CN102584974B - Acipenser sinensis antibacterial peptide and preparation method and application thereof - Google Patents
Acipenser sinensis antibacterial peptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an acipenser sinensis antibacterial peptide and a preparation method and an application thereof, and particularly discloses a novel acipenser sinensis natural antibacterial peptide pen-hepcidin, an amino acid sequence for encoding a gene and an application of a protein to preparation of a medicament for treating infectious diseases and a feed additive. An antibacterial peptide gene pen-hepcidin is cloned from acipenser sinensis blood with an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, and the DNA (Deoxyribose Nucleic Acid) sequence of the antibacterial peptide gene pen-hepcidin is shown as a sequence SEQ ID:1 in a sequence table. The amino acid sequence of a protein (pen-hepcidin) encoded by using the gene is shown as SEQ ID:3 in a sequence table. According to amino acid sequence analysis, a protein polypeptide which is similar to the antibacterial peptide is not found in a protein database, and belongs to a novel antibacterial peptide. The antibacterial peptide disclosed by the invention has antibacterial activity on a part of Gram-positive bacteria and Gram-negative bacteria, has a remarkable effect on drug-resistant strains, and can be used for preparing a medicament for treating infectious diseases or applied as antibacterial feed additives and the like.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of new natural antibacterial peptide, specifically refer to a kind of mandarin sturgeon antibacterial peptide and its preparation method and application.
Background technology
Antibacterial peptide is the small molecule polypeptide, and generally by forming less than 100 amino-acid residues, great majority are with a certain amount of positive charge.Antibacterial peptide has anti-bacteria, fungi, protobiont (Nicolas et al., 1995) and antiviral (Mohan et al., 2010), antitumor (Papo et al., 2005) isoreactivity, a class broad-spectrum antimicrobial polypeptide, and nontoxic to eukaryotic cell.Antibacterial peptide becomes the new focus of Recent study at present, and the content of research comprises: the isolation and purification of antibacterial peptide, the analysis of aminoacid sequence, the expression level of antibacterial peptide in each tissue, the relation of protein configuration and function, the mechanism of action of antibacterial peptide (Cammue et al., 1994; Cuesta et al., 2008), animals and plants Cecropin-GM gene engineering (Li Wenchu etc., 1996), transformation synthetic antibacterial peptide gene and using gene engineering are expressed antibacterial peptide gene.
Antibacterial peptide is owing to having unique bactericidal mechanism, stable physico-chemical property, broad spectrum antibiotic activity, be not easy to produce the characteristics such as resistance, the potential substitute that is considered to antibacterials, the antibacterial peptide that is present on animal body is immune important component part, has important biological action, be mainly manifested in have broad spectrum antibiotic activity, several aspects such as antivirus action, protozoacide effect, anti-tumor activity, immunological adjuvant.
Antibacterial peptide has broad spectrum antibiotic activity.The antibacterial of antibacterial peptide has been reported in a large amount of research, and nearly all antibacterial peptide has restraining effect to some gram-positive microorganisms and negative bacterium.The amino acid of antibacterial peptide is with positive charge, and the bacterial cell membrane lipid is rich in the phosphatide headgroup and with negative charge, therefore antibacterial peptide can optionally be adsorbed on the bacterial cell membrane surface, the degradation of cell film, antibacterial peptide epinecidin-1 and similar synthetics thereof have been proved, can destroy Gram-negative bacteria adventitia (Pan et al., 2009).Studies show that can the degrade adventitia (Pan et al., 2010) of riemerella anatipestifer of synthetic epinecidin-1, TH1-5 and TH2-3 polypeptide.
The result of study of Japan flatfish hepcidin shows that 26 amino acid whose JF2 small peptides have anti-microbial activity to intestinal bacteria, streptococcus aureus and Lactococcus garvieae, but Edwardsiella tarda is not had anti-microbial activity; Another 19 amino acid whose JF1 small peptides do not show anti-microbial activity (Hirono et al., 2005).Express the transgenic zebrafish of liver of hybrid tilapia antimicrobial polypeptide (TH) 2-3, the infection (Hsieh et al., 2010) that can resist the Gram-negative bacteria vibrio marinopraesens.Tilapia, zone (Douglas et al., 2003 of coding hepcidin multiple copied have been found on the fish of Atlantic salmon and some other kind; Huang et al., 2007).In addition, bacterial endotoxin and lipopolysaccharides (LPS) can be induced THs, the expression of TH2-3 gene, but but can not induce TH1-5, the expression of TH2-2 gene (Huang et al., 2007), may be that the multiple copied of hepcidin develops into different functions in the fish evolutionary process, and hepcidin main iron ion regulation and control and host defense of participating in Mammals.Different from Mammals is, the function of JF1 may be the iron ion regulation and control, and the function of JF2 may be antibiotic, certainly also requires further study and confirms this hypothesis.At present, do not have in Protein Data Bank to find about the protein and peptide similar to the mandarin sturgeon natural antibacterial peptide.
Summary of the invention
Exactly purpose of the present invention will provide a kind of mandarin sturgeon antibacterial peptide and this amino acid whose gene order of coding, total and method and the application of this protein polypeptide in preparation treatment infectious disease medicament and antiseptic feed additive of cloning acquisition this antibacterial peptide gene pen-hepcidin and recombinant expression protein thereof from mandarin sturgeon blood RNA.
The present invention is achieved through the following technical solutions: a kind of mandarin sturgeon antibacterial peptide provided by the invention, and its special character is: its nucleotide sequence is as shown in sequence table SEQ NO:1; The nucleotide sequence of insertion vector is as shown in sequence table SEQ NO:2; Its aminoacid sequence is as shown in sequence table SEQ NO:3; The primer of its amplification pen-hepcidin gene, its DNA sequence dna is as follows:
Forward primer: 5 ' ACAYCAGAACWAACAYTC3 ',
Reverse primer: 5 ' CYACTTTTAYAAGGCWTA3 '.
As preferred version, the aminoacid sequence of its DNA sequence dna and coding thereof is as follows:
DNA sequence dna: GAA TTC AGC AGA ACG AAC ACT CGA CAG AAA CAACAG AAA CAC AGG GAC AAA CAA ATC CCC TGG CAT TTT TCA GGACAA AAC GTC AAA GCC ATC TTT CCC TGT GCA GAT ACT GCT GCAACT GCT GTC GTA ACA AAG GCT GTG GAT ATT GCT GTA AAT TAA
The corresponding aminoacid sequence of coding:
SRTNTRQKQQKHRDKQIPWHFSGQNVKAIFPCADTAATAVVTKAVDIAVN
The present invention also provides a kind of method for preparing mandarin sturgeon antibacterial peptide Nucleotide, and its special character is that it comprises the following steps:
(1) design PCR primer, the DNA sequence dna of described primer pair is as follows:
Forward primer: 5 ' ACAYCAGAACWAACAYTC3 ',
Reverse primer: 5 ' CYACTTTTAYAAGGCWTA3 ';
(2) extract the total RNA of mandarin sturgeon blood, the laggard performing PCR amplification of reverse transcription, PCR product purification and order-checking obtain the nucleotide sequence as shown in sequence table SEQ ID NO:1.
The present invention also provides a kind of application of mandarin sturgeon antibacterial peptide, and its special character is described mandarin sturgeon antibacterial peptide in the medicine of preparation treatment gram-positive microorganism, Gram-negative bacteria or infection of drug-resistant bacteria disease or as the application of fodder additives.
The microorganism that the present invention uses:
Pichia pastoris GS115/pPICZ α A-pen-hepcidin Pichiapastoris GS115/pPICZ α A-pen-hepcidin, be deposited in Chinese Typical Representative culture collection center on January 11st, 2012, it is referred to as CCTCC, and deposit number is NO.M2012007.
The present invention's RT-PCR method, the clone obtains this antibacterial peptide gene pen-hepcidin and recombinant expression protein thereof from the total RNA of mandarin sturgeon blood.
According to the conservative property of sequence, design a pair of degenerate primer (F1, R1); Express primer (F2, R2) and add EcoR I and Not I restriction enzyme site, and add terminator codon in downstream primer.Design of primers is as follows:
F1：5’-ACAYCAGAACWAACAYTC-3’
R1：5’-CYACTTTTAYAAGGCWTA-3’
F2：5’-CGGAATTCAYCAGAACWAACAYTCG-3’
EcoR?I
R2：5’-TAGCGGCCGCTTAATTTACAGCAATATCCAC-3’
Not?I
With expressing primer (F2, R2) recombinant plasmid is carried out pcr amplification, purpose fragment and expression vector pPICZ α A pass through restriction enzyme EcoR I and Not I substep double digestion, 16 ℃ of connections of T4 ligase enzyme are spent the night, transform intestinal bacteria TOP10, build eukaryotic expression recombination plasmid pPICZ α A-antibacterial peptide, and send the order-checking of the large genome company of China to identify.
Recombinant expression plasmid pPICZ α A-antibacterial peptide is by the linearizing of BstX I single endonuclease digestion, electrophoresis, and purifying reclaims enzyme and cuts product.The Pichia yeast engineering GS115 of electricity transformed competence colibacillus, after transforming, coating contains the ZeocinTM substratum, the transformant that screening has high resistance.First use BMGY nutrient solution (containing glycerine) to activate and cultivate approximately 24h, OD
600When value reaches 2-6, collect bacterium liquid, change the BMMY nutrient solution, at 28 ℃, under the 250rpm condition, the beginning methanol induction is expressed.Every 24h, adding final concentration in the Erlenmeyer flask is 0.8% methyl alcohol, and replenishes appropriate sterilized water, induces simultaneously pPICZ α A empty carrier yeast.Collect the supernatant liquor after above-mentioned methanol induction is expressed after 72h, be that the concentrated yeast of 85% ammonium sulfate induces bacterium to express supernatant with saturation ratio, the yeast expressed supernatant liquor of recombinating is carried out bacteriostatic activity, inhibitory potency and thermal stability determination, tentatively judge the biological activity of mandarin sturgeon antibacterial peptide.
Antibacterial peptide of the present invention all has bacteriostatic activity to part gram-positive microorganism and negative bacterium, and Resistant strain is also had obvious effect, can be used for preparing the medicine for the treatment of infectious diseases or as application such as fodder additivess.
Description of drawings
Fig. 1: be mandarin sturgeon antibacterial peptide pen-hepcidin gene RT-PCR amplified production of the present invention.
Fig. 2: be in the present invention recombinant antibacterial peptide to the streptococcus aureus Antibacterial Activity
Fig. 3: be in the present invention recombinant antibacterial peptide to the intestinal bacteria Antibacterial Activity
Fig. 4: be in the present invention recombinant antibacterial peptide to the streptococcus agalactiae Antibacterial Activity
Fig. 5: be in the present invention recombinant antibacterial peptide to the Aeromonas sobria Antibacterial Activity
Fig. 6: be in the present invention recombinant antibacterial peptide to the Aeromonas hydrophila Antibacterial Activity
Fig. 7: the sequence table SEQ NO:1 that is nucleotide sequence in the present invention
Fig. 8: the sequence table SEQ NO:2 that is the nucleotide sequence of insertion vector in the present invention
Fig. 9: the sequence table SEQ NO:3 that is aminoacid sequence in the present invention
In figure: M:DL2000DNA standard in Fig. 1; 1,2: mandarin sturgeon antibacterial peptide pen-hepcidin amplified production.
In Fig. 2, recombinant antibacterial peptide is to the streptococcus aureus Antibacterial Activity: 1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-pen-hepcidin transformant induced product I; 4:pPICZ α A-pen-hepcidin transformant induced product II.
In Fig. 3, recombinant antibacterial peptide is to the intestinal bacteria Antibacterial Activity: 1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-pen-hepcidin transformant induced product I; 4:pPICZ α A-pen-hepcidin transformant induced product II.
In Fig. 4, recombinant antibacterial peptide is to the streptococcus agalactiae Antibacterial Activity: 1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-pen-hepcidin transformant induced product I; 4:pPICZ α A-pen-hepcidin transformant induced product II.
In Fig. 5, recombinant antibacterial peptide is to the Aeromonas sobria Antibacterial Activity: 1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-pen-hepcidin transformant induced product I; 4:pPICZ α A-pen-hepcidin transformant induced product II.
In Fig. 6, recombinant antibacterial peptide is to the Aeromonas hydrophila Antibacterial Activity: 1: penbritin 10 μ g; 2:pPICZ α A yeast transformant induced product; 3:pPICZ α A-pen-hepcidin transformant induced product I; 4:pPICZ α A-pen-hepcidin transformant induced product II.
Embodiment
Embodiment 1: the clone of mandarin sturgeon antibacterial peptide gene
(1) design of primers
According to having logined fish hepcidin sequence information on NCBI/GenBank, according to the conservative property of sequence, with the upstream and downstream zone of Primer 5.0 softwares at cDNA sequence ORF, design a pair of degenerate primer (F1, R1); F1:5 '-ACAYCAGAACWAACAYTC-3 '
R1：5’-CYACTTTTAYAAGGCWTA-3’。
(2) the Trizol method is extracted the total RNA of blood
With aseptic disposable syringe draw blood, normal temperature is after standing for some time from the mandarin sturgeon caudal artery, and under normal temperature, the centrifugal 30min of 1000rpm, abandon supernatant liquor, and precipitation is hemocyte.
The glassware of experiment use is through 0.1% DEPC water treatment, 150 ℃ of baking 4h.Plastic ware spends the night through the 0.1%DEPC water soaking, 121 ℃, 20min autoclave sterilization.The metal apparatus soaks 2h through the NaOH of 1mol/L, after 0.01% DEPC water cleaning down, and 37 ℃ of oven dry.The 0.01%DEPC water configuration of each reagent without RNase.Total RNA extracting is undertaken by the Trizol specification sheets.
(3) the synthetic and pcr amplification of cDNA article one chain
1) synthetic the first chain of reverse transcription, carry out reverse transcription take the total RNA of mandarin sturgeon blood that extracts as template, and reaction system is 40 μ L, that is:
70 ℃ of lower 10min, ice bath is cooling immediately, adds respectively afterwards:
2) mixing, the reverse transcription reaction condition is: 42 ℃ of lower 20min, 70 ℃ of lower 15min, 4 ℃ of preservations.
3) pcr amplification goal gene carries out pcr amplification take reverse transcription product (cDNA) as template, and reaction system is 50 μ L, that is:
4) mixing.The pcr amplification reaction condition is: 94 ℃ of lower denaturation 3min; Then carry out 30 circulating reactions, its temperature cycle condition is 94 ℃ of lower sex change 30s, the 30s that anneals under 55 ℃, 72 ℃ of downward-extension 1min; Extend again 10min after loop ends under 72 ℃.
5) after amplification is completed, get PCR product 5 μ L with 6 * nucleic acid sample-loading buffer point sample, 1.0% sepharose, 1 * TAE, 120V, electrophoresis 20min observations.
(4) cloning and screening of goal gene
After getting pMD19-T carrier 1 μ L (50ng/uL) and the purpose fragment 3 μ L of glue recovery mixing, add 6 μ L Sultion I, the rearmounted 16 ℃ of connections of mixing are spent the night.Get 10 μ L and connect product, add under aseptic condition in intestinal bacteria TOP10 competent cell, repeatedly blow and beat mixing with the pipettor gentleness, ice bath is placed 30min.42 ℃ of water-baths, heat shock 90s, ice bath 3min makes it cooling immediately afterwards, notes not rocking.Shift bacterium liquid to 500 μ L are housed are preheated in the LB nutrient solution of 37 ℃, 37 ℃ of gentle vibration 45min of 150rpm make bacterium recover resistance.Add 40 μ L X-gal (20mg/mL), 4 μ LIPTG (200mg/mL) on the LB agar plate that contains AMP (100 μ g/mL), evenly coating, 37 ℃ place half an hour after, get 150 μ L bacterium liquid and coat on flat board.Be inverted plate and cultivate 12-16h in 37 ℃ of constant incubators, be placed in 4 ℃ blueness is fully manifested, choose white colony and be inoculated in the LB nutrient solution that contains 150 μ g/mLAMP, identify after violent jolting (230rpm) 12-16h.The clone that pcr amplification is positive gets bacterium liquid and send China large gene biological engineering service company, checks order with dideoxy chain termination.The CDS sequence that to obtain a length be 327bp.
(5) the DNA sequence dna homology search is identified
By the American National biotechnology (NCBI of information center, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) BLAST (the Basic Local Alignment Search Tool) software of website is analyzed survey antibacterial peptide aminoacid sequence.With the Megalign software in the DNAstar software package, by nucleotide sequence similarity between ClustalW method different plant species relatively.Simultaneously, carry out 1000 times with the method for bootstrapping (Bootstrap) and repeat, obtain consistent genealogical tree, and weigh the reliability of analytical results with index of conformity (consistency index, CI).
Sequence table SEQ ID: the aminoacid sequence that is the present invention's mandarin sturgeon antibacterial peptide gene of expressing;
Mandarin sturgeon pen-hepcidin nucleotide sequence:
ACAGCAGAACGAACACTCGACAGAAACAACAGAAACACAGGGACAAACAAATCCCCTGGCATTTTTCAGGACAAAACGTCAAAGCCATCTTTCCCTGTGCAGATACTGCTGCAACTGCTGTCGTAACAAAGGCTGTGGATATTGCTGTAAATTCTGATCACATGGGCATTTGATGTGCACTTGGGAAAAGAGTTTCTTTGAAACATTAACTTATTTGGAATTCTGTCTTCAAAATCCCTGCTGAAATGATTTTCCCTGTCGCACCATGTTGTTTAAAACGGGTATAAATGCAGGCTGTGTCTCCATGCA
TATGCCTTGTAAAAGTTG
The expression vector sequencing result:
GTGGAAACGACTTTTACGACACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACG?ATG?AGA?TTT?CCT?TCA?ATT?TTT?ACT?GCT?GTT?TTA?TTCGCA?GCA?TCC?TCC?GCA?TTA?GCT?GCT?CCA?GTC?AAC?ACT?ACA?ACAGAA?GAT?GAA?ACG?GCA?CAA?ATT?CCG?GCT?GAA?GCT?GTC?ATC?GGTTAC?TCA?GAT?TTA?GAA?GGG?GAT?TTC?GAT?GTT?GCT?GTT?TTG?CCA?TTTTCC?AAC?AGC?ACA?AAT?AAC?GGG?TTA?TTG?TTT?ATA?AAT?ACT?ACT?ATTGCC?AGC?ATT?GCT?GCT?AAA?GAA?GAA?GGG?GTA?TCT?CTC?GAG?AAAAGA?GAG?GCT?GAA?GCT?GAA?TTC?
AGC?AGA?ACG?AAC?ACT?CGA?CAG AAA?CAA?CAG?AAA?CAC?AGG?GAC?AAA?CAA?ATC?CCC?TGG?CAT?TTT TCA?GGA?CAA?AAC?GTC?AAA?GCC?ATC?TTT?CCC?TGT?GCA?GAT?ACT GCT?GCA?ACT?GCT?GTC?GTA?ACA?AAG?GCT?GTG?GAT?ATT?GCT?GTA AAT?TAA?GCG?GCC?GCC?AGC?TTT?CTA?GAA?CAA?AAA?CTC?ATC?TCAGAA?GAG?GAT?CTG?AAT?AGC?GCC?GTC?GAC?CAT?CAT?CAT?CAT?CATCAT?TGA
GTTTGTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCGGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGTGTCGTATTACCGTACTAAAGGTTTGAAAAGAAAAGAGACGCTCGTTCTTTTTCTCGTCGAAAAGCATAAAATTTTATCACGTTCTTTCTGAAATTTTTTTTTTGATTTTCCCTTCGATGACTCATGAATTAGTATACGGCTCATCCCAAGGTTCAGGTTCCATCGCCATGTCGATTAACAC
Embodiment 2: expression and the Analysis of Antimicrobial Activity of recombinant eukaryon expression vector in pichia spp
(1) linearizing of recombinant plasmid
Recombinant expression plasmid pPICZ α A-pen-epcidin and pPICZ α A empty carrier are cut with restriction enzyme BstX I enzyme respectively, and system is as follows:
45 ℃ of reaction 5h, enzyme is cut product and is carried out purifying with DNA cleaning agents box, and 20 μ LddH2O carry out wash-out ,-20 ℃ of preservations.
(2) linearizing recombinant plasmid electricity transformed yeast competent cell
Pichia pastoris GS115 competent cell (Pichia pastoris) 80 μ L are mixed mutually with pPICZ α A-antibacterial peptide plasmid (20 μ L) after BstX I linearizing, be transferred in the 0.2cm electricity revolving cup (Bio-Red) of precooling, be placed in 5min on ice, 2kV, 25 μ F, 400 Ω, shock by electricity after 8 milliseconds, the 1mol/L sorbyl alcohol that adds immediately the 1mL precooling, getting 200 μ L coats respectively on the YPDS flat board that contains ZeocinTM (100 μ g/mL, 500 μ g/mL, 1000 μ g/mL), cultivate 72h for 28 ℃, be cultured to single bacterium colony and occur.Electricity transforms the pPICZ α A empty carrier that does not add any foreign gene simultaneously.
(3) screening of positive yeast colony
After the positive bacterium colony of picking is used YPD+ZeocinTM 250rpm shaking culture, adopt to boil and freeze cooking method extraction recombination yeast genomic dna.With the centrifugal 5min of 1mL bacterium liquid 2500g, abandon supernatant, add 500 μ L TE to suspend in precipitation and precipitate; The centrifugal 3min of 2500g abandons supernatant; Repeat step once; Precipitation is dissolved in 100 μ L TE, boiling water bath 10min ,-80 ℃ of freezing 30min, boiling water bath 10min again at last; The centrifugal 5min of 1500g; Getting supernatant 2 μ l is that template is PCR.Carry out pcr amplification take the recombination yeast genome as template, reaction system is 20 μ L, that is:
Mixing.The pcr amplification reaction condition is: 94 ℃ of lower denaturation 3min; Then carry out 30 circulating reactions, its temperature cycle condition is 94 ℃ of lower sex change 30s, the 30s that anneals under 55 ℃, 72 ℃ of downward-extension 45min; Extend again 10min after loop ends under 72 ℃.
After amplification is completed, get PCR product 5 μ L with 6 * nucleic acid sample-loading buffer point sample, 1.0% sepharose, 1 * TAE, 120V, electrophoresis 20min observations.
(4) abduction delivering of high copy positive strain
Select PCR to be accredited as positive recombination yeast bacterium colony and be inoculated in the 250mL Erlenmeyer flask that contains 20mL BMGY substratum, 28 ℃, 250rpm shaking culture 12-16h makes OD
600=2-6, centrifugal collection thalline discards the BMGY substratum.Appropriate thalline is transferred in the 1000mL Erlenmeyer flask that contains 100mL BMMY substratum, and 4 layers of gauze wrapping bottleneck make initial OD
600It is 1 left and right.At 28 ℃, under the 250rpm condition, the beginning methanol induction is expressed.Every 24h, adding final concentration in the Erlenmeyer flask is 0.8% methyl alcohol, and replenishes appropriate sterilized water, induces simultaneously pPICZ α A empty carrier yeast.
(5) In Vitro Bacteriostasis experiment
After methanol induction 72h, supernatant is expressed in the centrifugal collection of 10000rpm, 0.45 μ m membrane filtration is expressed supernatant, stirs on ice to add solid ammonium sulfate, making saturation ratio is 85%, after ammonium sulfate dissolves fully, 4 ℃ of hold over night, the centrifugal 30min of 14000rpm Superfreezing abandons supernatant, with 5mLPBS solution dissolution precipitation, 4 ℃ of preservations.Same method concentrates the yeast expressed supernatant of pPICZ α A empty carrier.
Adopt the Oxford agar diffusion method to carry out Antibacterial Activity to the yeast expressed supernatant liquor of recombinating: line streptococcus aureus and intestinal bacteria on the LB substratum, rule on the BHI substratum streptococcus agalactiae, Aeromonas hydrophila and Aeromonas sobria are cultured to mono-clonal and grow.Dilute bacterial strain to be measured to OD with 0.65% stroke-physiological saline solution
600Be 0.5-1, draw 50 μ L bacterium liquid to be measured add to the liquid LB that is cooled to below 50 ℃ and BHI solid medium and in, be down flat plate after mixing.Place sterilized Oxford cup on the flat board that solidifies, add the abduction delivering supernatant of concentrated 20 times of 100 μ L in the cup of Oxford (if no special instructions, specimen in use is all supernatants of concentrated 20 times, lower summary), to add equal volume AMP (0.1mg/mL), the concentrated negative contrast of the yeast expressed supernatant of pPICZ α A empty carrier.Be cultured to thalline and grow, observe the inhibition zone size.Streptococcus aureus and intestinal bacteria culture temperature are 37 ℃, and streptococcus agalactiae, Aeromonas hydrophila and Aeromonas sobria culture temperature are 28 ℃.Antibacterial effect is seen accompanying drawing 2-6.
Claims (3)
1. mandarin sturgeon antibacterial peptide, its aminoacid sequence is as shown in SEQ ID NO:3.
2. the gene of coding claim 1 described mandarin sturgeon antibacterial peptide, its nucleotide sequence is as shown in SEQ ID NO:2.
3. the application of mandarin sturgeon antibacterial peptide claimed in claim 1 in the medicine of preparation treatment Staphylococcus, Colibacter, streptococcus, Aeromonas infectious diseases.
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