CN108948163A - Queensland nut plant alexin and its application - Google Patents
Queensland nut plant alexin and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses Queensland nut plant defense fibroin and its applications, and amino acid sequence is as shown in SEQ ID No.1.And the nucleotide of albumen described in coding claim 1 is disclosed, degenerate sequence of the nucleotide sequence as shown in SEQ ID No.2 or with SEQ ID No.2 coding same protein.Australia plant defense fibroin of the invention can promote the proliferation of lymphocyte and leucocyte, red blood cell is maintained to stablize, inhibit the growth of pathogenic microorganisms, enhances humoral immunity (IgG, IgG1, IgG2a and specific antibody) and cellular immunity (CD4+, CD8+T cell), the immunocompetence and anti-microbial infection ability that improve people and animal.
Description
Technical field
The invention belongs to biotechnologys and genetic engineering field, and in particular to Queensland nut plant defense plain gene it is excellent
Change, saccharomyces cerevisiae expression building, gene expression technique and its people and animal disease resistant health care promote wound reparation, anti-inflammatory application
Method.
Background technique
Alexin (Defensin) is that the intracorporal cationoid of animal and plant that is widely present in of discovered in recent years resists
Bacterium active peptide.They are usually all 28~54 amino acid residue compositions, and intramolecular is formed rich in arginine and by cysteine
Intramolecular disulfide bond.They have the bactericidal activity of broad-spectrum high efficacy, can effectively kill gram-positive bacteria, Gram-negative
The microorganisms such as bacterium, certain fungies, conveyor screw, envelope virus, thus their research is had become one in current international academic community
A noticeable research hotspot.
Plant alexin molecular weight is small, isolates and purifies and examine that there are certain difficulties.Plant alexin in scientific research at present
Main source be chemical synthesis, but chemical synthesis is at high cost, measures small, is unfavorable for large-scale application.
INVSC1 (Thermofisher matches silent fly) is Wine brewing yeast strain, belongs to eukaryocyte.It is general to be directed to protokaryon
Biology antibiotic such as Ka Na and ammonia benzyl be invalid to yeast, therefore in order to prevent the prokaryotes such as Escherichia coli to yeast
Bacterial strain pollution is cultivated, the antibiotic of some ammonia benzyls and kanamycins is added, in the medium often to inhibit the pollution of bacterium
And growth.The suitable growth temperature of saccharomyces cerevisiae is 28-30 degree, and generally using 30 degree of cultures, temperature, which is more than 32 degree, be may cause
The death of cell.INVSC1 saccharomyces cerevisiae is the amphiploid yeast cells of fast growing, is the desirable strain of protein expression.
INVSC1 saccharomyces cerevisiae is His, Leu, Trp and Ura auxotrophic strain, in shortage histidine, leucine, tryptophan and urine
It can not be grown in the SC minimal media of pyrimidine.INVSC1 saccharomyces cerevisiae is ideal protein expression strain.
The mating carrier of INVSC1 is pYES3/CT, pYC2, pYES2 serial carrier.
Also gradually increase in relation to plant defense plain gene and other Cloning of Genes Related and the report of expression, but rarely found has
It closes and optimizes plant alexin small peptide gene by Codon degeneracy, and the highly expressed report in saccharomyces cerevisiae.
Summary of the invention
The technical problems to be solved by the invention are as follows: how a kind of new plant defense fibroin and nucleotides sequence are provided
Column, and the high efficient expression in saccharomyces cerevisiae.
The technical solution of the present invention is as follows: Queensland nut plant defense fibroin, amino acid sequence such as SEQ ID No.1 institute
Show.
The nucleotide of albumen shown in SEQ ID No.1 is encoded, nucleotide sequence is as shown in SEQ ID No.2 or and SEQ
The degenerate sequence of ID No.2 coding same protein.
A kind of recombinant vector, it includes nucleotide shown in SEQ ID No.2.Preferably, the recombinant vector is pYCa.
A kind of recombinant bacterium, it includes recombinant vector described above.Preferably, the recombinant bacterium is INVSC1 wine brewing ferment
It is female.
Albumen shown in SEQ ID No.1 or the nucleotide or above-mentioned recombination load for encoding albumen shown in SEQ ID No.1
The application of body or recombinant bacterium in preparation raising animal immune ability product.
Further, in application described above, the animal immune ability that improves is at least one in following M1-M5
Kind:
M1: inhibit the growth of pathogenic microorganisms；
M2: promote the increase of immunocyte；
M3: promote the immune response of vaccine；
M4: promote cellular immunity and/or humoral immunity；
M5: animal development and growth-weight gain are improved；
M6: promote wound reparation.
Further, the pathogenic microorganisms be Escherichia coli, salmonella, staphylococcus aureus or Pseudomonas aeruginosa,
The immunocyte is lymphocyte or leucocyte.
The biological agent of albumen containing amino acid sequence shown in SEQ ID No.1.
It is demonstrated experimentally that Queensland nut plant alexin protein molecular of the invention has the effect that promotion animal lymph
The increase of cell, red blood cell and leucocyte, the animal body endolymph for applying Queensland nut plant defense fibroin of the invention are thin
Born of the same parents, red blood cell and leucocyte content can be improved 8% or more；The obvious growth for inhibiting pathogenic microorganisms, applies Australia of the invention
The amount of the pathogenic microorganisms of Nut-Bearing Plants alexin protein can be reduced up to 20% or more；Promotion non-specific antibody (IgG, IgG1,
IgG2a) and the secretion of disease specific antibody, apply non-specific in the animal body of Queensland nut plant defense fibroin of the invention
40%-60% can be improved in the content of property antibody；Promote the expression of gene involved in immunity, and then improves animal and be immunized anti-infective energy
Power, the animal for applying Queensland nut plant defense fibroin of the invention attack malicious survival rate and significantly improve at least 60%, and growth increases
10% or more rate again.
Detailed description of the invention
Fig. 1 is the digestion and PCR result of target gene in AZJG-DEFENSIN building and transformant；
Fig. 2 is AZJG-DEFENSIN recombination yeast upgrading grain agarose gel electrophoresis verification result；
Fig. 3 is AZJG-DEFENSIN gene eukaryon Yeast expression RT-PCR electrophorogram；
Fig. 4 is that Validation in vitro AZJG-DEFENSIN stimulates mouse lymphocyte proliferation experiment result；
Fig. 5 is that bacteriostatic experiment verifies AZJG-DEFENSIN to Escherichia coli, salmonella, staphylococcus aureus, hammer
Bacterium inhibitory effect；
Fig. 6 is that peripheral white blood cells sum changes after experiment mice takes orally AZJG-DEFENSIN recombination yeast fermentation liquid；
Fig. 7 is that peripheral red blood cells sum changes after experiment mice injects AZJG；
Fig. 8 be experiment mice take orally AZJG-DEFENSIN recombination yeast fermentation liquid after peripheral blood CD4+ CD8+T cell it is total
The variation of several levels ratio；
Fig. 9 is that experiment mice takes orally peripheral blood serum IgG after AZJG-DEFENSIN recombination yeast fermentation liquid, IgG1, IgG2a water
Flat variation；
Figure 10 is that experiment mice takes orally changes of weight after AZJG-DEFENSIN recombination yeast fermentation liquid；
Figure 11 is that immunogene TNF-a changes after experiment mice takes orally AZJG-DEFENSIN recombination yeast fermentation liquid；
Figure 12 is that experiment mice takes orally inherent immunity pattern recognition receptors after AZJG-DEFENSIN recombination yeast fermentation liquid
The variation of TOLs protein level；
Figure 13 is that cytokine interleukin becomes after experiment mice takes orally AZJG-DEFENSIN recombination yeast fermentation liquid
Change；
Figure 14 is that gamma interferon changes after experiment mice takes orally AZJG-DEFENSIN recombination yeast fermentation liquid；
Figure 15 is to attack malicious lethality after experiment mice takes orally AZJG-DEFENSIN recombination yeast fermentation liquid；
Figure 16 is that external application promotes the reparation of mouse wound.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
PYESCTa carrier in following embodiments is Life technologies Products.
Escherichia coli standard bacteria (G in following embodiments-) it is ATCC (America Type Culture
Collection) product, catalog number 25922.
Escherichia coli drug-fast bacteria (G in following embodiments-) it is Sichuan University's Field of Animal Epidemic Disease Control and food safety Sichuan
Key lab of province provides, and the public can obtain the biomaterial from applicant.
Staphylococcus aureus standard bacteria (G in following embodiments+) it is ATCC (America Type Culture
Collection) product, catalog number 29213.
Staphylococcus aureus resistance bacterium (G in following embodiments+) it is that Sichuan University's Field of Animal Epidemic Disease Control and food are pacified
Full Key Laboratory of Sichuan Province provides, and the public can obtain the biomaterial from applicant.
Female ICR mice in following embodiments is Sichuan University's Experimental Animal Center.
Bacterial strain:
INV.SC1 saccharomyces cerevisiae auxotrophic strain is purchased from Invertrogene company
Escherichia coli Mach1-T1Phage Resistant is purchased from TransGene company
F-φ80(lacZ)ΔM15ΔlacX74hsdR(rkmk+)ΔrecA1398endA1tonA。
Carrier: empty plasmid pYES2/CT: it is purchased from Invertrogene company
Main agents
High fidelity PCR PRIME premierDNA polymerase and dNTP mixture: it is purchased from Fermentas；
Restricted type restriction endonuclease (Xho I, Xbal I): it is purchased from Fermentas；
IN-fusion links homologous recombination enzyme: praising purchased from Nanjing Nowe；
DNA Mini Kit (DNA MINI Extraction Kit): it is purchased from Omiga company；
LB, SOB, SOC, YPD culture medium are purchased from Transgene company；
2 expression plasmid of PYesCT is purchased from Invertrogene company；
SD-U uracil lacks selective medium and is purchased from Invertrogene company；
Saccharomyces cerevisiae competent reagent preparation box is purchased from Invertrogene company.
The clone of 1 Queensland nut plant alexin protein gene of embodiment
The sequence of Queensland nut plant alexin protein gene are as follows:
ATTCAGGGGTTTGTGCTTAAGCCACGGAACTGTGCTAATGTGTGTCGGACTGAGGGTTTTCCCGGCGGT
ACTTGCAAAGGGTTCCGGCGTCGTTGCTTCTGTGAGAAGAATTGTCAT
The amino acid sequence of the albumen Queensland nut plant alexin of coding are as follows:
IQGFVLKPRNCANVCRTEGFPGGTCKGFRRRCFCEKNCH (shown in SEQ ID No.1)
There is shortcoming when using Yeast System Expressing Heterogene destination protein, such as internal degradation, product are uneven
One, polymer formed, signal peptide plus not exclusively etc., cause expression protein structure significant difference.The mistake of expression product simultaneously
Degree glycosylation will lower the activity of albumen.The study found that the gene can not express in yeast, therefore utilize biological information credit
Analysis tool, further calculates expression efficiency, has carried out the optimization of gene order, sequence is as follows after optimization:
ATCCAAGGTTTCGTTTTGAAGCCAAGAAACTGTGCTAACGTTTGTAGAACTGAAGGTTTCCCAGGTGGTACTTGTAA
GGGTTTCAGAAGAAGATGTTTCTGTGAAAAGAACTGTCAC (shown in SEQ ID No.2), is named as AZJG-DEFENSIN
Gene.
Expression efficiency, which is promoted, after optimization causes 70%.
The amplification of AZJG-DEFENSIN gene
PCR primer design
Three primers, respectively P1, P2 and P3 are devised, their nucleotide sequence is as follows:
P1:5 '-GAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCT-3 ' (SEQ ID No.3)
P2:5’-AAAAGAGAGGCTGAAGCTATCCAAGGTTTCGTTTTGAAGCCAAGAAACTGTGCTAACGTTTGT
AGAACTGAAGGTTTCCCAGGTGGTACTTGTAAGGGTTTCAGAAGAAGATG-3’(SEQ ID No.4)
P3:5’-ACCTTCGAAGGGCCCTCTAGATCAGTGACAGTTCTTTTCACAGAAACATCTTCTTCTGAAACC
CTTACA-3’(SEQ ID No.5)
AZJG-DEFENSIN gene is overlapped amplification gained from primer P1+ (P2+P3) primer PCR；PCR reaction system
(template: 50ng, 20umol/L upstream and downstream primer each 2uL, 2xprime primer Mastermix25uL, dd water are extremely by 50uL
50uL).Reaction condition is as follows: 94 DEG C of 3min, 98 DEG C of 10s, 55 DEG C, 10s, 72 DEG C, 15s, totally 35 circulations；Last 72 DEG C of extensions
2min, 4 DEG C of preservations.Pcr amplification product takes 2uL to run electrophoresis verifying, residue plastic recovery kit on 1.5% Ago-Gel
Purifying.
First step reaction is by primer (P2+P3) mutually amplification gained P2+3, then second step reaction is by P1 and P3 primer amplification
Template P2+3, products therefrom P1+2+3。
By final pcr amplification product through 2% cool sepharose electrophoresis, the results showed that substantially 117bp has been obtained, with
It is expected that plant alexin gene size is consistent.
The building and identification of 2 pYCT2-a recombinant expression carrier of embodiment
By the clone products of AZJG-DEFENSIN gene XhoI and XbaI double digestion plasmid, target fragment and same is recycled
The secreted expression carrier pYCa of sample double digestion is connected under IN-FUSION connection enzyme effect, electroporated INVSC1 wine brewing ferment
Female competent cell, obtains pYCa-AZJG-DEFENSIN positive restructuring saccharomycete, is named as AZJG-DEFENSIN recombination ferment
Mother obtains positive restructuring transformant with yeast Selective agar medium SD-U plate screening；Plasmid PCR and XhoI are made in culture bacterium colony extraction
(Fig. 1) is identified with XbaI double digestion, and gel electrophoresis, which is shown at size, single band occurs, and size meets expection, it was demonstrated that
Recombinate pYCa plasmid construction success (Fig. 2)；Sequencing turns out to be correct recombinant plasmid.
Detection of expression of the target gene on rna level in 3 recombinant yeast of embodiment
AZJG-DEFENSIN recombination yeast is seeded in uracil auxotrophy culture medium 28 DEG C, 200rpm is overnight
Culture, centrifugation, obtains AZJG-DEFENSIN recombination yeast thallus.Thallus total serum IgE is extracted, with primer pair (F:5 '-
ATGTATAAGATGCAGCTCTTGT-3 ' (SEQ ID No.6) and R:5'-CTAATGGTGATGGTGATGAT-3'(SEQ ID
No.7 the expression quantity of target gene in AZJG-DEFENSIN recombination yeast)) is detected.The results show that AZJG-DEFENSIN is recombinated
There is purpose stripe size (122bp) consistent near the section marker band 100-150 in yeast.Show AZJG-DEFENSIN
AZJG-DEFENSIN gene in recombinant yeast is expressed.Referring to Fig. 3.
4 recombinant yeast of embodiment expresses the Tricine-SDS-PAGE analysis of plant alexin
PYCa-AZJG-DEFENSIN positive restructuring saccharomycete (yCY3/AZJG-DEFENSIN, yCY3/) is carried out a small amount of
Shaking flask inducing expression, the method is as follows:
From in picking positive single colonie access liquid selective medium containing 5mLSD-U, 30 DEG C were cultivated on selective plate
Night, seed liquor (OD6001.5): latter 4 DEG C, 5000r/min is centrifuged 3min, and with PBS cleaning 3 times, 4 DEG C of 3000rpm are centrifuged 2min；
Collection part thallus is forwarded to 5mLS.Gal-U liquid selective medium and cultivates again, makes OD6000.8 or so；Continuous induction table
Under the same conditions up to destination protein 40 hours (30 DEG C, 200r/min), with the egg of empty plasmid yCY3 saccharomyces cerevisiae inducing expression
It is white to be used as negative control.
The inducer at each time point is collected respectively, is centrifugated supernatant；Tricine-SDS- can be carried out after boiling centrifugation
PAGE electrophoresis detection.Tricine-SDS-PAGE is slightly modified according to experiment demand.De- tail ion uses three (methylol) amino
Glycine (Tricine) replaces glycine, can significantly improve the resolution ratio to small peptide, reduce the disperse degree of banding pattern.This examination
The method taken high concentration glue and add glycerol is tested, the pattern making according to " glue is concentrated in one squeegee one of separation gel " is discontinuously terraced
It spends glue and separates micromolecule polypeptide.AZJG-DEFENSIN polypeptide contains 39 amino acid, and molecular weight is about 7.9kDa；Tricine-
SDS-PAGE electrophoresis result shows, the AZJG-DEFENSIN plant defense fibroin and calculated value of recombinant yeast expression
It is consistent.
The purifying of 5 recombinant plant alexin of embodiment
4 DEG C of 5,000r/min of recombination Engineering Yeast bacterium induction broth are centrifuged 10min, and yeast cells training is collected by centrifugation
Supernatant is supported, negative control is done in empty plasmid yCY3 expression.
1cm diameter glass column is filled with Ni-NTA agarose agarose (being purchased from Qiagen company), with 100mM NiSO4Clearly
Wash charging；Again with buffer I (20mMNa2HPO4(contain 0.1% (v/v) Triton X-100,5mM glucose, mM
Imidazole resin column) is balanced.It after upper Centrifuge A sample liquid, is sufficiently rinsed with buffer I, uses the imidazole's containing 20mM instead
After buffer I is sufficiently eluted, then eluted with the buffer I of the imidazole of mM containing 50mM；Finally use buffer II (20mM
Na2HPO4,5mMglucose,0.5M(NH4)2SO4, the plant alexin of (v/v) the Triton X-100 of and 0.1% elution absorption
Molecule.
The In Vitro Bacteriostatic of 6 AZJG-DEFENSIN expression of recombinant yeast albumen of embodiment detects
1, the preparation of AZJG-DEFENSIN recombination yeast fermented supernatant fluid:
(1) by AZJG-DEFENSIN recombination yeast be inoculated in 3mL culture medium 1 (culture medium 1 be uracil auxotrophy
Culture medium), 28 DEG C, 200rpm overnight incubation activated spawn.
(2) bacterium solution for taking 300 μ L steps (1) to obtain is inoculated in the 100mL triangular flask of the culture medium of YPD containing 30mL, and 28
DEG C, 200rpm shaker fermentation culture 48h (OD600For 25).
(3) bacterium solution for taking 5mL step (2) to obtain, is centrifuged 2min under 12 000 × g, and obtained supernatant is named as
AZJG-DEFENSIN fermented supernatant fluid.
(4) AZJG-DEFENSIN fermented supernatant fluid carries out enzymatic treatment: first being surveyed in AZJG-DEFENSIN fermentation with pH test paper
The pH of clear liquid, then adjust AZJG-DEFENSIN fermented supernatant fluid to trypsase respectively with 2mol/L NaOH and 1mo/L HCl
The most suitable action pH 2.0 of most suitable action pH 7.0 and pepsin, makes the final concentration of 0.5mg/mL of its enzyme, and 37 DEG C of water-bath enzymes are anti-
1h is answered, the AZJG-DEFENSIN fermented supernatant fluid of trypsin treatment and the AZJG-DEFENSIN hair of pepsin are obtained
Ferment supernatant, it is spare in -20 DEG C of refrigerators.
According to the method described above, AZJG-DEFENSIN recombination yeast is replaced with into empty bacterium (hereinafter referred to as C), other steps are equal
It is constant, blank yeast fermented supernatant fluid C is respectively obtained, it is spare in -20 DEG C of refrigerators.
2, the yeast expressed AZJG-DEFENSIN of measurement pYCa-AZJG-DEFENSIN positive restructuring is to Escherichia coli mark
Quasi- bacterium (G-) (hereinafter referred to as S-G-), staphylococcus aureus standard bacteria (G+) (hereinafter referred to as S-G+), streptococcus and sand
Men Shi bacillus antibacterial situation, the specific method is as follows:
4 kinds of bacterium bacterial strain inoculations are activated and cultivate it first and are in exponential phase of growth (OD600About 0.5), it then uses
LB culture solution is diluted to OD600About 0.005, the bacterium solution after dilution is inoculated into 96 porocyte culture plates, the 100 every holes μ L/, often
A kind of a bacterium of 96 porocyte culture plates.Shown in Fig. 5.
It handles: 100 μ L sample liquid being added real in the following way containing germy 96 porocyte culture plates for each
It is mixed gently in verifying, each sample sets 3 repeating holes, and wherein sample liquid is respectively in the AZJG-DEFENSIN fermentation of step 1
Clear liquid (i.e. untreated AZJG-DEFENSIN fermented supernatant fluid), every kind of bacterium are all provided with 2 kinds of antibiotic gradients as positive control,
Every kind of bacterium is all provided with only plus LB culture solution is as negative control, and sets not bacteria-containing LB culture solution as blank control.
After 96 porocyte culture plates are set 37 DEG C of incubators incubations 2h, 16h, microplate reader (Bio-Reader3350) is used respectively
Detect every hole OD600Value.
S-G-And S-G+Four kinds of antibiotic gradients it is as shown in table 1, R-G-And R-G+Four kinds of antibiotic gradients such as table
Shown in 2.
Table 1, reference culture antibiotic gradient
Note: in table 1, " --- " indicates to be free of kanamycins.
Table 2, antibody-resistant bacterium antibiotic gradient
The results show that for each bacterium in these four bacterium, the OD of the various sample liquids of C600With the OD of negative control600
2h and when 16h without significant difference, show that C acts on the equal unrestraint of these four bacterium；The various samples of AZJG-DEFENSIN
The OD of liquid600In the OD of 2h and the empty map being substantially less than when 16h under the corresponding time600(P < 0.05), result when 16h is as schemed
5, show that AZJG-DEFENSIN fermented supernatant fluid has inhibiting effect, and AZJG-DEFENSIN fermentation supernatant to these four bacterium
Fungistatic effect when liquid is combined with antibiotic (ammonia benzyl mycin and kanamycins) is more preferable.
7 AZJG-DEFENSIN recombinant yeast tunning of embodiment is on lymphopoietic influence
1, the preparation of mouse lymphocyte
Aseptically, the more eyeball total 5mL of vein peripheral blood are taken with heparin tube (anticoagulant containing EDTA-2K), according to
It is thin that lymphocyte separation medium (using preceding 37 DEG C of preheating separating liquids and mixing fullys shake) operating procedure carries out separating mouse lymph
Born of the same parents.
2, AZJG-DEFENSIN recombinant yeast tunning In vitro biological activity detects
(1) by after above-mentioned isolated lymphocyte culture for 24 hours, the Landrace lymphoblast in culture dish is transferred to dry
In net 15mL sterile centrifugation tube, 1500rpm room temperature is centrifuged 15min and collects cell body.
(2) cell is washed with RPMI1640 complete medium (dual anti-, the 10% tire Swine serum containing mycillin), 2 times repeatedly,
1500rpm room temperature is centrifuged 15min and collects cell precipitation.
(3) with the RPMI1640 complete medium gravity treatment cell of-MM of α containing 20mg/mL and adjust number of cells be about 6 ×
106A/mL, obtains cell suspending liquid.
(4) be added according to typesetting into the every hole of 96 porocyte plates the cell suspending liquids of 75 μ L steps (3), 45 μ L sample liquid and
The RPMI1640 complete medium of 30 μ L α containing 20mg/mL-MM (methyl mannoside).Wherein sample liquid is respectively embodiment 6
AZJG-DEFENSIN fermented supernatant fluid, a kind of every sample liquid in hole, three multiple holes of every kind of sample liquid.It is respectively adopted and contains only
Blank control is used as in the cell suspending liquid of the RPMI1640 complete medium of 20mg/mL α-MM, PBS and step (3).It is placed in
5%CO2, 37 DEG C of cell incubator culture 48h.
(5) 96 porocyte plates are taken out, every hole is added 15 μ L CCK8 (Guangzhou Yi Yuan Biotechnology Co., Ltd) and mixes gently
After be put into 5%CO2, 37 DEG C of cell incubators continue to cultivate 2h, 96 porocyte plates are taken out, with microplate reader (Bio-Reader3350)
Detect every hole OD450。
AZJG-DEFENSIN fermented supernatant fluid and the as shown in Figure 4 to stimulation lymphoblast proliferative conditions result of C (walk
Suddenly blank control is used as in the cell suspending liquid of (3)).Without Protease Treatment and through trypsase and pepsin
Under, compared with control group zero load saccharomyces cerevisiae C fermented supernatant fluid, AZJG-DEFENSIN fermented supernatant fluid stimulates lymphoblast
Obtained lymphocyte dramatically increases (P < 0.05).Show that AZJG-DEFENSIN recombination yeast can significantly stimulate lymph thin
Born of the same parents are proliferated (P < 0.05).In Fig. 4, untreated fermented supernatant fluid of the expression without Protease Treatment.
8 AZJG-DEFENSIN recombination yeast tunning biological activity research in Mice Body of embodiment
1, the preparation of tunning
AZJG-DEFENSIN recombination yeast is activated and is inoculated in the 100mL triangular flask of the culture medium of YPD containing 30mL,
30 DEG C, 48h is cultivated under 220rpm, makes OD600About 25, obtain AZJG-DEFENSIN recombination yeast fermentation liquid.
According to the method described above, the PYCa that AZJG-DEFENSIN recombination yeast is replaced with to 1 step 1 of embodiment, does not contain
The empty plasmid yeast transfection group (hereinafter referred to as C) of target gene, other steps are constant, obtain C fermentation liquid.
2, ICR mice group is tested
50 18-20g, 3 week old Healthy female ICR mouse is taken, random 5 groups of grouping, every group of 10 mouse, group, which is numbered, is
1-5, wherein group 1, group 4 are C negative control group, group 3 is vaccine negative control group, and group 4, group 5 are experimental group.
3, mouse is raised
According to grouping situation, fresh fermentation broth is sent to Mouse Stomach with gastric perfusion needle, only, first time stomach-filling is remembered by 0.6mL/
It is stomach-filling the 0th day, stomach-filling is primary every three days, 4 weeks continuous (i.e. respectively in stomach-filling the 0th day, the 3rd day, the 6th day, the 9th day, the 12nd
It, the 15th day, the 18th day, the 21st day, the 24th day and the 27th day corresponding fermentation liquid of stomach-filling, each stomach-filling amount is 0.6mL/
Only).Fresh recombinant Saccharomyces cerevisiae bacterium solution fermentation is carried out to guarantee that recombinant protein is not metabolized before each animal stomach-filling.
Table 3, intragastric administration on mice
In table 3, stomach-filling is the vaccination ways of fermentation liquid, and 0.6mL/ is only the dosage of inoculation of fermentation liquid；Intramuscular injection is
The vaccination ways of vaccine, 0.2mL/ are only the dosage of inoculation of vaccine.
Respectively before stomach-filling, take every mouse within stomach-filling the 7th day, stomach-filling the 14th day, stomach-filling the 21st day and stomach-filling the 28th day
Tail vein carries out following experiment content: doing blood routine in blood counting instrument after 30 μ L whole bloods and the mixing of 30 μ L physiological saline；50
μ L whole blood does Flow cytometry；100 μ L whole bloods extract real-time quantitative gene involved in immunity after RNA；100 μ L whole bloods add 1mL
TRIZOL acutely mixes -80 DEG C and saves backup；200 μ L whole blood low-speed centrifugals collect blood plasma and detect antibody.
4, challenge viral dosage
The lethal Escherichia coli of high drug resistance are inoculated in the benzyl mycin of ammonia containing 0.1mg/ml and 0.1mg/ml kanamycins LB liquid
Activated in culture medium, the fresh bacterium solution after activation be inoculated in LB liquid medium 37 DEG C, 1500rpm cultivate to logarithmic phase from
The heart collects thallus, is resuspended with fresh LB liquid medium to 5.0 × 105CFU/ml obtains escherichia coli fermented broth.In advance
It is 0.1ml to the half lethal dose of 3 week old Healthy female ICR mouse that escherichia coli fermented broth is groped in experiment.Stomach-filling the 30th day exists
5 mouse peritoneal Escherichia Coli Injection fermentation liquids are randomly selected in every group, the injection volume of every mouse is half lethal dose, abdomen
The chamber injection same day is to attack after poison the 0th day.Per the incidence of mouse of observation for 24 hours, and mouse survival rate is counted, dissection is seen
Examine the variation of dead mouse internal.
By the lethal S. aureus Inoculate of high drug resistance in the benzyl mycin of ammonia containing 0.1mg/ml and 0.1mg/ml kanamycins
It is activated in LB liquid medium, the fresh bacterium solution after activation is inoculated in LB liquid medium 37 DEG C, 1500rpm is cultivated to right
The number phase, thalline were collected by centrifugation, is resuspended with fresh LB liquid medium to 5.0 × 105CFU/ml obtains golden yellow grape
Coccus fermentation liquid.Preliminary experiment gropes S. aureus fermentation liquid and is to the half lethal dose of 3 week old Healthy female ICR mouse
0.2ml.Stomach-filling the 30th day 5 mouse peritoneals to non-Escherichia Coli Injection fermentation liquid remaining in every group inject golden yellow grape
Coccus fermentation liquid, the injection volume of every mouse are half lethal dose, and the intraperitoneal injection same day is also to attack after poison the 0th day.It is every to see for 24 hours
The incidence of a mouse is examined, and counts mouse survival rate, the variation of dead mouse internal is dissected and observed.
5, analysis of experimental results
Every group of mouse peripheral blood leucocyte and change of red blood cell are as shown in Figures 6 and 7, and blank control indicates C fermentation liquid.As a result
It has been shown that, the content of experimental mice peripheral white blood cells are significantly higher than C negative control group and vaccine negative control group (P < 0.05).
Illustrate that AZJG-DEFENSIN recombination yeast tunning can effectively stimulate immune cell propagation.
Sample is handled referring to fluidic cell operating procedure, is protected from light machine testing Th on low temperature (CD4+ lymphocyte), Tc (CD8+
Lymphocyte) cell quantity.Fig. 8, which is shown, randomly selects CD4+ and CD8+ lymphocyte in every 10000 cells of mouse peripheral blood
Variation.As seen from the figure, it is right to be all remarkably higher than C feminine gender for the content of experiment mice the peripheral blood CD4+ and CD8+ after immunity inoculation
According to group and vaccine negative control group (P < 0.05), all reach peak value within 21 or 28 days after immune.Illustrate that AZJG-DEFENSIN is recombinated
Yeast tunning has the function of stimulation test mouse immune response.
By low-speed centrifugal collect blood plasma according to ELISA kit operating procedure detection non-specific antibody IgG,
IgG1, IgG2a, and the antibody titer of specific antibody MP antibody is detected, the kit of use is respectively mouse immune globulin
G1 (IgG1) ELISA kit (article No. 69-210245), mouse immune globulin G (IgG) ELISA kit (article No. 59-
20037), mouse immune globulin G (IgG2a) ELISA kit (article No. 69-210250), porcine mycoplasmal antibody elisa reagent
Box (article No. 69-40349), above each kit are the silent picogram Biotechnology Co., Ltd product in Wuhan.Fig. 9 display is through immune
IgG, IgG1, IgG2a level are aobvious compared with C negative control group and vaccine negative control group in experiment mice peripheral blood serum after inoculation
It writes and increases (P < 0.05), all reach peak value within 14 or 28 days after immune.Illustrate AZJG-DEFENSIN recombination yeast tunning energy
It stimulates in immune Mice Body and generates more IgG, IgG1, IgG2a antibody.
As shown in Figure 10, experiment mice takes orally changes of weight after AZJG-DEFENSIN recombination yeast fermentation liquid；Figure 11 is real
Immunogene TNF-a changes after testing Mouse oral AZJG-DEFENSIN recombination yeast fermentation liquid.
As shown in figure 12, mouse inherent immunity expression of pattern recognition receptors level significantly improves, as shown in figure 13；Show to exempt from
Epidemic disease Mice Inoculated more rapidly can efficiently identify pathogenic microorganism, and inherent immunity ability gets a promotion, IL-1 principal biological function
Immunological regulation, collaboration stimulation APC and T cell activation can be carried out in local low concentration, promote B cell proliferation and secretory antibody.It is real
Mouse IL-1 secretion level (P < 0.05) can be obviously improved by testing novel antibacterial peptide gene as the result is shown.The rising explanation of IL-2
Novel defensin gene can promote inoculation animal the activation of immunocyte, promote the expression of cytokine profiles, especially
It is to promote cellular immunity.IL-7 significantly rises with IL-12, illustrates that immunological memory is likely to be obtained reinforcement.
Shown in Figure 14, at 21 days, IFN-γ reached peak, was higher than 10-20 times of control group (same time compares).CS abdomen
Chamber group is significantly higher than other group of average level.VAP, VADP are immunized mouse IFN-γ and significantly increase.New Fusion antibacterial peptide gene
Joint interleukin gene VAP2, VAP4/6 rise significantly, immune higher than the independent antibacterial peptide of VAP, VADP of same period
Group (P < 0.05).The rising of IFN-γ illustrates that novel antimicrobial peptide gene and interleukins joint coexpression vector are dynamic for being inoculated with
Lift is positively correlated in the result of object and above-mentioned CD4+T lymphocyte quantity and ratio, promotes 1 type immune response of Th.
Shown in Figure 15, challenge viral dosage uses two plant height drug-resistant bacterias: G+ bacterium: staphylococcus aureus, G- bacterium: Escherichia coli
It carries out after attacking poison, 12 hours, 24 hours, 48 hours to 5 days observation survival rates and lesion, death condition is spaced after intraperitoneal injection.
Immunized mice, each immunity inoculation experimental group pair can be effectively protected through New Fusion antibacterial peptide gene VADP as the result is shown
Staphylococcus aureus, Escherichia coli resistance significantly reinforce, the experimental group death rate 0/5 (G+) -0/5 (G-), and blank pair
According to group dead 4/5.
Fermentation liquid can promote mouse skin notch to repair rapidly, prevent wound infection and eliminate wound excessive inflammatory response.
From Figure 16, it is apparent that when AZJG external application concentration 20ug/ml, it can see that wound is formed a scab in third day, wound area is bright
It is aobvious to be less than control group.Prove that the molecule has the ability for promoting wound reparation.
Sequence table
<110>Sichuan hundred can fragrant Biology Pharmacy Co., Ltd
<120>Queensland nut plant alexin and its application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> PRT
<213> Macadamia ternifolia
<400> 1
Ile Gln Gly Phe Val Leu Lys Pro Arg Asn Cys Ala Asn Val Cys Arg
1 5 10 15
Thr Glu Gly Phe Pro Gly Gly Thr Cys Lys Gly Phe Arg Arg Arg Cys
20 25 30
Phe Cys Glu Lys Asn Cys His
35
<210> 2
<211> 117
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atccaaggtt tcgttttgaa gccaagaaac tgtgctaacg tttgtagaac tgaaggtttc 60
ccaggtggta cttgtaaggg tttcagaaga agatgtttct gtgaaaagaa ctgtcac 117
<210> 3
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaagaagggg tatctctcga gaaaagagag gctgaagct 39
<210> 4
<211> 113
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aaaagagagg ctgaagctat ccaaggtttc gttttgaagc caagaaactg tgctaacgtt 60
tgtagaactg aaggtttccc aggtggtact tgtaagggtt tcagaagaag atg 113
<210> 5
<211> 69
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
accttcgaag ggccctctag atcagtgaca gttcttttca cagaaacatc ttcttctgaa 60
acccttaca 69
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atgtataaga tgcagctctt gt 22
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ctaatggtga tggtgatgat 20
Claims (10)
1. Queensland nut plant defense fibroin, which is characterized in that its amino acid sequence is as shown in SEQ ID No.1.
2. encoding the nucleotide of albumen described in claim 1, which is characterized in that its nucleotide sequence is as shown in SEQ ID No.2
Or the degenerate sequence with SEQ ID No.2 coding same protein.
3. recombinant vector, which is characterized in that it includes nucleotide shown in SEQ ID No.2.
4. recombinant vector according to claim 3, which is characterized in that the recombinant vector is pYCa.
5. recombinant bacterium, which is characterized in that it includes recombinant vector described in claim 3 or 4.
6. recombinant bacterium according to claim 5, which is characterized in that the recombinant bacterium is INVSC1 saccharomyces cerevisiae.
7. albumen described in claim 1 or nucleotide as claimed in claim 2 or recombinant vector as claimed in claim 3 or power
Benefit require 4 described in recombinant bacterium or recombinant bacterium as claimed in claim 6 described in recombinant vector or claim 5 improved in preparation
Application in animal immune ability product.
8. application according to claim 7, which is characterized in that the raising animal immune ability is in following M1-M5
It is at least one:
M1: inhibit the growth of pathogenic microorganisms；
M2: promote the increase of immunocyte；
M3: promote the immune response of vaccine；
M4: promote cellular immunity and/or humoral immunity；
M5: animal development and growth-weight gain are improved；
M6: promote wound reparation.
9. application according to claim 8, which is characterized in that the pathogenic microorganisms is Escherichia coli, salmonella, gold
Staphylococcus aureus or Pseudomonas aeruginosa, the immunocyte are lymphocyte or leucocyte.
10. containing the biological agent of albumen described in claim 1.
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Cited By (2)
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| CN110577587A (en) * | 2018-06-07 | 2019-12-17 | 瑞鑫百奥生物科技（深圳）有限公司 | Isolated plant defensin polypeptide and preparation method and application thereof |
| CN114736308A (en) * | 2022-03-15 | 2022-07-12 | 魏于清 | Preparation and application of coccidian antigen peptide/IL 5 fusion protein gene engineering bacteria |
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| WO2003040297A2 (en) * | 2001-11-06 | 2003-05-15 | Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement (Cirad) | New protein of the defensin family from oil palm |
| CN101003809A (en) * | 2006-12-18 | 2007-07-25 | 广东省微生物研究所 | Method for preparing engineering bacterium for recombining plant alexin PD5 till its products |
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| CN110577587A (en) * | 2018-06-07 | 2019-12-17 | 瑞鑫百奥生物科技（深圳）有限公司 | Isolated plant defensin polypeptide and preparation method and application thereof |
| CN110577587B (en) * | 2018-06-07 | 2022-07-12 | 瑞鑫百奥生物科技（深圳）有限公司 | Isolated plant defensin polypeptide and preparation method and application thereof |
| CN114736308A (en) * | 2022-03-15 | 2022-07-12 | 魏于清 | Preparation and application of coccidian antigen peptide/IL 5 fusion protein gene engineering bacteria |
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