CN105647936A - Sparrow defensin gene and expression product - Google Patents
Sparrow defensin gene and expression product Download PDFInfo
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- CN105647936A CN105647936A CN201610095375.XA CN201610095375A CN105647936A CN 105647936 A CN105647936 A CN 105647936A CN 201610095375 A CN201610095375 A CN 201610095375A CN 105647936 A CN105647936 A CN 105647936A
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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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
Abstract
The invention discloses a sparrow defensin gene, an encoded amino acid sequence of the gene and mature peptide with an anti-bacterial function and further discloses a method for preparing defensin recombinant protein. The passwords of the defensin gene are modified and intercalated into an expression plasmid, and a recombinant expression plasmid is constructed; the recombinant expression plasmid is converted into escherichia coli, and genetically engineered bacteria efficiently expressing sparrow defensin are obtained. The sparrow defensin has good bacteriostatic activity for escherichia coli, staphylococcus aureus, candida albicans and the like, and efficient and environment-friendly antibacterial protein is provided for prevention and control of infection of livestock by pathogenic bacteria. The provided sparrow defensin is easy to prepare, has a wide antibacterial spectrum and is low in cost and high in safety.
Description
Field that the present invention belongs to:
The present invention relates to bioengineering field, specifically, the present invention relates to the preparation of a kind of Passeris montani saturati phylaxin gene and expression product thereof and application.
Background technology:
Alexin is a class Endogenous antimicrobial polypeptide, has the effect such as infection and immunomodulating, is the animal body natural weapons of resisting the invasion and attack of extraneous invasive organism. Alexinic antimicrobial mechanism is unique, is not likely to produce drug resistance, and safety is high, therefore has higher Development volue and good application prospect (higher primary school is gorgeous, 2010) in new drug development. Owing to the problems such as pathogen drug resistance, antibiotic remains take place frequently, develop one of antibacterial peptide medicament hot research direction having become biological technical field.
Difference according to alexinic aminoacid sequence and molecular structure, it is possible to Animal Defensins is divided into three major types: ��-alexin, beta-alexin and ��-alexin. Find have ��-alexin and beta-alexin in mammal body, in minority primate body, find there is ��-alexin. The Avian Defensins having been found that belongs to beta-alexin. Therefore, Avian Defensins is otherwise known as avian ��-defensins. The precursor of Avian Defensins generally comprises signal peptide, front fragment and mature peptide three part, mature peptide molecular weight about 3��4kDa. Wherein, mature peptide part is the part finally playing biologic activity. Avian Defensins has good antibacterial action, but the antimicrobial spectrum of different Avian Defensins and antibacterial activity are variant. At present, it has been reported in the poultry bodies such as chicken, duck, goose and has been found that multiple Avian Defensins or its gene. These alexinic expression have certain tissue specificity. Natural Avian Defensins expression is less, not easily obtains, and the alexinic cost of synthetic is too high again. Therefore, technique for gene engineering is adopted to prepare the research direction that Avian Defensins becomes desirable. Research shows, some Avian Defensins of gene engineering expression has antibacterial activity (flood victory brightness etc., 2010) equally, possesses the potentiality developing into antibiotic preparation.
China's livestock breeding industry is badly in need of the antibacterials Substitute For Partial antibiotic of environment-friendly high-efficiency, to alleviate the threat that environment and food safety are constituted by tradition antibacterials. Alexin etc.NovelThe research and development of antibacterials, can solve this problem well.At present, the alexinic research in China fowl source is started late, and pays close attention to face also narrower, is concentrated mainly on the alexin aspect in poultry source. Search out the fowl source alexin of antibacterial activity height, has a broad antifungal spectrum, and solve the problems such as its little, purification difficult of expression existed in artificial expression, be the key successfully developing fowl alexin medicine. In order to improve alexin expression, the conventional multi-copy in tandem of researcher express or with other protein fusion expression, but face expressing protein activity reduce, without antibacterial activity, need enzyme action to remove the problems such as fusion protein.
Inventive technique content:
An object of the present invention is in that to provide the aminoacid sequence of a kind of Passeris montani saturati phylaxin gene and coding thereof.
It is a further object to provide the high alexinic engineering strain of efficient expression Passeris montani saturati and construction method thereof, escherichia coli, staphylococcus aureus, Candida albicans etc. are had good bacteriostatic activity by the expression product of this bacterial strain, infect, for preventing and treating pathogen of domestic animal and poultry, the antibacterial protein providing a kind of high-efficiency environment friendly.
It is also another object of the present invention to provide and prepare the alexinic method of Passeris montani saturati with this engineered strain.
4th purpose of the present invention is the preventing and treating that Passeris montani saturati alexin is applied to that pathogen of domestic animal and poultry infects.
The invention discloses a kind of new Passeris montani saturati phylaxin gene SBD10, it is characterised in that the nucleotides sequence of this gene is classified as shown in SEQIDNO:1. Passeris montani saturati phylaxin gene SBD10 is obtained by RT-PCR technology: 1) according to the poultry alexin sequence included in GenBank, select sequence preservative section therein, with Primer5.0 software design primer. Forward primer is shown in SEQIDNO:4, and downstream primer is as shown in for SEQIDNO:5; 2) using Passeris montani saturati renal tissue total serum IgE as template, obtaining Passeris montani saturati phylaxin gene through two-step method RT-PCR amplification, the aminoacid sequence of gene code is such as shown in SEQIDNO:2, and its mature peptide playing antibacterial functions is shown in SEQIDNO:3.
The invention also discloses a strain capable of high-efficiency and express the alexinic genetic engineering bacterium of this Passeris montani saturati and construction method thereof. Passeris montani saturati phylaxin gene is obtained as template through two-step method RT-PCR amplification by Passeris montani saturati nephridial tissue total serum IgE, codon transformation insertion vector PET28a (+) in obtain recombinant expression plasmid PET28a-SBD10, convert the genetic engineering bacterium that e. coli bl21 obtains, strain name is escherichia coli PET28a-SBD10/BL21, EscherichiacoliPET28a-SBD10/BL21. This genetic engineering bacteriumPreservationIn Chinese Typical Representative culturePreservationCenter, preservation address is Wuhan, China university,PreservationNumbering CCTCCNO:M2016058,PreservationDate is on January 22nd, 2016.
Escherichia coli PET28a-SBD10/BL21,PreservationThe construction method of numbering CCTCCNO:M2016058 is: includes design of primers, construction of recombinant expression plasmid and host strain and converts, it is characterised in that:
1) design of primers described in is according to the poultry alexin sequence included in GenBank, selects sequence preservative section therein, with Primer5.0 software design primer,
Forward primer is such as shown in SEQIDNO:4, and downstream primer is such as shown in SEQIDNO:5;
2) construction of recombinant expression plasmid described in is using passeris montani saturati renal tissue total serum IgE as template, two-step method RT-PCR amplification is used to obtain Passeris montani saturati phylaxin gene SBD10, with Codon-adaptationtool software, SBD10 gene is transformed, remove the signal peptide in optimized gene, and BamH I and Hind III restriction enzyme site is designed at new gene fragment two ends, the improved gene of synthetic, with BamH I enzyme and Hind III enzyme double digestion, and digestion products is connected to carrier PET28a (+) double enzyme site on obtain recombinant expression plasmid PET28a-SBD10;
3) it is exactly be transformed in e. coli bl21 by recombinant expression plasmid PET28a-SBD10 that the host strain described in converts, and obtains expressing the engineered strain PET28a-SBD10/BL21 of Passeris montani saturati alexin SBD10 after resistance screening and checking,PreservationIt is numbered CCTCCNO:M2016058.
In the present invention, according to e. coli codon preference, with Codon-adaptationtool software optimization Passeris montani saturati phylaxin gene so that it is be more suitable at E. coli; The new gene that synthetic optimizes, removes signal peptide sequence, and designs BamH I and Hind III restriction enzyme site at gene two ends; Gene through BamH I and Hind III restriction enzymes double zyme cutting, reclaims the genetic fragment after enzyme action, with ligase connect expression vector PET28a (+); Product will be connected and convert DH5a escherichia coli, prepare recombinant expression carrier PET28a-SBD10 in a small amount; Recombinant expression carrier PET28a-SBD10 is converted BL21 escherichia coli, after resistance screening and enzyme action are identified, obtains expressing the engineered strain PET28a-SBD10/BL21 of SBD10.
The invention also discloses the preparation alexinic method of Passeris montani saturati, the method comprise the following steps: to be inoculated in engineered strain PET28a-SBD10/BL21 in the NaCl high salt LB culture medium containing 8g/L 37 DEG C of constant-temperature tables be cultured to OD600 be 0.6��0.8 time, add the derivant IPTG of final concentration of 0.4��0.8mM, 37 DEG C of abduction deliverings 3.5 hours, centrifugal collect thalline. Thalline is washed with the PBS of the pH8.0 of pre-cooling, then with Tris-NaCl buffer (20mMTris, 500mMNaCl, pH8.0) the resuspended thalline of pre-cooling, ultrasonication thalline, centrifugal collecting precipitation. With the resuspended resolution of precipitate inclusion body protein of the Tris-NaCl buffer containing 6M guanidine hydrochloride, centrifugal 10 minutes of 12000rpm. Its supernatant is the sample before purification, and supernatant, through Ni-Agarose media filtration, by the Tris-NaCl eluting target protein containing 100mM imidazoles renaturation, obtains Passeris montani saturati alexin recombiant protein SBD10.
The invention also discloses the Passeris montani saturati alexin SBD10 antibacterial activity to staphylococcus aureus, escherichia coli, streptococcus agalactiae and Candida albicans: when restructuring alexin concentration is 50 �� g/mL, the antibacterial activity of escherichia coli, streptococcus agalactiae and staphylococcus aureus can be reached more than 90%.
The invention have the advantage that
1) the Passeris montani saturati alexin of the present invention is the alexin in newfound a kind of wild fowl source, has broad spectrum antibiotic activity. The process building this Passeris montani saturati alexin expression vector optimizes gene order, and introduces two little purification tags, His label and T7 label, substantially increase the expression of target protein, it is ensured that the activity of albumen, make purification more convenient simultaneously.
2) the Passeris montani saturati alexin that the present invention expresses can produce in a large number, does not need special handling, directly apply after extraction;
3) the alexinic has a broad antifungal spectrum of Passeris montani saturati that the present invention expresses, is respectively provided with good inhibition to polytype pathogen such as escherichia coli, staphylococcus aureus and streptococcus agalactiaes; This Passeris montani saturati alexin has good effect to treating and preventing animal bacteria disease, the bacterial disease of chicken can be prevented, mammitis of cow also shows that good therapeutic effect, and this Passeris montani saturati alexin provides a kind of efficient new medicine for developing poultry anti-infectives and feed additive etc.
Accompanying drawing explanation
Fig. 1It it is the RT-PCR amplification electrophoresis of Passeris montani saturati alexin SBD10Figure.Wherein M represents DNAmarker2000, and 1 and 2 represent hepatic tissue, and 3 and 4 represent nephridial tissue, and 5 and 6 represent muscular stomach tissue, and 7 and 8 represent lung tissue.
Fig. 2The structural representation of recombiant plasmidFigure
Fig. 3It it is the enzyme action qualification of recombinant expression plasmid PET28a-SBD10Figure, wherein M represents DNALadder10000; 1 represents that recombinant expression plasmid is through BamH I and Hind III double digestion
Fig. 4It it is the western-blot qualification of recombiant protein SBD10Figure, wherein 1 represents target protein SBD10 specific band; 2 represent negative control
Fig. 5Represent the bacteriostatic activity detection of restructuring SBD10 albumenFigure, E.coli represents that escherichia coli, S.aureus represent that staphylococcus aureus, S.agalacttiae represent streptococcus agalactiae, and C.albicans represents Candida albicans.
Detailed description of the invention
Below in conjunction with experimental data and specific embodiment, the present invention is expanded on further. It should be noted that these embodiments are only for the present invention rather than restriction the scope of the present invention. Bacillus coli DH 5 alpha used in the present invention from China type culturePreservationCenter is enough bought.
The preparation of embodiment 1 reagent
Tris-Nacl buffer: containing 20mMTris, 500mMNacl, pH value 8.0.
Renaturation solution: containing 0.5M arginine, 1mMEDTA, 1mM reduced glutathion and 1mM oxidized form of glutathione, with above-mentioned Tris-Nacl buffer, adjusts pH value to 7.5.
Low concentration culture medium: commercial TSB culture medium, dilutes 1000 times with sterilized water.
PBS:NaCl8g, KCl0.2g, Na2HPO41.44g, KH2PO40.24g, adds ddH2O is settled to 1000ml, adjusts pH value to 7.2.
The clone and sequence of embodiment 2 Passeris montani saturati alexin SBD10 gene
(1) design of primers
According to the poultry alexin sequence included in GenBank, select sequence preservative section therein, with Primer5.0 software design primer. Forward primer is such as shown in SEQIDNO:4, and downstream primer is such as shown in SEQIDNO:5. The size of expection amplified production is about 207bp.
(2) total tissue RNA is extracted
Passeris montani saturati tissue sample total serum IgE is extracted: every 50mg tissue adds 500 �� LTrizol reagent, and room temperature places 5min so that it is fully crack with reference to Trizol method; 12000r/min is centrifuged 15min; Aspirate supernatant, is transferred in another EP pipe, adds the chloroform of 1/5 volume, and after vibration mixing, room temperature places 5min, 4 DEG C of centrifugal 10min of 12000r/min; Drawing upper strata aqueous phase, to another EP pipe, add the mixing of equal-volume isopropanol, room temperature places 30min; 4 DEG C of centrifugal 10min of 12000r/min; Abandoning supernatant, RNA is sunken at the bottom of pipe; Add 75% ethanol of lmL pre-cooling, 4 DEG C of centrifugal 5min of 7500r/min; Supernatant is abandoned in suction as far as possible, and low temperature drains 5��10min; Precipitation is dissolved in 50 �� LDEPC water; Measuring RNA concentration with nucleic acid-protein analyzer ,-80 DEG C frozen standby.
(3) synthesis of cDNA
The total serum IgE extracted with Oligo (dt) primer pair carried in RT-PCR kit carries out reverse transcription, reaction system is: dNTPMixture (10mMeach) 1.0 �� L, Oligo (dt) primer 1.0 �� L, RNA template 2.0 �� L, RNaseFreedH2O filling-in, total system 10.0 �� L.
After reaction system prepares, 65 DEG C of degeneration 5min in PCR instrument, 4 DEG C of annealing. Take out the reaction tube centrifugal several seconds, make reactant be gathered in bottom reaction tube. Then in above-mentioned 10.0 �� L solution, the reactant liquor of reverse transcription is added: 5 �� PrimScriptBuffer4.0 �� L, RNaseInhibitor (40U/ �� L) 0.5 �� L, PrimeScriptRTase0.5 �� L, RNaseFreedH2O5.0 �� L, total system 20.0 �� L.By centrifugal for the reaction tube several seconds, PCR instrument carries out reverse transcription: 42 DEG C of reaction 20min, 95 DEG C of reaction 5min, 4 DEG C of annealing.
(4) pcr amplification Passeris montani saturati alexin SBD10
Take in step (3) solution after reverse transcription reaction as the PCR template reacted, by following composition preparation PCR reactant liquor: above-mentioned transcribe after reactant liquor 4.0 �� L, 10 �� PCRBuffer5.0 �� L, dNTPMixture2.0 �� L, forward primer 0.5 �� L, downstream primer 0.5 �� L, TaKaRaExTaqHS
0.5 �� L, RNaseFreedH2O mends to overall 50.0 �� L.
Reaction tube is put the enterprising performing PCR reaction of PCR instrument: 95 DEG C of denaturation 4min; 94 DEG C of degeneration 30sec, 56 DEG C of annealing 30sec, 72 DEG C extend 40sec, totally 30 circulation; 72 DEG C extend 10min. PCR primer separates with the agarose gel electrophoresis of 1%, then PHOTOGRAPHIC ANALYSIS in gel imaging system. Result display obtain genetic fragment size be about 207bp (Fig. 1), wherein in nephridial tissue, the gene band of amplification is the most obvious, is consistent with expection.
(5) gene cloning and sequencing analysis
Reclaim test kit with DNA gel and reclaim the genetic fragment that amplification is arrived, product will be reclaimed and be connected with pMD18-T carrier. Connect product and convert bacillus coli DH 5 alpha, screening positive clone, send Wuhan to hold up the order-checking of Kechuang neoplasm Technology Co., Ltd.. Sequencing result is such as shown in SEQIDNO:1. With blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), sequencing result is compared, the homology of discovery and known Avian Defensins 10 is between 65%��81%, relatively low with the homology of other Animal Defensins, called after Passeris montani saturati alexin 10 (SBD10) then. Deriving aminoacid sequence with DNAMAN software is SEQIDNO:2), it has been found that this alexin has the characteristic amino acid structure of Avian Defensins, i.e. 6 conservative cysteine and " GXC " structure. Finding with SignalP4.1server software analysis, this alexinic precursor is made up of signal peptide, front fragment and mature peptide 3 part, wherein plays the sequence of mature peptide of antibacterial functions such as shown in SEQIDNO:3.
The structure of embodiment 3 recombinant expression carrier
(1) optimization of Passeris montani saturati alexin SBD10 gene and synthesis
With software Codon-AdaptationTool, the nucleotide sequence of SBD10 gene is optimized transformation, makes gene be more suitable at E. coli; Nucleotide fragments after synthetic optimization, comprises mature peptide but without signal peptide sequence, and introduces BamH I and Hind III restriction enzyme site at fragment two ends. New gene fragment sequence after optimization is such as shown in SEQIDNO:6
(2) structure of recombinant expression plasmid PET28a-SBD10
The genetic fragment of synthesis and pET-28a (+) plasmid carries out double digestion with BamH I and Hind III respectively, with DNA gel reclaim test kit reclaim the genetic fragment after enzyme action and pET-28a (+) plasmid. It is attached reaction: the plasmid 1.0 �� L of recovery, phylaxin gene fragment 4.0 �� L, T4DNALigase1.0 �� L, connection Buffer1.0 �� L, the ddH of 10 times by following system and condition2O2.0 �� L, cumulative volume 10.0 �� L, connect overnight in 16 DEG C of waters bath with thermostatic control. Recombiant plasmid called after PET28a-SBD10 after connection, its structural representationFigure is such as Fig. 2Shown in.
(3) screening is identified with enzyme action
Take bacillus coli DH 5 alpha competent cell 100 �� L, add the above product 5 �� L that connects and also mix, put on ice after 10min, 42 DEG C of heat shocks 90 seconds, ice bath 2min, then coats on the LB agar plate containing Kan.Just put 1h, then Flat plate turnover is being come for 37 DEG C, be inverted cultivation 12��16h, until obvious bacterium colony occurs. The single bacterium colony grown on picking flat board, is inoculated in the LB culture medium containing Kan, constant-temperature table shaken cultivation 8h. Take the bacterium solution after cultivation, extract recombiant plasmid with the little extraction reagent kit of plasmid, with restricted enzyme BamH I and Hind III carry out double digestion qualification (Fig. 3)��
The engineering bacteria of the high efficient expression Passeris montani saturati alexin SBD10 of embodiment 4 builds and abduction delivering
Extract the recombinant expression plasmid PET28a-SBD10 built in embodiment 2, convert e. coli bl21 (DE3) competent cell, screen positive bacterium colony with the LB agar plate containing Kan. The single bacterium colony grown on picking LB flat board, is inoculated in containing in the antibiotic LB fluid medium of Kan, reaches 0.8��1.0 in 37 DEG C of shaken cultivation to bacterium solution OD600 value, places 8��12 hours in 4 DEG C of refrigerators. Next day, by culture in 2% ratio (volume ratio) inoculate containing in the antibiotic fresh LB fluid medium of Kan, be about 3.0h in 37 DEG C of shaken cultivation, to OD600nm value reach 0.6��0.8 time, add derivant IPTG to final concentration of 0.4��0.8mmol/L, continue to cultivate about 3.5h. Centrifugal collect the thalline after induction, carry out western-blot detection with anti-His tag antibody as primary antibodie, observe albumen whether successful expression. Target protein size be about 9.1KD (Fig. 4)��
Embodiment 5 is recombinated the extraction of Passeris montani saturati alexin SBD10
Centrifugal for bacterium solution 12000r/min after abduction delivering in embodiment 3 5min is collected thalline, then extracts the recombiant protein of expression by the following method:
1) wash thalline with the PBS (pH8.0) of pre-cooling, then with the resuspended thalline of Tris-NaCl buffer of pre-cooling, ultrasonication thalline, the centrifugal 15min of 12000rpm collects precipitation.
2) resuspended with the Tris-NaCl buffer containing 6M guanidine hydrochloride of 1/10 volume, ice bath 2h dissolves inclusion body, and period shakes 4 times, centrifugal 10 minutes of 12000rpm. Take supernatant, use 0.45um membrane filtration.
3) supernatant is through Ni-Agarose media filtration, with the Tris-NaCl eluting target protein containing 100mM imidazoles, obtains Passeris montani saturati alexin recombiant protein SBD10.
4) recombiant protein SBD10 is loaded bag filter, 2 days renaturation of 4 DEG C of dialysis in renaturation solution.
Embodiment 6 recombinate alexin SBD10 antibacterial activity detection
Adopt the colony counting method inspection restructuring alexin SBD10 antibacterial activity to staphylococcus aureus (S.aureus), escherichia coli (E.coli), streptococcus agalactiae (S.agalacttiae) and Candida albicans (C.albicans). Bacterium source is in Chinese Typical Representative culturePreservationCenter catalogue, researcher can be toPreservationThis experiment of repetition is asked at center. Detection method is as follows:
(1) taking above bacterium and be inoculated in TSB fluid medium, 37 DEG C or the concussion of 28 DEG C of (Candida albicans) shaking tables are cultured to exponential phase, with low concentration culture medium dilution antibacterial to about 2.5 �� 10 after count of bacteria6CFU/mL��
(2) by PBS dilution restructuring alexin to 25 �� g/mL and 50 �� g/mL, draw in 250 �� L to 96 porocyte culture plates, then draw the 10 �� L antibacterial diluted and join in each hole. Arranging without alexinic PBS control group, the every hole of matched group adds the 10 �� L antibacterial diluted simultaneously. Every kind of antibacterial is cooked 3 repeating holes.
(3) sample adds after well, is placed on by culture plate in 37 DEG C of incubators and stands 2h. Then, with the PBS of sterilizing, sample in hole is made 10 times of gradient dilutions, take each 100 �� L of different dilution bacterium solution and be seeded on TSA flat board, 37 DEG C of incubators are cultivated 12��18h, observes and record the colony count on each TSA flat board.
(4) every kind of bacterium takes 3 and repeats on TSA flat boards the meansigma methods of clump count as the clump count (CFU) of this sample, the survival rate of each antibacterial after computing. The clump count of survival rate (the %)=sample of antibacterial/PBS control group clump count �� 100%. The survival rate assessment alexinic antibacterial activity of Passeris montani saturati according to antibacterial.
When restructuring alexin concentration is 50 �� g/mL, the bactericidal activity of escherichia coli, streptococcus agalactiae and staphylococcus aureus can be reached more than 90% by 37 DEG C of effect 2h, seesFig. 5��
The embodiment 7 alexin SBD10 that recombinates compares with the alexinic antibacterial activity in similar fowl source
With sterilized water, alexin SBD10 is diluted to 50 �� g/mL, checks the SBD10 bacteriostatic activity to staphylococcus aureus (S.aureus), escherichia coli (E.coli), streptococcus agalactiae (S.agalacttiae) and Candida albicans (C.albicans) by the colony counting method in embodiment 6. Set the chicken alexin 10 of synthetic, duck alexin 10 and goose alexin 10 as a comparison, different alexin 10 is all diluted to 50 �� g/mL simultaneously. Using PBS control group clump count as reference, calculate the bacteriostasis rate of each experimental group. Bacteriostasis rate (%)=(clump count of 1-experimental group/PBS control group clump count) �� 100%. Result shows, the inhibition of streptococcus agalactiae and Candida albicans is significantly stronger than the alexin 10 in other 3 kinds of poultry sources by alexin SBD10, also shows that stronger bacteriostasis rate to suppressing escherichia coli and staphylococcus aureus. It is specifically shown inTable 1��
Table 1The bacteriostasis rate of 4 kinds of antibacterials is compared by different fowl sources alexin 10
| Alexin | E.coli | S.aureus | S.agalacttiae | C.albicans |
| SBD10 | 96.4% | 90.0% | 92.1% | 86.2% |
| Chicken alexin 10 | 90.4% | 84.0% | 62.4% | 46.8% |
| Duck alexin 10 | 94.2% | 90.2% | 68.8% | 52.5% |
| Goose alexin 10 | 96.4% | 78.4% | 60.2% | 54.6% |
Embodiment 8 is recombinated the alexin SBD10 prevention for chicken bacterial disease
1 monthly age white meat-type chickens is randomly divided into experimental group and matched group, often group 100. Adding 0.4g alexin SBD10 in the every kilogram of feed of experimental group, matched group adds the fish flour of Isodose. Two groups of chickens are raised under same environment, same feeding method, observe with or without morbidity chicken and ill chicken and the chicken that dies of illness are made bacteriology checking, the incidence of the bacterial diseasees such as record chicken bacterial diarrhea, colibacillosis, staphylococcosis, Bacillus pasteurii disease, candidiasis, adds up the total incidence of two groups of chickens. Continuous Observation 6 months, the bacterial disease sickness rate of result display experimental group, significantly lower than matched group, illustrates that chicken bacterial disease is had preventive effect by alexin SBD10. ResultSuch as table 2��
Table 2Experimental chicken incidence is added up
Embodiment 9 is recombinated the alexin SBD10 treatment for mammitis of cow
Select the milch cow 10 suffering from clinic mastitis, be randomly divided into experimental group and matched group, often group 5. Experimental group adopts perfusion dose regimen in breast to inject alexin SBD10, the Mei Ru district 10ml of 100 �� g/mL. Matched group only irrigates normal saline. Treatment 7 days continuously, observe the PD situation of the mastitis of two groups of milch cows, add up cure rate. Result shows, experimental group has 4 and suffers from cattle and get well in 7 days, cure rate is apparently higher than 0 of matched group; The symptom of other 1 cattle of experimental group also alleviates to some extent, and matched group suffers from the symptom of cattle then without significant change. Illustrate that mammitis of cow is had good therapeutic effect by SBD10. Concrete outcome is shown inTable 3��
Table 3Alexin SBD10 treats the statistics of mammitis of cow
Claims (7)
1. a Passeris montani saturati phylaxin gene SBD10, it is characterised in that the nucleotides sequence of this gene is classified as shown in SEQIDNO:1.
2. the Passeris montani saturati alexin protein sequence of claim 1 coding is such as shown in SEQIDNO:2.
3. claim 1 encodes, and the Passeris montani saturati alexin mature peptide playing antibacterial functions is shown in SEQIDNO:3.
4. express the alexinic genetic engineering bacterium of Passeris montani saturati in claim 1 for one kind, it is characterized in that: obtained Passeris montani saturati phylaxin gene as template through two-step method RT-PCR amplification by Passeris montani saturati nephridial tissue total serum IgE, codon transformation insertion vector PET28a (+) in obtain recombinant expression plasmid PET28a-SBD10, convert the genetic engineering bacterium that e. coli bl21 obtains, strain name is escherichia coli PET28a-SBD10/BL21, EscherichiacoliPET28a-SBD10/BL21, this genetic engineering bacterium is preserved in China typical culture collection center, deposit number CCTCCNO:M2016058.
5. the method expressing the genetic engineering bacterium of Passeris montani saturati phylaxin gene SBD10 in preparation claim 4, converts including design of primers, construction of recombinant expression plasmid and host strain, it is characterised in that:
1) design of primers described in is according to the poultry alexin sequence included in GenBank, selects sequence preservative section therein, with Primer5.0 software design primer,
Forward primer is such as shown in SEQIDNO:4, and downstream primer is such as shown in SEQIDNO:5;
2) construction of recombinant expression plasmid described in is using Passeris montani saturati renal tissue total serum IgE as template, two-step method RT-PCR amplification is used to obtain Passeris montani saturati phylaxin gene SBD10, with Codon-adaptationtool software, SBD10 gene is transformed, remove the signal peptide in optimized gene, and BamH I and Hind III restriction enzyme site is designed at new gene fragment two ends, the improved gene of synthetic, with BamH I enzyme and Hind III enzyme double digestion, and digestion products is connected to carrier PET28a (+) double enzyme site on obtain recombinant expression plasmid PET28a-SBD10;
3) it is exactly be transformed in e. coli bl21 by recombinant expression plasmid PET28a-SBD10 that the host strain described in converts, obtaining expressing the engineered strain PET28a-SBD10/BL21 of Passeris montani saturati alexin SBD10 after resistance screening and checking, deposit number is CCTCCNO:M2016058.
6. the method that the genetic engineering bacterium described in claim 4 prepares Passeris montani saturati alexin SBD10, including inoculation, cultivation, induction, broken, purification and renaturation, it is characterized in that: first engineered strain PET28a-SBD10/BL21 is inoculated in high salt (NaCl containing 8g/L) LB culture medium, 37 DEG C of shaking tables are cultured to OD600 when being 0.6��0.8, add the derivant IPTG of final concentration of 0.4��0.8mM, 37 DEG C of abduction deliverings 3.5 hours, centrifugal collect thalline; The PBS (pH8.0) of pre-cooling washs thalline, then with Tris-NaCl buffer (20mMTris, 500mMNaCl, pH8.0) the resuspended thalline of pre-cooling, ultrasonication thalline, centrifugal collecting precipitation; With the resuspended resolution of precipitate inclusion body protein of the Tris-NaCl buffer containing 6M guanidine hydrochloride, centrifugal 10 minutes of 12000rpm, its supernatant is the sample before purification. Supernatant, through Ni-Agarose media filtration, with the Tris-NaCl eluting target protein containing 100mM imidazoles dialysis renaturation, obtains Passeris montani saturati alexin recombiant protein SBD10.
7. the effect that Passeris montani saturati alexin described in claim 2 infects at preventing and treating pathogen of domestic animal and poultry.
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