CN105288586A - Antibacterial peptide plectasin film-forming agent and preparation method and application thereof - Google Patents
Antibacterial peptide plectasin film-forming agent and preparation method and application thereof Download PDFInfo
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- CN105288586A CN105288586A CN201510539838.2A CN201510539838A CN105288586A CN 105288586 A CN105288586 A CN 105288586A CN 201510539838 A CN201510539838 A CN 201510539838A CN 105288586 A CN105288586 A CN 105288586A
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Abstract
The invention discloses an antibacterial peptide plectasin film-forming agent and a preparation method and application thereof. The antibacterial peptide plectasin film-forming agent prepared from antibacterial peptide plectasin is simple in technology, high in encapsulation efficiency and stability and low in cost, can be produced on a large scale and is capable of playing a role in protection directly to kill bacteria rapidly and prevent further infection of inflammation, thereby treating infection effectively. The antibacterial peptide plectasin film-forming agent has no significant difference in influence on dairy cow milk yield, milk protein content, milk sugar content, butter-fat percentage and nonfat milk solid content and has notable prevention effect when being applied to subclinical mastitis of dairy cows.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of antibacterial peptide plectasin film former and preparation method thereof and application.
Background technology
Mammitis of cow (BovineMastitis) is one of large disease of harm milk cow production three, mainly causes milk production of cow decline, milk-quality reduces and fertility performance declines.It is reported that the sickness rate of external mammitis of cow is 20% ~ 60%, and domestic sickness rate is up to 30% ~ 70%, be wherein 21% ~ 23% at clinic mastitis average attack rate, recessive mastitis Incidence is then up to 50% ~ 70%.Being caused damage by mastitis every year, very large if the U.S. is up to 2,000,000,000 dollars, Britain reaches 2.67 hundred million pounds, and Japan reaches 500,000,000 dollars, and Germany reaches 1,000,000,000 Euros etc.And the loss that causes because of mastitis every year of China approximately every cattle 1200 ~ 3600 yuan.As can be seen here, mammitis of cow brings huge economic loss to animal husbandry, and the food safety of people in serious threat, governs the sustainable health development of dairy.
Main pathogen of bovine mastitis is staphylococcus aureus, streptococcus and escherichia coli, and its sickness rate is very high.Current treatment mammitis of cow, from original adoption antibiotic therapy, changes herbal treatment into, but treatment by Chinese herbs mammitis of cow consumption is large, curative effect is slow, has not been most ideal treatment method.Therefore, finding antibiotic that a kind of efficient, safe new drug replaces generally using at present and Chinese medicine is the important means of preventing and treating bovine mastitis at present, is also the hot issue of animal husbandry R & D and manufacture.
Antibacterial peptide (antibacterialpeptides) is also known as antimicrobial peptide (antimicrobialpeptides, AMPs), be that multiple biology produces the bioactive molecule with innate immune reaction, play cellular host defense effect by invasion pathogenic microorganism.Antibacterial peptide has broad spectrum antibiotic activity, low toxicity and low drug resistance, noresidue and pollution-free, is considered to have future and potential antibiotic replacement candidates medicine a kind of future simultaneously, belongs to environment-friendly type antibiotic as higher organism immunomodulator.Except antimicrobial acivity function, antibacterial peptide is also as drug carrier system, antitumor agent, mitogen, contraceptive and signal transducers and feed additive.Therefore, antibacterial peptide is with a wide range of applications, but due to it self, the impact mixing with other adjuvants, in granulation process easily by various trace elements, soda acid, exogenous protease is very big, and granulation process TRANSIENT HIGH TEMPERATURE also can affect its biological activity simultaneously; In addition, antibacterial peptide is soluble in water, greatly reduces its prevention and therapy active.
In order to well improve the stability of antibacterial peptide, utilize existing polypeptide preparation technology to be made into the dosage forms such as enteric coated formulation, capsule, micropill, microemulsion, liposome, microgranule, nanoparticle simultaneously, but microemulsion, emulsion preparation are poor in envelop rate and stability, the encapsulating demand of antibacterial peptide cannot be met, and liposome is due to shortcomings such as dosage is large, envelop rate is low, poor stabilities, the preparation requirement of antimicrobial peptide preparation can not be met.And enteric coated formulation, microgranule, microcapsule and nanoparticle exist complicated process of preparation, cost is higher, is difficult to realize large-scale production, is not suitable for the preparation of antimicrobial peptide preparation equally.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of antibacterial peptide plectasin film former and preparation method thereof and application, be intended to solve that existing antimicrobial peptide preparation preparation method envelop rate is low, poor stability and the high problem of cost.
Technical scheme of the present invention is as follows:
A preparation method for antibacterial peptide plectasin film former, wherein, comprises step:
The structure of the expression vector of A, antibacterial peptide plectasin
Plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
After carrying out restriction enzyme digestion to recombiant plasmid, electricity forwards in Pichia sp., after then cultivating by culture medium in Pichia sp., filters out positive transformant;
Above-mentioned positive transformant is carried out shaking table cultivation, and the part bacterium liquid after then being cultivated by shaking table carries out inducing culture, centrifugal again after cultivation, collects supernatant, obtains plectasin fermentation liquid;
The preparation of B, antibacterial peptide plectasin film former
Get 1 ~ 3mL plectasin fermentation liquid, add PVA fully stir, swelling, heating for dissolving and cross screen cloth, then leaves standstill the filtrate that obtains under room temperature;
Add Tween-80 and glycerol in filtrate after leaving standstill, after fully stirring, treat room temperature, add azone, stir, finally add the CMC-Na infiltrated with dehydrated alcohol, fully stir until dissolve completely, obtain antibacterial peptide plectasin film former; Wherein, PVA:Tween-80: glycerol: CMC-Na is 3 ~ 6:1.4 ~ 1.6:5 ~ 8:1.9 ~ 2.1 by volume.
The preparation method of described antibacterial peptide plectasin film former, wherein, described steps A specifically comprises:
A1, albumen coded sequence according to plectasin, design Pichia sp. Preference codon, synthesis plectasin gene order, is then cloned in pUC57 by plectasin gene order, uses
xhoI,
avrIIafter double digestion, plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
A2, restriction enzyme digestion is carried out to recombiant plasmid after electricity forward in Pichia sp., then cultivate based on after cultivating 2 ~ 3d at 25 ~ 30 DEG C with the YPD containing 500 ~ 1000 μ g/mL bleomycin, filter out positive transformant;
A3, be inoculated in the centrifuge tube containing 3mLBMGY culture medium by above-mentioned positive transformant, at 25 ~ 30 DEG C, 20 ~ 25h cultivated by 220r/min shaking table; Then the part bacterium liquid after being cultivated by shaking table is transferred in BMMY inducing culture, at 25 ~ 30 DEG C, 220r/min carries out inducing culture, and every 24h adds volumetric concentration than the absolute methanol being 1%, more every 24h is in the centrifugal 5 ~ 10min of 12000r/min, collect supernatant, obtain plectasin fermentation liquid.
The preparation method of described antibacterial peptide plectasin film former, wherein, in described step B, described PVA:Tween-80: glycerol: CMC-Na is 5:1.5:7:2.1 by volume.
The preparation method of described antibacterial peptide plectasin film former, wherein, in described step B, the order number of described screen cloth is 35 ~ 45 orders.
The preparation method of described antibacterial peptide plectasin film former, wherein, also step is comprised: test the adhesion of antibacterial peptide plectasin film former, adhesion testing procedure is: respectively 9 groups of antibacterial peptide plectasin film former are uniformly coated on 3 × 4cm after described step B
2the abdominal part film forming of rabbit, then will fold 0.5cm near rabbit abdomen inner opposite end film, at the uniform velocity drip in plastic bag with clip one plastic bag blend rubber pipe, and till film rupture, record weight, gained weight is the adhesion of antibacterial peptide plectasin film former.
The preparation method of described antibacterial peptide plectasin film former, wherein, also step is comprised: test the breathability of antibacterial peptide plectasin film former, breathability testing procedure is: antibacterial peptide plectasin film former is spread evenly across 6 × 6cm after described step B
2rabbit abdominal part film forming, the film then choosing thickness 0.1 ~ 0.2 μm is tested; By anhydrous for 6g CaCl
2put into gas permeable devices, with formed film, bottle sealing is placed in the calorstat of 37 DEG C, weigh and record 1 time/h; Map with mass change and the relation of time, obtain the slope S of figure cathetus, and obtain the Air permenbility Q of thin film according to following formula;
Wherein, Q=(S/A) (g/h m
2), in formula: S is the slope of straight line; A is the test area of film.
The preparation method of described antibacterial peptide plectasin film former, wherein, also step is comprised: the release of antibacterial peptide plectasin film former is tested after described step B, release testing procedure is: fill rabbit lower inner ring border with normal saline, rabbit is fixed in single chamber Transdermal diffusion cell, and control single chamber Transdermal diffusion cell and be in 37 DEG C of water-baths, 2mL antibacterial peptide plectasin film former is evenly coated in rabbit 3 × 4cm
2on the surface, draw 5mL liquid every 1/2h from opening for getting medicine, then supplement with 5mL normal saline; The liquid of sucking-off is injected quartz colorimetric utensil, measures absorbance OD value at wavelength X=595nm place, calculated the release concentration of antibacterial peptide plectasin film former by linear equation.
The preparation method of described antibacterial peptide plectasin film former, wherein, described linear equation is drawn by following steps: bovine serum albumin standard substance being diluted with distilled water into concentration is 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL solution; Then measure absorbance OD value at λ=595nm place respectively to the solution of above-mentioned each concentration, with absorbance OD value for vertical coordinate, protein standard substance concentration is abscissa drawing standard curve, obtains the linear equation y=0.0069x+0.0419 of standard curve, R
2=0.9983, wherein, x is protein standard substance concentration, and y is absorbance OD value.
A kind of antibacterial peptide plectasin film former, wherein, described antibacterial peptide plectasin film former adopts the preparation method of as above arbitrary described antibacterial peptide plectasin film former to be prepared from.
An application for antibacterial peptide plectasin film former, wherein, is used for preventing mammitis of cow by antibacterial peptide plectasin film former described above.
Beneficial effect: antibacterial peptide plectasin is made antibacterial peptide plectasin membrane by the present invention; not only technique is simple; envelop rate is high; good stability, with low cost, can large-scale production be carried out; and antibacterial peptide plectasin membrane can directly play a protective role; kill antibacterial rapidly, prevent inflammation from infecting further, have good treatment to infection.
Accompanying drawing explanation
Fig. 1 is mass change and the canonical plotting of time in breathability of the present invention test.
Fig. 2 is the canonical plotting of absorbance OD value and protein concentration during the present invention linearly tests.
Fig. 3 is the graph of relation of antibacterial peptide plectasin film former concentration and drug release time in release of the present invention test.
Fig. 4 is that antibacterial peptide plectasin film former of the present invention affects schematic diagram to milk production of cow.
Fig. 5 is that antibacterial peptide plectasin film former of the present invention affects schematic diagram to content of milk protein.
Fig. 6 is that antibacterial peptide plectasin film former of the present invention affects schematic diagram to lactose content.
Fig. 7 is that antibacterial peptide plectasin film former of the present invention affects schematic diagram to butterfat percnetage.
Fig. 8 is that antibacterial peptide plectasin film former of the present invention affects schematic diagram to nonfat milk solids content.
Detailed description of the invention
The invention provides a kind of antibacterial peptide plectasin film former and preparation method thereof and application, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The present invention discloses a kind of preparation method of antibacterial peptide plectasin film former, and it comprises step:
The structure of the expression vector of A, antibacterial peptide plectasin
Plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
After carrying out restriction enzyme digestion to recombiant plasmid, electricity forwards in Pichia sp., after then cultivating by culture medium in Pichia sp., filters out positive transformant;
Above-mentioned positive transformant is carried out shaking table cultivation, and the part bacterium liquid after then being cultivated by shaking table carries out inducing culture, centrifugal again after cultivation, collects supernatant, obtains plectasin fermentation liquid;
The preparation of B, antibacterial peptide plectasin film former
Get 1 ~ 3mL plectasin fermentation liquid, add PVA fully stir, swelling, heating for dissolving and cross screen cloth, then leaves standstill the filtrate that obtains under room temperature;
Add Tween-80 and glycerol in filtrate after leaving standstill, after fully stirring, treat room temperature, add azone, stir, finally add the CMC-Na infiltrated with dehydrated alcohol, fully stir until dissolve completely, obtain antibacterial peptide plectasin film former; Wherein, PVA:Tween-80: glycerol: CMC-Na is 3 ~ 6:1.4 ~ 1.6:5 ~ 8:1.9 ~ 2.1 by volume.
By technique scheme, the present invention can obtain that Dispersion of Solute Matter is even, viscosity good, the antibacterial peptide plectasin film former of good fluidity and the advantage such as film formation time is shorter.
In the present invention, described steps A specifically comprises:
A1, albumen coded sequence according to plectasin, design Pichia sp. Preference codon, synthesis plectasin gene order, is then cloned in pUC57 by plectasin gene order, uses
xhoI,
avrIIafter double digestion, plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
Particularly, above-mentioned steps A1 is:
Protein sequence is as follows:
Antibacterial peptide Plectasin
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY
Plectasin NZ2114
GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY
According to above-mentioned protein sequence, design corresponding Pichia sp. Preference codon, submit biotech firm synthesis NZ2114 gene order to.The NZ2114 gene order of synthesis is cloned in pUC57, uses
xhoI,
avrIIafter double digestion, NZ2114 gene order is inserted yeast expression vector pPicZ α MF57, be positioned at the downstream of alpha factor signal peptide mutant sequence, obtain recombiant plasmid pPicZ α MF57-NZ2114.
A2, restriction enzyme digestion is carried out to recombiant plasmid after electricity forward in Pichia sp., then cultivate based on after cultivating 2 ~ 3d at 25 ~ 30 DEG C with the YPD containing 500 ~ 1000 μ g/mL bleomycin, filter out positive transformant;
The present invention also takes to carry out restriction enzyme digestion and order-checking qualification to this recombiant plasmid pPicZ α MF57-NZ2114.By recombiant plasmid pPicZ α MF57-NZ2114 after restriction enzyme digestion (PmeI enzyme action), electricity forwards in Pichia pastoris GS115, then cultivate based on after cultivation 2 ~ 3d under 25 ~ 30 DEG C (as 30 DEG C) with the YPD containing 500 ~ 1000 μ g/mL bleomycin (Zeocin), filter out the positive transformant of multicopy.Above-mentioned steps tentatively can determine that plectasin (NZ2114) gene order is recombinated on Pichia sp. chromosome set.In addition, the present invention also carries out antibacterial activity detection and pcr amplification Molecular Identification to positive transformant.Detect and qualification result, by recombinant plectasin Pichia sp. called after pPicZ α MF57-NZ2114 best for antibacterial activity according to it.
A3, be inoculated in the centrifuge tube containing 3mLBMGY culture medium by above-mentioned positive transformant, at 25 ~ 30 DEG C, 20 ~ 25h cultivated by 220r/min shaking table; Then the part bacterium liquid after being cultivated by shaking table is transferred in BMMY inducing culture, at 25 ~ 30 DEG C, 220r/min carries out inducing culture, and every 24h adds volumetric concentration than the absolute methanol being 1%, more every 24h is in the centrifugal 5 ~ 10min of 12000r/min, collect supernatant, obtain plectasin fermentation liquid.
Above-mentioned steps A3 is, the multiple positive transformants above-mentioned screening obtained are inoculated in supports in the centrifuge tube in base containing 3mLBMGY or 500 μ g/mLZeocin, and under 25 ~ 30 DEG C (as 30 DEG C), 220r/min shaking table cultivates 20 ~ 25h(as 24h).Then the part bacterium liquid after being cultivated by shaking table is transferred in BMMY inducing culture, and under 25 ~ 30 DEG C (as 30 DEG C), 220r/min proceeds inducing culture, and every 24h adds the absolute methanol that concentration ratio is 1%.Every 24h by fermentation liquid in the centrifugal 5 ~ 10min(of 12000r/min as 5min), collect supernatant, obtain plectasin fermentation liquid.
The present invention also takes the formula of orthogonal test to synthetic antibacterial peptide plectasin film former to be optimized.
(1), the screening of PVA dosage: when PVA percentage by volume is 5%, the performance of the antibacterial peptide plectasin film former of acquisition is best.
(2), the suitable amount scope of Tween-80 dosage choice: Tween-80 is 0.6 ~ 0.8mL(by volume percent basis, and the suitable amount scope of Tween-80 is 1.4 ~ 1.6%); Wherein, when Tween-80 consumption is 0.7mL, the performance of the antibacterial peptide plectasin film former of acquisition is best.
(3), glycerol dosage choice: the suitable amount scope of glycerol is 6.0 ~ 8.0mL(by volume percent basis, and the suitable amount scope of glycerol is 6 ~ 8%); Wherein, when glycerol consumption is 7.0mL, the performance of the antibacterial peptide plectasin film former of acquisition is best.
(4), the suitable amount scope of CMC-Na dose screening: CMC-Na by volume percent be 1.9 ~ 2.1%.By volume percent basis, when the consumption of CMC-Na is 2%, the performance of the antibacterial peptide plectasin film former of acquisition is best.
(5), orthogonal test selection optimum formula
Orthogonal Testing factors table
Choose the orthogonal test of L9 (3) 3, using film formation time and presentation quality as evaluation.Orthogonal Testing factors is carried out to Tween-80, glycerol and CMC-Na tri-factors and level is screened, choose optimal dose by orthogonal test.
The impact of orthogonal test result display: factor c is comparatively large, and factor b takes second place, and the optimum combination of each factor is tentatively defined as a
2b
2c
3, i.e. by volume percent basis, glycerol accounts for 7%, Tween-80 account for 1.5% and CMC-Na account for 2.1%.Therefore, in described step B, described PVA:Tween-80: glycerol: CMC-Na when being 5:1.5:7:2.1 by volume, the performance of the antibacterial peptide plectasin film former obtained is best, and described antibacterial peptide plectasin film former has that Dispersion of Solute Matter is even, viscosity good, good fluidity and the advantage such as film formation time is shorter.
Preferably, in described step B, the order number of described screen cloth is 35 ~ 45 orders.More preferably, the order number of described screen cloth is 40 orders.
Based on the antibacterial peptide plectasin film former of above-mentioned preparation, the performance of the present invention to described antibacterial peptide plectasin film former is tested.Specific performance test comprises following:
(1), the adhesion test of antibacterial peptide plectasin film former
Also step is comprised: test the adhesion of antibacterial peptide plectasin film former, adhesion testing procedure is: respectively 9 groups of antibacterial peptide plectasin film former are uniformly coated on 3 × 4cm after described step B
2the abdominal part film forming of rabbit, then will fold 0.5cm near rabbit abdomen inner opposite end film, at the uniform velocity drip in plastic bag with clip one plastic bag blend rubber pipe, and till film rupture, record weight, gained weight is the adhesion of antibacterial peptide plectasin film former.
In above-mentioned adhesion test, the optimum formula that antibacterial peptide plectasin film former goes out according to orthogonal test screen obtains.And repeat above-mentioned adhesion and test 3 times.3 test results display: this antibacterial peptide plectasin film former adhesion scope is 153.41 ~ 230.36g, show the absorption of this antibacterial peptide plectasin film former and action time all longer.
(2), the breathability test of antibacterial peptide plectasin film former
Also step is comprised: test the breathability of antibacterial peptide plectasin film former, breathability testing procedure is: antibacterial peptide plectasin film former is spread evenly across 6 × 6cm after described step B
2rabbit abdominal part film forming, the film then choosing thickness 0.1 ~ 0.2 μm is tested; By anhydrous for 6g CaCl
2put into gas permeable devices, bottle sealing is placed in the calorstat (control of calorstat wet scope: RH>=75%) of 37 DEG C with formed film, weighs and record 1 time/h; Map with mass change and the relation of time, obtain the slope S of figure cathetus, and obtain the Air permenbility Q of thin film according to following formula;
Wherein, Q=(S/A) (g/h m
2), in formula: S is the slope of straight line; A is the test area of film.
Above-mentioned breathability test result is shown in Fig. 1, can be obtained by Fig. 1: take time as abscissa, and mass change is vertical coordinate, gained standard curve y=0.0049x-0.0001, R
2=0.9999, there is good linear relationship in 0 ~ 4h.This illustrates and the speed that gas permeable devices changes along with temporal quality namely reflects the transmission rates of steam in film.Analyze and find: antibacterial peptide plectasin film former good air permeability of the present invention, can not affect skin function.
(3), the release test of antibacterial peptide plectasin film former
Also step is comprised: the release of antibacterial peptide plectasin film former is tested after described step B, release testing procedure is: fill rabbit lower inner ring border with normal saline, then rabbit is fixed on single chamber Transdermal diffusion cell (as, TK-12B type Franz single chamber Transdermal diffusion cell, be purchased from Shanghai Xie Kai trade Co., Ltd) on, skin corium towards receiving chamber, and controls single chamber Transdermal diffusion cell and is in 37 DEG C of water-baths, and 2mL antibacterial peptide plectasin film former is evenly coated in rabbit 3 × 4cm
2on the surface, draw 5mL liquid every 1/2h from opening for getting medicine, then supplement with 5mL normal saline; The liquid of sucking-off is injected quartz colorimetric utensil, measures absorbance OD value at wavelength X=595nm place, calculated the release concentration of antibacterial peptide plectasin film former by linear equation.Fig. 2 is the variation relation figure of release concentration with drug release time of antibacterial peptide plectasin film former, analyze discovery from Fig. 2: antibacterial peptide plectasin film former release amount increases progressively in time gradually, after 300min, this antibacterial peptide plectasin film former release amount reaches balance.
Wherein, described linear equation is obtained by following steps: bovine serum albumin standard substance being diluted with distilled water into concentration is 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL solution; Then measure absorbance OD value at λ=595nm place respectively to the solution of above-mentioned each concentration, with absorbance OD value for vertical coordinate, protein standard substance concentration is abscissa drawing standard curve.Standard curve is shown in Fig. 3, and test result shows: with absorbance OD value for vertical coordinate, protein standard substance concentration is abscissa, and the linear equation of gained standard curve is y=0.0069x+0.0419, R
2=0.9983, wherein, x is protein standard substance concentration, and y is absorbance OD value; Found by standard curve, be in 10 ~ 100 μ g/mL in protein standard substance concentration, absorbance OD value and protein standard substance concentration have good linear relationship.
(4), the primary stability test of antibacterial peptide plectasin film former
Primary stability test comprises: centrifugal test; Illumination is tested; High temperature test; High humidity is tested; The primary stability test of medicine; Accelerated test.Test result shows: I, antibacterial peptide plectasin film former color and luster are consistent, homogeneous, pH value about 6.21, antibacterial peptide plectasin film former content is about >=35 μ g/mL, film formation time is about 6min, and microbial limit meets the standard (2010 editions pharmacopeia) about film former microbial limit; After II, centrifugal 30min, medicine is without layering, demulsifying phenomenon; III, antibacterial peptide plectasin film former carried out illumination test, high temperature test, high humidity test, stability test and accelerated test after, antibacterial peptide plectasin film former stable in properties, without denaturalization phenomenon.
(4), the skin of antibacterial peptide plectasin film former swashs test, mucosa irritation test
Skin irritation is tested: choose healthy rabbit (3-4 monthly age) 12 (each 6 of male and female) in children's age, with depilatory, rabbit is carried on the back abdominal part depilation.Random point 2 groups (often organizing 6): one group is carried out intact skin and stimulates test (being lost hair or feathers both sides, rabbit back), another group is carried out damaging skin irritation test and (is lost hair or feathers both sides, rabbit back, skin degerming, then left and right sides skin is scratched with aseptic syringe needle with " # " font, skin is made to occur oozing of blood, damage zone area 9cm
2, cut spacing 0.5cm).District lose hair or feathers on the right side of rabbit every 12h to antibacterial peptide plectasin film former once, each 1mL, is uniformly distributed depilation district.Left side normal saline makes negative control.
Single is tested to antibacterial peptide plectasin film former: give after antibacterial peptide plectasin film former first, and after perusal 1/2,1,24,48 and 72h, coating part is with or without the situation such as erythema and edema.
Repeatedly give the test of antibacterial peptide plectasin film former: each remove antibacterial peptide plectasin film former after 1h and observing before again sticking, whether record erythema and edema, coating part have pigmentation, petechia, pachylosis or epidermatic atrophy situation and generation thereof and regression time.Continuous antibacterial peptide plectasin film former 7d, after last sticks, after perusal 1/2,1,24,48 and 72h, coating part is with or without the situation such as erythema and edema.
Irritation on mucous membrane is tested: first-selected rabbit 6 (be divided into 2 groups at random, often organize 3), carries out fluorescein sodium check (having the animal of eye irritation symptom, corneal defect and conjunctival damage can not be used for test) before test in 24h to the eyes of every animal.Test adopts consubstantiality left and right sides self-contrast method, the antibacterial peptide plectasin film former of 100mg on experimental group every eyes, and eyelid of then gently sleeping is about 10s, does not need to rinse eyes; The instillation of matched group every eyes is with the normal saline of dosage.Single to the test of antibacterial peptide plectasin film former Eye irritation, to give after antibacterial peptide plectasin film former 1,2,4,24,48 and 72h eye is checked.
Result shows: by single with repeatedly give antibacterial peptide plectasin film former test display this antibacterial peptide plectasin film former to no skin irritation.Non-stimulated to irritation on mucous membrane, there is slight stimulation to eye.
(5), antibacterial peptide plectasin film former clinical practice test
1. test animal and grouping: choose milch cow (4 ~ 6 years old age, 3 ~ 5 months puerperal), screens 20 healthy cows according to CMT method and 20 latent mammitis milch cows are tested.Treatment group: hidden property mammitis of cow 20, is divided into two groups, experimental group 10 test antibacterial peptide plectasin film former, matched group 10 precious No. II (Xi'an Han Long Chemical Industry Science Co., Ltd, lot number 140415, the 5L/ bucket) of breast; Prevention group: healthy cow 20, is divided into two groups, experimental group 10 test antibacterial peptide plectasin film former, matched group 10 precious No. II (Xi'an Han Long Chemical Industry Science Co., Ltd, lot number 140415, the 5L/ bucket) of breast.
2. therapeutic scheme: treatment group and prevention group first clean disease Lac Bovis seu Bubali district with warm water, then after drying with dry towel, each newborn district's coating control mammitis of cow antibacterial peptide plectasin film former 5g, matched group precious No. II of breast, often organize 2 times/d, 7d are a course for the treatment of.Adopt milk sample in 0,3,5 and 7d respectively, full milk analysis is carried out to milk sample for two groups, calculate the prevention rate of bovine subclinical mastitis cure rate and mammitis of cow according to somatic number.
Test result shows: filter out positive totally 21, the milk district for the treatment of group latent mammitis by CMT method, cure 11 Ge Ru districts (change according to somatic number is observed) after medication 7d altogether, Primary treatment rate reaches 52.38%; Positive totally 25, the milk district of matched group latent mammitis, after 7d, You35Ge Ru district is positive.Wherein prevention group is after the prevention of 7d, and have 2 Ge Ru districts infected, preliminary prevention rate can reach 95%; After 7d, You15Ge Ru district is infected is positive for matched group.
Therefore; antibacterial peptide plectasin is made antibacterial peptide plectasin membrane by the present invention; not only technique is simple, and envelop rate is high, good stability; with low cost; large-scale production can be carried out, and antibacterial peptide plectasin membrane directly can play a protective role, and kills antibacterial rapidly; prevent inflammation from infecting further, have good treatment to infection.And containing wetting agent composition in antibacterial peptide plectasin membrane, Absorbable rod affected part transudate and moisture, play good moisture-keeping function, and antibacterial peptide plectasin membrane has good affinity and breathability to skin.
Based on said method, the present invention also provides a kind of antibacterial peptide plectasin film former, and wherein, described antibacterial peptide plectasin film former adopts the preparation method of as above arbitrary described antibacterial peptide plectasin film former to be prepared from.Antibacterial peptide plectasin is made antibacterial peptide plectasin membrane by the present invention, efficiently solves low, the poor stability of envelop rate that existing antimicrobial peptide preparation exists and the large problem of consumption.
Based on above-mentioned antibacterial peptide plectasin film former, the present invention also provides a kind of application of antibacterial peptide plectasin film former, wherein, is used for preventing mammitis of cow by antibacterial peptide plectasin film former described above.
Particularly, by SPSS software analysis, the antibacterial peptide plectasin film former prepared by the present invention has investigated is on the impact of milk production of cow, content of milk protein, lactose content, butterfat percnetage and nonfat milk solids content.Fig. 4 ~ 8 are analyzed and are found, antibacterial peptide plectasin film former of the present invention is on the impact of above-mentioned milk production of cow, content of milk protein, lactose content, butterfat percnetage and nonfat milk solids content there are no significant difference.Illustrate that this antibacterial peptide plectasin film former is applied to cow recessive mastitis and has significant preventive effect thus.
In sum; antibacterial peptide plectasin film former provided by the invention and preparation method thereof and application, antibacterial peptide plectasin is made antibacterial peptide plectasin membrane by the present invention, and not only technique is simple; envelop rate is high; good stability, with low cost, can large-scale production be carried out; and antibacterial peptide plectasin membrane can directly play a protective role; kill antibacterial rapidly, prevent inflammation from infecting further, have good treatment to infection.Antibacterial peptide plectasin film former of the present invention is on the impact of above-mentioned milk production of cow, content of milk protein, lactose content, butterfat percnetage and nonfat milk solids content there are no significant difference, and antibacterial peptide plectasin film former is applied to cow recessive mastitis significant preventive effect.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Protein sequence is as follows:
Antibacterial peptide Plectasin
GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY
Plectasin NZ2114
GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCY
Claims (10)
1. a preparation method for antibacterial peptide plectasin film former, is characterized in that, comprises step:
The structure of the expression vector of A, antibacterial peptide plectasin
Plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
After carrying out restriction enzyme digestion to recombiant plasmid, electricity forwards in Pichia sp., after then cultivating by culture medium in Pichia sp., filters out positive transformant;
Above-mentioned positive transformant is carried out shaking table cultivation, and the part bacterium liquid after then being cultivated by shaking table carries out inducing culture, centrifugal again after cultivation, collects supernatant, obtains plectasin fermentation liquid;
The preparation of B, antibacterial peptide plectasin film former
Get 1 ~ 3mL plectasin fermentation liquid, add PVA fully stir, swelling, heating for dissolving and cross screen cloth, then leaves standstill the filtrate that obtains under room temperature;
Add Tween-80 and glycerol in filtrate after leaving standstill, after fully stirring, treat room temperature, add azone, stir, finally add the CMC-Na infiltrated with dehydrated alcohol, fully stir until dissolve completely, obtain antibacterial peptide plectasin film former; Wherein, PVA:Tween-80: glycerol: CMC-Na is 3 ~ 6:1.4 ~ 1.6:5 ~ 8:1.9 ~ 2.1 by volume.
2. the preparation method of antibacterial peptide plectasin film former according to claim 1, it is characterized in that, described steps A specifically comprises:
A1, albumen coded sequence according to plectasin, design Pichia sp. Preference codon, synthesis plectasin gene order, is then cloned in pUC57 by plectasin gene order, uses
xhoI,
avrIIafter double digestion, plectasin gene order is inserted yeast expression vector, obtains recombiant plasmid;
A2, restriction enzyme digestion is carried out to recombiant plasmid after electricity forward in Pichia sp., then cultivate based on after cultivating 2 ~ 3d at 25 ~ 30 DEG C with the YPD containing 500 ~ 1000 μ g/mL bleomycin, filter out positive transformant;
A3, be inoculated in the centrifuge tube containing 3mLBMGY culture medium by above-mentioned positive transformant, at 25 ~ 30 DEG C, 20 ~ 25h cultivated by 220r/min shaking table; Then the part bacterium liquid after being cultivated by shaking table is transferred in BMMY inducing culture, at 25 ~ 30 DEG C, 220r/min carries out inducing culture, and every 24h adds volumetric concentration than the absolute methanol being 1%, more every 24h is in the centrifugal 5 ~ 10min of 12000r/min, collect supernatant, obtain plectasin fermentation liquid.
3. the preparation method of antibacterial peptide plectasin film former according to claim 1, is characterized in that, in described step B, described PVA:Tween-80: glycerol: CMC-Na is 5:1.5:7:2.1 by volume.
4. the preparation method of antibacterial peptide plectasin film former according to claim 1, is characterized in that, in described step B, the order number of described screen cloth is 35 ~ 45 orders.
5. the preparation method of antibacterial peptide plectasin film former according to claim 1, it is characterized in that, also step is comprised: test the adhesion of antibacterial peptide plectasin film former, adhesion testing procedure is: respectively 9 groups of antibacterial peptide plectasin film former are uniformly coated on 3 × 4cm after described step B
2the abdominal part film forming of rabbit, then will fold 0.5cm near rabbit abdomen inner opposite end film, at the uniform velocity drip in plastic bag with clip one plastic bag blend rubber pipe, and till film rupture, record weight, gained weight is the adhesion of antibacterial peptide plectasin film former.
6. the preparation method of antibacterial peptide plectasin film former according to claim 1, it is characterized in that, also step is comprised: test the breathability of antibacterial peptide plectasin film former, breathability testing procedure is: antibacterial peptide plectasin film former is spread evenly across 6 × 6cm after described step B
2rabbit abdominal part film forming, the film then choosing thickness 0.1 ~ 0.2 μm is tested; By anhydrous for 6g CaCl
2put into gas permeable devices, with formed film, bottle sealing is placed in the calorstat of 37 DEG C, weigh and record 1 time/h; Map with mass change and the relation of time, obtain the slope S of figure cathetus, and obtain the Air permenbility Q of thin film according to following formula;
Wherein, Q=(S/A) (g/h m
2), in formula: S is the slope of straight line; A is the test area of film.
7. the preparation method of antibacterial peptide plectasin film former according to claim 1, it is characterized in that, also step is comprised: the release of antibacterial peptide plectasin film former is tested after described step B, release testing procedure is: fill rabbit lower inner ring border with normal saline, rabbit is fixed in single chamber Transdermal diffusion cell, and control single chamber Transdermal diffusion cell and be in 37 DEG C of water-baths, 2mL antibacterial peptide plectasin film former is evenly coated in rabbit 3 × 4cm
2on the surface, draw 5mL liquid every 1/2h from opening for getting medicine, then supplement with 5mL normal saline; The liquid of sucking-off is injected quartz colorimetric utensil, measures absorbance OD value at wavelength X=595nm place, calculated the release concentration of antibacterial peptide plectasin film former by linear equation.
8. the preparation method of antibacterial peptide plectasin film former according to claim 7, it is characterized in that, described linear equation is drawn by following steps: bovine serum albumin standard substance being diluted with distilled water into concentration is 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL solution; Then measure absorbance OD value at λ=595nm place respectively to the solution of above-mentioned each concentration, with absorbance OD value for vertical coordinate, protein standard substance concentration is abscissa drawing standard curve, obtains the linear equation y=0.0069x+0.0419 of standard curve, R
2=0.9983, wherein, x is protein standard substance concentration, and y is absorbance OD value.
9. an antibacterial peptide plectasin film former, is characterized in that, the preparation method of the antibacterial peptide plectasin film former as described in described antibacterial peptide plectasin film former employing is as arbitrary in claim 1 ~ 8 is prepared from.
10. an application for antibacterial peptide plectasin film former, is characterized in that, will be used for preventing mammitis of cow by antibacterial peptide plectasin film former as claimed in claim 9.
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