CN104774867A - Method for producing chicken beta antibacterial peptide Gal-3 by using alfalfa as bioreactor - Google Patents
Method for producing chicken beta antibacterial peptide Gal-3 by using alfalfa as bioreactor Download PDFInfo
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- CN104774867A CN104774867A CN201510039259.1A CN201510039259A CN104774867A CN 104774867 A CN104774867 A CN 104774867A CN 201510039259 A CN201510039259 A CN 201510039259A CN 104774867 A CN104774867 A CN 104774867A
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Abstract
The invention discloses a method for producing a chicken beta antibacterial peptide Gal-3 by using alfalfa as a bioreactor, and belongs to the technical field of plant transgenes. A plant expression vector pCAMBIA330-Gal-3 containing the chicken beta antibacterial peptide Gal-3 is obtained through substituting a plant expression vector pCAMBIA3301 by the optimized beta-Gal-3 gene; and the chicken beta antibacterial peptide Gal-3 is produced by planting transgenic alfalfa plants containing the recombinant chicken beta antibacterial peptide Gal-3. The transgenic alfalfa plants containing the recombinant chicken beta antibacterial peptide Gal-3 can be used as a feed additive.
Description
Technical field
The invention belongs to technical field of plant transgene, particularly a kind ofly utilize clover to produce the method for chicken β antibacterial peptide Gal-3 as bio-reactor.
Background technology
Antimicrobial peptide (antimicrobial peptides, AMPs), also known as antibacterial peptide (antibacterialpeptides; Or peptide antibiotic (peptide antibiotics) ABPs), it is the small active peptides that the class be extensively present in animal, plant and microbe has defense function, there is congenital immunity function, the effect of Developing restraint and deactivation can be produced pathogens such as the bacterium in intrusive body, fungi, virus and protozoons.
The research of chicken β antibacterial peptide starts from 1994, and Harwig etc. find 4 kinds of antibacterial peptides with Evans etc. at the white corpuscle of chicken, called after " gallinacins " (Gal-1, Gal-2, Gal-3 and Gal-1 α).Chicken β antibacterial peptide does not singly have antibacterium, mould, virus, spirochete activity, also has the function of antitumor cell toxicity, and take part in endogenous immunity and adaptive immunity.Because bird neutrophil cell does not have oxidation mechanism, so beta-defensin plays prior effect in bird congenital immunity.Oxidation mechanism relates to the effect of super oxidative ionic, hydrogen peroxide and myeloperoxidase, and non-oxidative mechanism is only completed by a few enzyme, cationic protein and polypeptide.The not super oxidative ionic of bird neutrophil cell and myeloperoxidase, they rely on non-oxidative mechanism more and play a role, and comprise the effect of N,O-Diacetylmuramidase, cationic protein and polypeptide.Chicken β antibacterial peptide is exactly a kind of cationic polypeptide, and it is the important immune factor of bird thus.
Leguminous plants is the main vegetable protein source of people and animals, alfalfa is a kind of excellent leguminous forage, have the laudatory title of " King of Pasture ", its suitable planting in extensive range, various abiotic stress can be tolerated, and have that biomass is large concurrently simultaneously, reproducibility is strong, can the speciality such as Multiple harvests, be therefore the desirable acceptor material carrying out plant bioreactor transgenic research and production.
Summary of the invention
For overcoming the shortcoming and deficiency that exist in prior art, primary and foremost purpose of the present invention is to provide a kind of plant expression vector containing recombination chicken β antibacterial peptide Gal-3.
Another object of the present invention is to the preparation method that a kind of transgenic alfalfa containing recombination chicken β antibacterial peptide Gal-3 is provided.
Another object of the present invention is to the transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 providing above-mentioned preparation method to obtain.
Another object of the present invention is to provide a kind of utilizes clover to produce the method for chicken β antibacterial peptide Gal-3 as bio-reactor.
Another object of the present invention is to provide the above-mentioned application of transgenic alfalfa plants in fodder additives containing recombination chicken β antibacterial peptide Gal-3.
Object of the present invention is achieved through the following technical solutions: a kind of plant expression vector containing recombination chicken β antibacterial peptide Gal-3, β-Gal-3 the gene be optimized according to alfalfa codon preference by coded amino acid replaces the gus gene between upper NcoI and BstEII of plant expression vector pCAMBIA3301, obtains the plant expression vector pCAMBIA330-Gal-3 containing recombination chicken β antibacterial peptide Gal-3;
The gene order of the β-Gal-3 gene that described coded amino acid is optimized according to alfalfa codon preference is as shown in SEQ ID NO.1; The amino acid whose sequence of its coding is as shown in SEQ ID NO.2.
A kind of preparation method of the transgenic alfalfa containing recombination chicken β antibacterial peptide Gal-3, concrete steps are for passing through agrobcterium-mediated transformation, the T-DNA region of the plant expression vector pCAMBIA330-Gal-3 containing recombination chicken β antibacterial peptide Gal-3 according to claim 1 is imported the blade of alfalfa, then screen on screening/regeneration culture medium, obtain the transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 of resistance regeneration.
The formula of described screening/regeneration culture medium is: MS substratum 4.74g/L, sucrose 30g/L, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L, KT 0.25mg/L, 125mg/L cef, 125mg/L carb, 1.5mg/Lbasta, pH=5.8, agar powder 8g/L.
A kind of transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 is obtained by above-mentioned preparation method.
Utilize clover to produce a method of chicken β antibacterial peptide Gal-3 as bio-reactor, by planting the above-mentioned transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3, producing chicken β antibacterial peptide Gal-3, obtaining the clover containing chicken β antibacterial peptide Gal-3.
The transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 of above-mentioned acquisition is applied as fodder additives.
The present invention has following advantage and effect relative to prior art:
The present invention utilizes alfalfa to produce the method for chicken β antibacterial peptide Gal-3 as bio-reactor, utilize agriculture bacillus mediated alfalfa genetic transformation that the T-DNA region of carrier pCAMBIA330-Gal-3 is imported the blade of alfalfa, and selection systems is carried out to the 3142 strain transfer-gen plants obtained, final acquisition 2 strain list copies and high expression level plant, identifies that the chicken β antibacterial peptide Gal-3 that it is expressed accounts for 0.17% and 0.26% of total soluble protein through Elisa.Through molecular weight cut-off be the poly (ether sulfone) film ultrafiltration of 3K and 10k centrifugal after obtain the chicken β antibacterial peptide Gal-3 that molecular weight is about 8KDa, antibacterial activity in vitro qualification shows that it significantly can suppress gold-coloured staphylococci, colon bacillus, Salmonella typhimurium bacteria growing.The alfalfa turning β-Gal-3 gene to be fed mouse as foodstuff additive, its mean body weight not genetically modified alfalfa of comparatively feeding obviously increases as the mouse of foodstuff additive, but intestinal microflora is without considerable change.
Accompanying drawing explanation
Fig. 1 is the structure iron of plant expression vector pCAMBIA330-Gal-3.
Fig. 2 is agriculture bacillus mediated alfalfa genetic transformation schema; Wherein, A is preculture, and B is infecting of callus, and C is the screening of embryoid, and D is differentiation culture, and E is root culture.
Fig. 3 is the southern results of hybridization figure of alfalfa transgenic plant; Wherein, M is trans15K DNA molecular amount ,+be positive plasmid ,-be negative plant, 1 is transformation event A91, and 2 is transformation event A375,3, be transformation event C19,4 is transformation event D57, and 5 is transformation event E245.
Fig. 4 is the Protein Detection result figure of single copy alfalfa transgenic plant; Wherein, M is Blue Plus II protein standard ,-be negative plant, 1 is transformation event A91, and 2 is transformation event A375,3, be transformation event C19,4 is transformation event D57, and 5 is transformation event E245.
Fig. 5 is that alfalfa turns β-Gal-3 gene plant bacteriostatic activity test result figure; Wherein, A is that the chicken β antibacterial peptide Gal-3 of alfalfa production is to the inhibition of gold-coloured staphylococci; B is that the chicken β antibacterial peptide Gal-3 of alfalfa production is to the inhibition of colon bacillus; C is that the chicken β antibacterial peptide Gal-3 of alfalfa production is to the inhibition of salmonella typhi.
Fig. 6 is that the chicken β antibacterial peptide Gal-3 of alfalfa expression is to the effect diagram of mouse intestinal flora.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1: the structure of plant expression vector pCAMBIA330-Gal-3
Under the prerequisite keeping aminoacid sequence (see sequence table SEQ ID NO.2) not change, the optimization design of gene order is carried out according to alfalfa codon preference, obtain the gold and silver sequence shown in SEQ ID NO.1, and the restriction enzyme site (NcoI and BstEII) carrying out vector construction is convenient in interpolation at gene two ends, the large fragment that this fragment discharges with the pCAMBIA3301 plasmid cut by same enzyme after NcoI with BstEII double digestion is connected.Connect at the enterprising row filter of LB solid medium containing kantlex after product conversion competent escherichia coli cell, picking positive colony and carry out confirmation sequence correctly after, called after pCAMBIA330-Gal-3 (see Fig. 1).
Embodiment 2: agriculture bacillus mediated alfalfa genetic transformation
2.1 substratum:
2.1.1 bacteria culture medium:
YEB liquid nutrient medium (1L): 5g extractum carnis, 1g Tryptones, 1g yeast extract, 5g/L sucrose, PH is 7.4; MgSO is added after sterilizing
4be 2mM to final concentration; 100mM MgSO
4solution, uses after high-temperature sterilization.
YEB solid medium: add the agar powder of 1.5% in YEB liquid nutrient medium.
2.1.2 plant tissue culture substratum
Infect substratum (1L): MS substratum 4.74g, sucrose 30g pH=5.6.
Preculture/Dual culture substratum (1L): MS substratum 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L, KT 0.25mg/L, pH=5.8.
Micro-organisms base (1L): MS substratum 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L, KT 0.25mg/L, 125mg/L cef, 125mg/L carb, pH=5.8, agar powder 8g/L.
Screening/regeneration culture medium (1L): MS substratum 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D2.00mg/L, KT 0.25mg/L, 125mg/L cef, 125mg/L carb, 1.5mg/L basta, pH=5.8, agar powder 8g/L.
Sprouting/root media (MSO substratum 1L): MS substratum 4.74g, sucrose 30g, 2mg/L basta, pH=5.8.
2.2 operation steps
2.2.1 preculture:
Alfalfa-leaves scalpel or operation are sheared or be cut into two halves, is inoculated on precultivation medium, culturing room's illumination cultivation 7 days (see Fig. 2 A).
Be ready for the engineering bacteria bacterium liquid of dip-dye simultaneously, EHA105 Agrobacterium with destination carrier is coated in containing 50mg/L rifampicin, 50mg/L kanamcin respectively, the YEB flat board of pH7.0 activates, picking list bacterium colony, OD600 value is cultured between 0.6-1.0 with YEB liquid nutrient medium, collect thalline, with dip-dye substratum resuspended to OD600 value be 1.0.
2.2.2 transform
By with cultivate after explant put into During Agrobacterium liquid and soak 10min.Discard bacterium liquid, be put on Dual culture substratum, light culture 3 days (see Fig. 2 B).
2.2.3 antibacterial:
After Dual culture, the MSO substratum of explant containing 250mg/L cef, 250mg/L carb is washed 3 times, blots, be inoculated on micro-organisms base with aseptic filter paper, light cultivates (16 h light 8 h dark) 7 days.
2.2.4 screen, regenerate:
Explant is transferred on screening/regeneration culture medium and screens, every two weeks subcultures once (see Fig. 2 C).
2.2.5 sprout, take root:
Treat that embryoid grows to about 1cm (usually oneself to come off from embryoid clump), be inoculated into germination medium and sprout (see Fig. 2 D).After about 20 days, embryoid is sprouted and is obtained regrowth, is cut by regrowth, is inoculated on root media take root (see Fig. 2 E).
Embodiment 3: the screening turning Gal-3 gene alfalfa high expression level plant
The blade turning Gal-3 gene Medicago sativa is cut, grind into powder in liquid nitrogen, add PBS damping fluid (137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/LNa in 1:2 (W/V) ratio
2hPO
4, 2mmol/L KH
2pO4), ice bath is to melting completely.Whirlpool 30sec; 4 DEG C, the centrifugal 20min of 15000r/min, get supernatant and be total soluble protein.
The leaves total protein coating buffer of extraction is diluted 100 times.Chicken β antibacterial peptide Gal-3 protein standard sample coating buffer (1.59g/L Na
2cO
3, 2.93g/L Na
2cO
3) be diluted to 2,500,1,250,625,312,156,78,39pg/mL (drawing standard curve).Get 100 μ l respectively in enzyme plate, 4 DEG C of bags are spent the night; Within second day, outwell sample solution, wash 3 times, each 4min with PBST damping fluid [PBS containing 1% (V/V) Tween20]; Add confining liquid [PBS containing 10% (M/V) skim-milk] 150 μ l, 37 DEG C of closed 2h; Outwell confining liquid, add primary antibodie (the mouse-anti chicken β antibacterial peptide Gal-3) working fluid of 100 μ l confining liquid dilutions as stated above after washing, 37 DEG C of bags are by 1h; Outwell primary antibodie working fluid, as preceding method washing, add two anti-(sheep anti mouses) of 100 μ l confining liquid dilutions, wrap by 1h; Outwell two anti-working fluids, as front method is washed, add developer 100 μ l, room temperature half-light reaction 15min; Add 50 μ l 2mol/L H
2sO
4termination reaction.And the 450nm light absorption value read in microplate reader is substituted into the content that typical curve calculates fibroblast growth factor in counter sample.According to the light absorption value drawing standard curve of different concns protein standard sample, as the R of typical curve
2during >0.98, calculate chicken β antibacterial peptide Gal-3 protein content in counter sample.
From 3142 strain transfer-gen plants, select according to Elisa detected result 5 strains that β antibacterial peptide Gal-3 expression amount is the highest, chicken β antibacterial peptide Gal-3 of its expression accounts for 0.07% (A91), 0.17% (A375) of total soluble protein, 0.26% (C19), 0.04% (D57) and 0.05% (E245).
Embodiment 4: the copy number analysis turning Gal-3 gene alfalfa high expression level plant
The blade 5 strains being turned Gal-3 gene alfalfa high expression level plant is cut, and in liquid nitrogen, grind into powder CTAB method extracts STb gene.Get 10 μ g DNA EcoRI enzymes to cut, 0.8% agar gel 80V electrophoresis is transferred to after 3 hours on nitrocellulose filter, and UV-crosslinked twice, each 30 seconds, 2 minutes, interval.The method provided according to DIGHigh Prime DNA Labeling and Detection Starter Kit I test kit carries out Southern blot analysis, and hybridization probe is that between 35S Promoter, β-Gal-3, NOS terminater, size is DNA fragmentation and the Trans15KMarker of 0.8kb.As shown in Figure 3, all transformation events except transformation event D57 are and singly copy transfer-gen plant results of hybridization.
Embodiment 5: the expression analysis and the separation and purification of chicken β antibacterial peptide that turn β-Gal-3 gene alfalfa high expression level plant
Wild-type alfalfa and the blade turning β-Gal-3 gene Medicago sativa are cut, grind into powder in liquid nitrogen, adds PBS damping fluid (137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na in 1:2 (W/V) ratio
2hPO
4, 2mmol/L KH
2pO
4), ice bath is to melting completely.Whirlpool 30sec; 4 DEG C, the centrifugal 20min of 15000r/min, get supernatant and be total soluble protein.
Get 10 μ l protein solutions to mix with equivalent 2 × sample-loading buffer, boiling water temperature bath 5min, after carry out 16.5%Tricine-SDS-PAGE electrophoresis detection, and to dye with coomassie brilliant blue R250.And the gel not carrying out coomassie brilliant blue R250 dyeing under the same terms is transferred on the nylon membrane of 0.2 μm by half-dried transferring film instrument under 200mA constant current conditions, 6 times are washed, each 5min with PBST damping fluid [PBS containing 1% (V/V) Tween20]; Add confining liquid [PBS containing 10% (M/V) skim-milk] 100mL, 37 DEG C of closed 2h; Outwell confining liquid, add primary antibodie (the mouse-anti chicken β antibacterial peptide Gal-3) working fluid of 100mL confining liquid dilution as stated above after washing, 37 DEG C of bags are by 1h; Outwell primary antibodie working fluid, as preceding method washing, add two anti-(alkali phosphatase enzyme mark, sheep anti mouses) of 100mL confining liquid dilution, wrap by 1h; Outwell two anti-working fluids, as front method is washed, add 10mL developer BCIP/NBT, the reaction of room temperature half-light is until after there is expection hybridization signal intensities, wash film 5min, with termination reaction with 50ml distilled water or TE damping fluid.Western is result as shown in Figure 4 again.
By total soluble protein after 0.22 μm of filter membrane suction filtration, be placed in the centrifugal 15min of ultra-filtration centrifuge tube 4000g that the molecular weight that dams is 10KD, getting effluent liquid, to be placed in the molecular weight that dams again be after the centrifugal 60min of ultra-filtration centrifuge tube 5000g of 3KD, retains partially liq and be chicken β antibacterial peptide Gal-3 purifying.Show that wherein chicken β antibacterial peptide Gal-3 purity reaches more than 90% through Elisa detected result, purification efficiency is greater than 40%.
Embodiment 6: the bacteriostatic activity analysis of the chicken β antibacterial peptide Gal-3 that transgenic alfalfa is expressed
It is 10 that bacterium liquid to be measured is adjusted concentration
6getting 1mL after individual/ml is coated on LB solid medium plate, and with punch tool, thick for 1mm filter paper is broken into the disk of diameter 5mm, it is immersed in respectively in the total protein of the wild-type alfalfa total protein of extraction and the transfer-gen plant of difference list copy transformation event, taking-up is placed on and fills gold-coloured staphylococci, colon bacillus, on the LB solid medium plate of salmonella typhi, 37 DEG C of incubated overnight, observe bacterial restrain, result as shown in Figure 5, the chicken β antibacterial peptide Gal-3 that the transgenic alfalfa of different single copy transformation event is expressed all can produce inhibition to three kinds of bacteriums.
Chicken β antibacterial peptide Gal-3 after purified is configured as certain storage liquid in order to using.Colony counting method is utilized to measure the minimal bactericidal concentration of several antibacterial peptide.Using 10mM sodium radio-phosphate,P-32 solution (pH 7.4) as diluent, doubling dilution is used to configure the antibacterial peptide solution of graded series successively.Get above-mentioned solution 100 μ l and be placed in 96 porocyte culture plates, then add isopyknic bacterium liquid (10 to be measured respectively
6individual/ml) in each hole.37 DEG C of constant temperature culture 12h.Then be placed in by Tissue Culture Plate in microplate reader and measure OD600 light absorption value, the less explanation fungistatic effect of light absorption value is better, measures the chicken β antibacterial peptide Gal-3 of alfalfa production according to this to the minimal bactericidal concentration (MIC) of different bacterium.Experimental result shows that the chicken β antibacterial peptide Gal-3 utilizing alfalfa to produce to the minimal inhibitory concentration of gold-coloured staphylococci, colon bacillus, salmonella typhi is according to this: 4,2,16 μMs.
Embodiment 7: the mouse experiment of the chicken β antibacterial peptide Gal-3 that transgenic alfalfa is expressed
By wild-type alfalfa with turn chicken β antibacterial peptide Gal-3 gene alfalfa and add in mouse feed according to the ratio of 1:250 (M/M), show through 30 days feeding experiment results, with the addition of in feed turn chicken β antibacterial peptide Gal-3 gene alfalfa Mouse Weight comparatively control group on average increase by 0.428 ± 0.162g/ days, and the Mouse Weight that with the addition of wild-type alfalfa in feed comparatively control group only on average increase by 0.167 ± 0.085g/ days.
Respectively using the tap water that the sterilized water containing 16 μMs of chicken β antibacterial peptide Gal-3 and 16 μM kantlex is tested with mouse as two groups, after one week, compare intestinal microflora.Intestinal microflora count results as shown in Figure 6, the mouse intestinal flora adding chicken β antibacterial peptide Gal-3 group in tap water comparatively control group without considerable change, and larger change has appearred in the mouse intestinal flora adding kantlex group in tap water, especially add white under its effect.Therefore, can think that the chicken β antibacterial peptide Gal-3 turning chicken β antibacterial peptide Gal-3 gene Medicago sativa and expressed by it has the potentiality of alternative conventional antibiotic as fodder additives.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. the plant expression vector containing recombination chicken β antibacterial peptide Gal-3, it is characterized in that: the β-Gal-3 gene be optimized according to alfalfa codon preference by coded amino acid replaces the gus gene between upper NcoI and BstEII of plant expression vector pCAMBIA3301, obtains the plant expression vector pCAMBIA330-Gal-3 containing recombination chicken β antibacterial peptide Gal-3;
The gene order of the β-Gal-3 gene that described coded amino acid is optimized according to alfalfa codon preference is as shown in SEQ ID NO.1.
2. the preparation method containing the transgenic alfalfa of recombination chicken β antibacterial peptide Gal-3, it is characterized in that: concrete steps are for passing through agrobcterium-mediated transformation, the T-DNA region of the plant expression vector pCAMBIA330-Gal-3 containing recombination chicken β antibacterial peptide Gal-3 according to claim 1 is imported the blade of alfalfa, then screen on screening/regeneration culture medium, obtain the transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 of resistance regeneration.
3. the preparation method of the transgenic alfalfa containing recombination chicken β antibacterial peptide Gal-3 according to claim 2, it is characterized in that: the formula of described screening/regeneration culture medium is: MS substratum 4.74g/L, sucrose 30g/L, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L, KT 0.25mg/L, 125mg/L cef, 125mg/L carb, 1.5mg/L basta, pH=5.8, agar powder 8g/L.
4. one kind is obtained by the preparation method described in Claims 2 or 3 containing the transgenic alfalfa plants of recombination chicken β antibacterial peptide Gal-3.
5. one kind utilizes clover to produce the method for chicken β antibacterial peptide Gal-3 as bio-reactor, it is characterized in that: by planting the transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 according to claim 4, produce chicken β antibacterial peptide Gal-3, obtain the clover containing chicken β antibacterial peptide Gal-3.
6. the transgenic alfalfa plants containing recombination chicken β antibacterial peptide Gal-3 according to claim 4 is applied as fodder additives.
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| CN109485704A (en) * | 2018-11-27 | 2019-03-19 | 温州大学 | A kind of expression system of meningococcus fHbp albumen |
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Application publication date: 20150715 |