feed for piglets.
Compared with prior art, the present invention has following remarkable advantage and advantageous effect：
(1) by the anion anti-oxidation peptide of amalgamation and expression animal origin, the positive charge of antibacterial peptide is offset, mitigates antibacterial peptide
Inhibition to host strain；Anti-oxidation peptide is conducive to increase the storage stability of antibacterial peptide, and 4 DEG C of the fusion protein places suppression in 20d
Bacterium antibacterial activity only declines 7.7%.It only loses 15.4% to the vigor of Escherichia coli (ATCC 25922) after 30d.
(2) Pichia anomala expression obtains the fusion protein of Bovine lactoferricin and human lysozyme, has without cracking anti-
Bacterium activity, and antibacterial activity is more stronger than Bovine lactoferricin and human lysozyme, antimicrobial spectrum is wider.
(3) right after pepsin extracorporeal hydrolysis in fusion protein sequence containing easily by the site of acid and pepsin hydrolysis
The bacteriostatic activity of Escherichia coli and staphylococcus aureus is respectively increased 38.9% and 18.5%, therefore it is as feed addictive
Bacteriostatic activity can enhance after being cracked into animal alimentary canal.
(4) the Pichia pastoris product containing the fusion protein can improve piglet growth performance, reduce grice diarrhoea rate.
(4) it illustrates
Fig. 1 is the structure schematic diagram (b) of fusion protein sequence (a) and plasmid.Seq.1 and 3 is pig fribrillin in a
Anti-oxidation peptide, Seq.4 are Bovine lactoferricin, and Seq.2 is chicken egg white anti-oxidation peptide, and Seq.7 is flexible peptide linker,
Seq.5 is human lysozyme sequence, and the NGDPE and EDPNG between above-mentioned sequence are easy to by acid, azanol and pepsin hydrolysis
Amino acid sites.
Fig. 2 is 1 tunning inhibition zone of embodiment.A is Escherichia coli (ATCC 25922) inhibition zone, and b is golden yellow Portugal
Grape coccus (ATCC 25923) inhibition zone.The recombination fermented liquid of 1 α of pGAPZ containing empty plasmid；2P.pastoris GS115 bacterial strains
Zymotic fluid；The zymotic fluid of 3 and 4 recombinant bacteriums；
Fig. 3 is that the sample at different fermentations time point carries out SDS-PAGE electrophoresis.
Fig. 4 is to crack and the cycle after dialysing prepares chromatogram, and No. 1 peak is uncracked LfcinB-hLY fusion proteins, 2
Number peak is human lysozyme (hLY), and No. 3 peaks are Bovine lactoferricin (LfcinB).
Fig. 5 is the Tricine-SDS-PAGE protein adhesives of different samples.M：Marker；1：Sample before cracking, i.e. Fig. 3's
The electrophoretogram at No. 1 peak；2：The electrophoretogram of No. 2 peak eluents in Fig. 3；3：The electrophoretogram of No. 3 peak eluents in Fig. 3.
Fig. 6 LfcinB-hLY fusion proteins are after different temperatures and time-triggered protocol to Escherichia coli (ATCC 25922)
Antibacterial circle diameter.
Fig. 7 LfcinB-hLY fusion proteins are after different pH processing to Escherichia coli (ATCC 25922) bacteriostatic activity.
Fig. 8 LfcinB-LhY fusion proteins bacteriostatic activity after different digestion enzymatic treatments.
The storage stability of Fig. 9 LfcinB-hLY fusion proteins.
(5) specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
1 Bovine lactoferricin of embodiment-fusion table of the human lysozyme hybrid protein (LfcinB-hLY) in Pichia pastoris
It reaches
Pig fribrillin anti-oxidation peptide (SEQ ID NO.1), DPNG catenation sequences, Bovine lactoferricin (SEQ ID
NO.4), NGDPE catenation sequences, chicken egg white anti-oxidation peptide (SEQ ID NO.2), pig myogen myosin anti-oxidation peptide
(SEQ ID NO.3), flexible peptide linker (SEQ ID NO.7), EDPNG catenation sequences, human lysozyme (SEQ ID NO.5) are successively
Bovine lactoferricin in series-human lysozyme hybrid protein (SEQ ID NO.6) (a in Fig. 1).According to Pichia pastoris codon
LfcinB-hLY hybrid protein gene coded sequence SEQ ID NO.8 after optimization transfer to gene chemical synthesis company to carry out full genome
Synthesis.
The DNA sequence dna of SEQ ID NO.8 is cloned on plasmid pGAPZ α (b in Fig. 1), uses restriction enzyme Bln
After I carries out plasmid linearization, electrotransformation Pichia pastoris (P.pastoris) GS115 bacterial strains (Invitrogen companies) are contained
The recombinant bacterium of LfcinB-hLY hybrid protein genes.The bacterium solution of 100 μ L recombinant bacteriums is taken to be spread evenly across containing 100 μ g/mL Zeocin
YPDS solid plates on, 30 DEG C of constant temperature incubations 2 days.By the positive recombinant GS-LfcinB-hLY confirmed by PCR in YPD
30 DEG C of cultures are used as seed culture fluid for 24 hours in culture medium.Seed liquor is accessed into fermentation medium with 10% (v/v) inoculum concentration,
200rpm, 30 DEG C of cultures to 72h.Fermented liquid supernatant is collected, measures bacteriostatic activity, and use 10%SDS-PAGE detection expression productions
Object.Under similarity condition, turn P.pastoris GS115's with the plasmid pGAPZ α electricity without LfcinB-hLY hybrid protein genes
Supernatant and P.pastoris GS115 fermented supernatant fluids without LfcinB-hLY hybrid proteins gene and plasmid pGAPZ α
For control.
YPDS solid plate quality final concentrations form：1% yeast powder, 2% peptone, 2% glucose, 18.2% sorb
Alcohol, 2% agar powder, solvent are deionized water, and pH value is natural.When cultivating the Pichia pastoris host containing target gene, it is added dense eventually
Degree is the Zeocin resistances of 100 μ g/mL.
YPD culture medium quality final concentrations form：1% yeast powder, 2% peptone, 2% glucose, solvent are deionized water,
PH value is natural.
Fermentation medium quality final concentration forms：5% glucose, 0.5% peptone, 1%NH4AC, 2% yeast powder,
0.3%KH2PO4, 0.03%MgSO4, 0.05%MnSO4, 0.05%CaCl2, solvent is deionized water, and pH value is natural.
Fusion protein Antibacterial Activity detects：Escherichia coli Escherichia coli (ATCC 25922) and gold are selected respectively
Staphylococcus aureus Staphylococcus aureus (ATCC 25923) single bacterium is fallen in LB liquid medium, 37 DEG C,
Taking out to survey OD600 and adjusted with sterile water after 200rpm shaking table cultures 12-16h makes the OD600 of bacterium solution be 0.6 or so.Xiang Weining
Solid solid LB media in, according to bacterium solution：Culture medium=1 LB：E.coli bacterium solutions are added in 1000 volume ratios, gently shake up,
It pours into rapidly afterwards in the tablet after sterilizing, pouring volume is 25mL or so, thickness 4-5mm, and standing 20-30min makes its solidification.It waits for
After solidification, 4 holes are made a call to the midpoint of culture medium upper edge culture medium radius is equidistant with the card punch of a diameter of 8mm after sterilizing.
The fermented liquid supernatant 100uL for measuring its albumen concentration by Coomassie Brilliant Blue is taken, is added in the hole of culture medium, is placed on 37
DEG C constant temperature biochemical cultivation case culture 8-12h.Each sample makees 3 tablets, separately takes a tablet that sterile water is added to make blank control.
As shown in Fig. 2, empty plasmid and P.pastoris GS115 bacterial strain fermentation liquors supernatants that progress electricity turns are not shown
The strain fermentation supernatant of antibacterial activity, GS-LfcinB-hLY recombinant plasmids is shown to Escherichia coli and staphylococcus aureus
Antibacterial activity, antibacterial circle diameter is respectively 15mm and 25mm.Using methanol extraction method concentration different time points (72h, 68h,
64h, 56h, 52h, 44h, 36h and 28h) fermented sample, obtain the SDS-PAGE electrophoresis of different sample points, as shown in figure 3,
Ferment 64h after, can gradually see doubtful band, doubtful item is gradually deepened as time increases, this also with detect antibacterial peptide
Activity present positive correlation, illustrate that foreign protein is expressed.
The antibacterial potency assay method of fusion protein is dilution 2nTimes without inhibition zone, then potency AU：2n× 0.1 × 1/c,
Middle n is the extension rate of minimal inhibitory concentration, and 0.1 is that the volume 0.1mL, c of fusion protein is added as the concentration of fusion protein, list
Position is mg/mL.Potency unit is AU/mg.Mole potency is defined as：AU/M.Wherein M is the relative molecular mass (kD) of sample.
According to the inoculum concentration inoculation recombinant bacterium seed liquor extremely 5L fermentation tanks containing 3L fermentation mediums of volumetric concentration 8%.Oxyty
Setting 20% doses supplemented medium 10mL/h after inoculation for 24 hours.It waits for that biomass is not further added by after 72h, terminates fermentation.Feed supplement is trained
Supporting matrix amount final concentration group becomes 5% glucose, 2% yeast powder, and solvent is deionized water, and pH value is natural.Measure foreign protein
Expression quantity is 35mg/L, and fermented liquid supernatant antibacterial circle diameter reaches 41mm, potency 11.66AU/mg.
2 fusion protein LfcinB-hLY's of embodiment isolates and purifies
(1) zymotic fluid that embodiment 1 obtains passes through ion exchange resin SP Sepharose Fast after centrifugation
Flow is purified, and column volume 20mL, UV Detection wavelength is 220nm and 280nm.5mM acetic acid-acetic acid of flow velocity 1.5mL/min is received slow
Fliud flushing (pH 4.5) balances pillar.By fermented liquid supernatant 50mL loadings.After completion of the sample, balanced again using above-mentioned buffer solution
Pillar.After balance, it is used as and is washed using the 50mM acetic acid-sodium acetate buffer solutions (pH 4.5) of the NaCl of 0.2mol/L containing concentration
De- liquid is eluted, flow velocity 1.5mL/min.After having collected all eluting peaks, antibacterial activity is measured, obtains that there is antibacterial activity
Fusion protein LfcinB-hLY samples.The above-mentioned eluting peak for having antibacterial activity obtained after cation-exchange chromatography is freezed
Drying is concentrated into 1mL, as fusion protein LfcinB-hLY crude products, potency 17.62AU/mg.The product is on the one hand into traveling
One step purifies, and on the other hand carries out the hydroxylamine hydrochloride cracking in embodiment 3.
(2) it is further purified by recycling preparative chromatograph, filler JAIGEL-GS310；Sample size is 10mL；Flow velocity
For 5.0mL/min；UV Detection wavelengths are 280nm.Mobile phase is 5mmol/L sodium acetates (pH 4.5).After collecting all eluting peaks,
Antibacterial activity is measured, the eluting peak with antibacterial activity is obtained.The antibacterial potency of eluting peak is 39.68AU/mg, as merges egg
White LfcinB-hLY sterlings.
The hydroxylamine hydrochloride of 3 fusion protein LfcinB-hLY of embodiment cracks
Hydroxylamine cleavage buffer solution is that 25.80g hydroxylamine hydrochlorides and 4.8g Tris are dissolved in 100mL distilled waters, with 4M NaOH
Regulated value pH 9.0 is simultaneously dissolved to 200mL.
The dry hydroxylamine cleavage site (Asn-Gly) of promise is devised in fusion protein sequence.To 2 step of embodiment (1) by sun
The hydroxylamine cleavage buffer solution of two volumes, 45 DEG C of water-baths are added in fusion protein LfcinB-hLY samples after ion-exchange chromatography
After 4h, reacting liquid pH value is adjusted to 7 or so using hydrochloric acid and reduces reaction temperature to room temperature, terminates cracking reaction.Then use
1KD bag filters remove azanol and small-molecular peptides.It takes the permeate after dialysis treatment to be purified by recycling preparative chromatograph, grasps
Make consistent with embodiment 2.As shown in figure 4, No. 1 absorption peak near 21min, No. 2 absorption peaks near 24min are attached in 28min
Nearly No. 3 absorption peaks.According to the principle of the isolated polypeptide of cycle preparative chromatograph, three absorption peaks correspond to uncracked respectively
LfcinB-hLY fusion proteins, human lysozyme (hLY), Bovine lactoferricin (LfcinB).
Antibacterial activity will be measured after the eluent concentration of three absorption peaks.The eluent of above-mentioned No. 1 absorption peak is uncracked
LfcinB-hLY fusion proteins, No. 2 absorption peaks are hLY, and No. 3 absorption peaks are LfcinB.It is measured according to the method in embodiment 1
The front and back potency for inhibiting E.coli K 88 of fusion protein LfcinB-hLY cracking.Measurement result LfcinB-hLY (39.68AU/
Mg), hLY (17.3AU/mg), LfcinB (6.6AU/mg), the antibacterial potency of fusion protein LfcinB-hLY are respectively higher than independent
HLY and LfcinB.
The eluent that cycle prepares chromatography is collected, the protein sample after methanol extraction concentrates carries out Tricine-SDS-
PAGE protein electrophoresis.The methanol extraction method of protein is that 4 times of volumes, which are added, to albumen to be precipitated is pre-chilled pure methanol, 2 times of volumes
The distilled water of chloroform and the precooling of 3 times of volumes, violent mixing, 4 DEG C of standings 30min, 8600g centrifuge 1min.Remove upper strata aqueous phase.To
The pre- cold methanol of 1/2 volume is added in lower layer and intermediate protein precipitation, mixes well, 9000g centrifuges 5min.Supernatant is abandoned, it is dry,
It is precipitated using sample-loading buffer soluble protein.As shown in figure 5, No. 1 band is the polypeptide sample of No. 1 absorption peak in Fig. 4, i.e., do not split
The fusion protein LfcinB-hLY samples of solution, size about 24kD or so.No. 2 bands are the polypeptide samples of No. 2 absorption peaks in Fig. 4,
Size is 14kD or so, suitable with the molecular size range of human lysozyme.No. 3 bands are the polypeptide samples of No. 3 absorption peaks in Fig. 4,
Size is 4kD or so, the sizableness with Bovine lactoferricin.
The property research of 4 fusion protein LfcinB-hLY of embodiment
1, the research of LfcinB-hLY fusion proteins antimicrobial spectrum
Detect Antibacterial Activity of the LfcinB-hLY fusion proteins to listed bacterial strain in table 1.Various bacterium are incubated at 5mL respectively
Fluid nutrient medium shaking table culture, condition of culture is as listed in table 1.When thalli growth is to exponential phase, respectively according to implementation
The method of 1 method of example carries out the measurement of inhibition zone.It measures 3 times and is averaged.
Above-mentioned LB culture mediums quality composition is tryptone 1%, yeast extract 0.5%, NaCl1%, solvent be go from
Sub- water, pH value are natural.
Czapek's medium quality group become sucrose 3%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%,
Potassium chloride 0.05%, ferrous sulfate 0.001%, solvent are deionized water, and pH value is natural.
MRS culture mediums are purchased from OXOID companies.
LfcinB-hLY fusion proteins are to using staphylococcus aureus, bacillus subtilis, bacillus licheniformis as representative
Gram-positive bacteria and Escherichia coli be representative Gram-negative bacteria fungistatic effect have fungistatic effect, wherein to golden yellow
Staphylococcus effect is the most apparent.To being acted on by the mould unrestraint of representative of aspergillus oryzae, to saccharomyces cerevisiae unrestraint effect, and
There is very strong inhibiting effect to same Eukaryotic Candida.Therefore, it is different from lysozyme only to gram-positive bacteria
Bacteriostatic activity is stronger, and LfcinB-hLY fusion proteins have good antibacterial effect to gram-positive bacteria and Gram-negative bacteria
Fruit.
The fungistatic effect of the condition of culture and LfcinB-hLY fusion proteins of the different indicator bacterias of table 1
Note：++:Indicate that antibacterial circle diameter is more than 20mm；+ indicate that inhibition zone effect is more than 13mm；/ indicate no fungistatic effect.
2, the stability of LfcinB-hLY fusion proteins
2.1 thermostabilization
Fusion protein LfcinB-hLY prepared by 2 step of embodiment (1) is respectively at 60 DEG C, 80 DEG C, 100 DEG C, 120 DEG C
10min, 30min, 60min are handled respectively, using Escherichia coli as indicator bacteria, are tested Antibacterial Activity, are with untreated sample
Control, as a result as shown in Figure 6.
It will be appreciated from fig. 6 that LfcinB-hLY fusion proteins handle 60min by 60 DEG C and 80 DEG C, vigor is without being decreased obviously.
100 DEG C of heating water bath 10min vigor decline 2.8%, and with the increase of heating time, to heating 60min, vigor declines more
Significantly reach 8.6%.120 DEG C heated in autoclave 10min its vigor decline heating 60min in 8.6%, with boiling water have it is identical
Vigor loss, when heating 60min, vigor loss is up to 22.8%, it can be seen that, fusion protein has preferable thermostabilization
Property.
2.2 ph stability
By 2 step of embodiment (2) prepare fusion protein LfcinB-hLY respectively under the conditions of pH3,4,5,6,7,8,9 at
1h is managed, using Escherichia coli as indicator bacteria, tests antibacterial circle diameter, is control with untreated sample, as a result as shown in Figure 7.
As shown in Figure 7, antibacterial activity is stronger when pH is 4, and under conditions of sourer, vigor is compared with control group vigor
Decline, with the increase of pH, downward trend is presented to the fungistatic effect of Escherichia coli (ATCC 25922), when pH is increased to 7
When, vigor zero.It can be seen that when zymotic fluid nature pH is 4.5 or so, stability is preferable.
2.3 enzymolysis stability
Fusion protein LfcinB-hLY solution prepared by 2 step of embodiment (2) is added to the digestion of final concentration of 1mg/mL
Enzyme, and adjust to obtain the pH value of table 2 with 0.1M HCl and 0.2M NaOH.As handled 1h under conditions of table 2, then at boiling water bath
Manage 5min inactivation, then with corresponding acid-base accommodation pH be 5 or so, with untreated fusion protein be control, with Escherichia coli with
Staphylococcus aureus is indicator bacteria, measures inhibition zone, as a result sees Fig. 8.
The reaction condition of the different enzymes of table 2
Pepsin be can be seen that on Fig. 8 to LfcinB-hLY fusion proteins to staphylococcus aureus and Escherichia coli
Bacteriostatic activity have facilitation, it may be possible to the bacteriostatic activity of LfcinB, hLY for being released by pepsin hydrolysis have
Cooperate with synergistic effect.Trypsase and chymotrypsin, compared with the control group, antibacterial activity influences little.LfcinB-hLY melts
Hop protein has stronger tolerance to the various protease of animal gastrointestinal tract.
The storage stability of 2.4 fusion proteins containing LfcinB-hLY
By fusion protein LfcinB-hLY prepared by 2 step of embodiment (2) and No. 2 peaks that embodiment 3 obtains and No. 3
Peak mixed liquor is in 4 DEG C, using Escherichia coli (ATCC 25922) as indicator bacteria, measures the antibacterial activity after placing different time,
Two kinds of solution albumen concentration are equal, and are control with first day.As can be seen from Figure 9, LfcinB-hLY fusion proteins in 20d are placed
Bacteriostatic activity only decline 7.7%.It only loses 15.4% to the inhibition vigor of Escherichia coli (ATCC 25922) after 30d.And
The decline that hydroxylamine cleavage, dialysis remove hLY the and LfcinB mixed solution bacteriostatic activities obtained after anti-small molecule oxidation peptide wants bright
It is aobvious to be faster than LfcinB-hLY fusion proteins, and it only loses 38.8% to the vigor of Escherichia coli after 30d.
5 Pichia pastoris high density fermentation of embodiment expresses LfcinB-hLY fusion proteins
(1) the recombinant bacterium single bacterium colony of the gene of hybrid protein containing LfcinB-hLY built from picking embodiment 1 on YPD tablets
5mL YPD culture mediums are inoculated in, 30 DEG C of shaken cultivations are stayed overnight；With 1：Bacterium solution is seeded to 300mL YPD trainings by 100 volume ratio
Support base, 30 DEG C of shaken cultivations are overnight to OD600 up to 2~6, the seed liquor as inoculation fermentation tank.
(2) 10L fermentation mediums (composition is with embodiment 1) are added in domestic 30L fermentation tanks, and 121 DEG C sterilize 20 minutes, adjust
Temperature is saved to 30 DEG C, pH to 4.5 is adjusted with ammonium hydroxide.According to inoculum concentration inoculation step (1) seed liquor of volumetric concentration 8%, fermentation
Temperature control passes through phosphoric acid solution and ammonium hydroxide and carries out pH tune at 30 DEG C hereinafter, rotating speed control is between 300-800rpm in the process
It is whole, while ensureing that dissolved oxygen is not less than 20%.After inoculation for 24 hours, supplemented medium (composition is with embodiment 1) 10mL/h is dosed.After 72h
It waits for that biomass is not further added by, terminates fermentation.Blowing after fermentation, zymotic fluid is spray-dried, obtains fusion protein LfcinB-
HLY Pichia pastoris products.In addition also the Pichia pastoris GS115 host strain without containing LfcinB-hLY genes is similarly sent out
Ferment.
The piglet administering transgenic of the Pichia pastoris product of 6 LfcinB-hLY containing fusion protein of embodiment
Influence for verification recombinant protein Lfcin B-hLY to piglet growth performance and immunity, has carried out recombination egg
The piglet administering transgenic of white Lfcin B-hLY.It tests and is divided into experimental group I, experimental group II and control group, every group 12.Experimental group
Weanling pig similar in age in days, weight is selected with control group.Control group feeds basal diet；Experimental group I, feeding basal diet+
0.3% fusion protein LfcinB-hLY Pichia pastoris products of mass concentration.Experimental group II feeds basal diet+mass concentration
0.3% Pichia pastoris product (the Pichia pastoris GS115 host strain that i.e. prepared by embodiment 5 without fusion protein LfcinB-hLY
Fermented product).Test result is as follows：
| Experimental group I | Experimental group II | Control group | |
| Initial counterpoise/Kg | 7.6 | 7.5 | 7.4 |
| Terminate counterpoise/Kg | 24.6 | 22.9 | 21.8 |
| Test number of days | 45 | 45 | 45 |
| Daily gain | 377.8 | 342.2 | 320 |
| Feedstuff-meat ratio | 1.84 | 1.89 | 2.18 |
| Survival rate | 100% | 100% | 100% |
| Diarrhea rate | 3.6% | 5.6% | 7.0% |
Experimental result：
1. adding Pichia pastoris product in feed, piglet daily gain improves 22.2 grams than control group, when addition contains recombination
The Pichia pastoris product of albumen LFB-hLY, piglet daily gain further increase, and 57.8 grams are improved than control group, illustrate to recombinate egg
White LFB-hLY makes piglet growth performance be improved；
2. experimental group I feedstuff-meat ratios are less than control group 0.34, experimental group II feedstuff-meat ratios are less than control group 0.29, illustrate to finish red ferment
Female addition makes efficiency of feed utilization improve, and the addition of recombinant protein LFB-hLY slightly improves efficiency of feed utilization；
3, experimental group I and experimental group II diarrhea situation are less than control group, and finish red ferment containing recombinant protein LFB-hLY
Female product is to preventing the effect of grice diarrhoea to be better than the Pichia pastoris product without recombinant protein LFB-hLY.
Sequence table
<110>Zhejiang Polytechnical University
<120>A kind of Bovine lactoferricin-human lysozyme fusion protein, gene and its application
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ggcggcggcg gaagtgagga ccctaacggc aaagtcttcg agaggtgcga gcttgccagg 300
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ttagcaaaat gggagagtgg atacaacact agagctacga attacaacgc tggtgacagg 420
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cctggtgccg tcaacgcctg tcatttaagt tgctctgcat tactacaaga caacatagcc 540
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tga 663
Claims (6)
1. a kind of Bovine lactoferricin-human lysozyme fusion protein, it is characterised in that the fusion protein is by pig muscle fibril
Protein antioxidant peptide, Bovine lactoferricin, chicken egg white anti-oxidation peptide, pig myogen myosin anti-oxidation peptide, flexible connection
Peptide, human lysozyme are followed in series to form.
2. Bovine lactoferricin-human lysozyme fusion protein as described in claim 1, it is characterised in that the ammonia of the fusion protein
Base acid sequence is shown in SEQ ID NO.6.
3. a kind of Bovine lactoferricin described in claim 1-human lysozyme fusion protein encoding gene, it is characterised in that the volume
The nucleotides sequence of code gene is classified as shown in SEQ ID NO.8.
4. Bovine lactoferricin-human lysozyme fusion protein is in preparing antiseptic feed additive described in a kind of claim 1
Using.
5. application as claimed in claim 4, it is characterised in that the antiseptic feed additive is additive for farm animal feed.
6. application as claimed in claim 5, it is characterised in that the antiseptic feed additive is additive for feed for piglets.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094833A (en) * | 2020-03-24 | 2020-12-18 | 深圳市康佑生物技术有限公司 | Bacteriostatic protein, coding gene, application and strain method thereof |
| CN113214404A (en) * | 2020-01-21 | 2021-08-06 | 杭州芳韵生物科技有限公司 | Epidermal growth factor-lysozyme fusion protein and application thereof |
| CN114230672A (en) * | 2021-11-23 | 2022-03-25 | 华侨大学 | Bovine lactoferrin peptide fusion protein, coding gene and application of bovine lactoferrin peptide fusion protein in seafood preservation and/or preservation |
| CN114437238A (en) * | 2022-01-26 | 2022-05-06 | 浙江工业大学 | Collagen peptide-bovine lactoferrin peptide fusion protein, gene and expression method thereof |
| CN117024604A (en) * | 2023-08-09 | 2023-11-10 | 广西福莱明生物制药有限公司 | Recombinant biological defense fusion protein and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005024002A1 (en) * | 2003-09-11 | 2005-03-17 | Novozymes A/S | Recombinant production of antimicrobial agents |
| CN1673370A (en) * | 2005-01-28 | 2005-09-28 | 扬州大学 | Method for preparing transient expression recombination protein chicken salpingo biological reactor |
| US20070037165A1 (en) * | 2000-09-08 | 2007-02-15 | Applera Corporation | Polymorphisms in known genes associated with human disease, methods of detection and uses thereof |
| CN101899469A (en) * | 2010-05-14 | 2010-12-01 | 上海艾魁英生物科技有限公司 | Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes |
| US20100316643A1 (en) * | 2009-02-05 | 2010-12-16 | The Regents Of The University Of California | Targeted antimicrobial moieties |
| CN104031965A (en) * | 2014-06-05 | 2014-09-10 | 西北农林科技大学 | Preparation method of goat cheese protein source antibacterial peptide |
| US20180080013A1 (en) * | 2015-04-21 | 2018-03-22 | Novartis Ag | Rna-guided gene editing system and uses thereof |
-
2018
- 2018-04-04 CN CN201810300197.9A patent/CN108794635B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070037165A1 (en) * | 2000-09-08 | 2007-02-15 | Applera Corporation | Polymorphisms in known genes associated with human disease, methods of detection and uses thereof |
| WO2005024002A1 (en) * | 2003-09-11 | 2005-03-17 | Novozymes A/S | Recombinant production of antimicrobial agents |
| CN1673370A (en) * | 2005-01-28 | 2005-09-28 | 扬州大学 | Method for preparing transient expression recombination protein chicken salpingo biological reactor |
| US20100316643A1 (en) * | 2009-02-05 | 2010-12-16 | The Regents Of The University Of California | Targeted antimicrobial moieties |
| CN101899469A (en) * | 2010-05-14 | 2010-12-01 | 上海艾魁英生物科技有限公司 | Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes |
| CN104031965A (en) * | 2014-06-05 | 2014-09-10 | 西北农林科技大学 | Preparation method of goat cheese protein source antibacterial peptide |
| US20180080013A1 (en) * | 2015-04-21 | 2018-03-22 | Novartis Ag | Rna-guided gene editing system and uses thereof |
Non-Patent Citations (6)
| Title |
|---|
| HUANG H C等: "The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 * |
| QIANG DENG等: "Preparation of An Antibacterial Peptide Bovine Lactoferricin by Genetic Engineering", 《INTERNATIONAL CONFERENCE ON BIOMEDICAL ENGINEERING AND INFORMATICS》 * |
| ULVATNE H 等: "Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes", 《FEBS LETTERS》 * |
| 李忠清等: "抗菌肽牛乳铁蛋白肽衍生肽在毕赤酵母中的表达及活性研究", 《资源化学研究室》 * |
| 李忠清等: "牛乳铁蛋白肽的研究现状与应用前景", 《食品科学》 * |
| 陈洪生等: "蛋清蛋白对猪肉肌原纤维蛋白功能性的影响", 《中国食品学报》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113214404A (en) * | 2020-01-21 | 2021-08-06 | 杭州芳韵生物科技有限公司 | Epidermal growth factor-lysozyme fusion protein and application thereof |
| CN112094833A (en) * | 2020-03-24 | 2020-12-18 | 深圳市康佑生物技术有限公司 | Bacteriostatic protein, coding gene, application and strain method thereof |
| CN112094833B (en) * | 2020-03-24 | 2022-12-27 | 深圳市康佑生物技术有限公司 | Bacteriostatic protein, and coding gene, application and strain thereof |
| CN114230672A (en) * | 2021-11-23 | 2022-03-25 | 华侨大学 | Bovine lactoferrin peptide fusion protein, coding gene and application of bovine lactoferrin peptide fusion protein in seafood preservation and/or preservation |
| CN114230672B (en) * | 2021-11-23 | 2023-08-08 | 华侨大学 | Bovine lactoferrin peptide fusion protein, encoding gene and application thereof in seafood fresh-keeping and/or preservative |
| CN114437238A (en) * | 2022-01-26 | 2022-05-06 | 浙江工业大学 | Collagen peptide-bovine lactoferrin peptide fusion protein, gene and expression method thereof |
| CN114437238B (en) * | 2022-01-26 | 2024-03-26 | 浙江工业大学 | Collagen peptide-bovine lactoferrin peptide fusion protein, gene and expression method thereof |
| CN117024604A (en) * | 2023-08-09 | 2023-11-10 | 广西福莱明生物制药有限公司 | Recombinant biological defense fusion protein and application thereof |
| CN117024604B (en) * | 2023-08-09 | 2024-03-29 | 广西福莱明生物制药有限公司 | Recombinant biological defense fusion protein and application thereof |
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