CN105274134A - Preparation method and application of scylla paramamosain antimicrobial peptide SCY2 - Google Patents
Preparation method and application of scylla paramamosain antimicrobial peptide SCY2 Download PDFInfo
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Abstract
The invention relates to a preparation method and application of scylla paramamosain antimicrobial peptide SCY2. The preparation method comprises the following steps: establishing a scylla paramamosain antimicrobial peptide SCY expression vector, namely, designing an upstream primer, adding a base sequence corresponding to a amino acid sequence of a Kex2 enzyme digestion site before a 5'-end EcoRI enzyme digestion site when the upstream primer is designed, performing PCR amplification on the SCY2 gene, and inserting the SCY2 gene into a pPIC9K vector, thereby obtaining a pPIC9K-SCY2 recombinant expression vector; transferring the obtained recombinant expression vector into a host cell, performing inducible expression on the host cell, thereby obtaining an expression product; separating and purifying the obtained expression product, and performing N-end sequencing, thereby confirming that the obtained expression product is SCY2 with an N-end identical to that of natural protein. The scylla paramamosain antimicrobial peptide SCY2 can be used for preparing a bacterium growth inhibitor, and can be also used for preparing an animal feed additive and an anti-pathogenic microorganism medicine.
Description
Technical field
The present invention relates to Scylla paramamosain, especially relate to the expression vector establishment of Scylla paramamosain antibacterial peptide SCY2 and the preparation method of Pichia anomala expression product.
Background technology
Scylla paramamosain is the important mariculture kind of China's southeastern coast, and it has the features such as individuality is large, meat is good, nutritious, is loved by the people.In recent years, along with cultivation scale constantly expand, the appearance of the problem such as environmental pollution increases the weight of, a large amount of hyperplasia of pathogenic bacterium, Scylla paramamosain there occurs large-scale death, makes China's mariculture industry suffer huge financial loss.
Antibacterial peptide is the important Immunity active factor in Crustacean innate immunity, and the study of antimicrobial peptide from crustacea reported mainly comprises: penaeidin
[1,2], Crustin
[3-5], coagulogen (ALF)
[6-8], and the antibacterial peptide Scygonadin of the special high expression level of mud crab sexual gland
[9].Antibacterial peptide has broad application prospects as the medicine of anti-drug resistance bacterium and fodder additives in the diseases prevention and treatment of marine fish, crustaceans etc.Research, development and application antibacterial peptide, have important scientific meaning and using value to problems such as the generation and fishery products drug residue that solve the drug tolerant bacteria caused due to antibiotic abuse in sea farming exceed standard.
The applicant clones and obtains a novel antimicrobial peptide SCY2 in Scylla paramamosain, and use the display of ProtParam tool analysis result, SCY2 mature peptide contains 100 amino-acid residues, and molecular-weight average is 10925.4Da, PI is 4.84, is Anionic Antimicrobial Peptides.The applicant studies discovery simultaneously, and Scylla paramamosain antibacterial peptide SCY2 may play an important role in the reproduction immunity of mud crab
[10].
Reference:
[1]CuthbertsonBJ,BüllesbachEE,FievetJ,BachèreE,GrossPS.Anewclass(penaeidinclass4)ofantimicrobialpeptidesfromtheAtlanticwhiteshrimp(Litopenaeussetiferus)exhibitstargetspecificityandanindependentproline-rich-domainfunction.BiochemJ,2004,381(Pt1):79-86.
[2]LiCY,YanHY,SongYL.Tigershrimp(Penaeusmonodon)penaeidinpossessescytokinefeaturestopromoteintegrin-mediatedgranulocyteandsemi-granulocyteadhesion.FishShellfishImmunol,2010,28(1):1-9.
[3]ImjongjirakC,AmparyupP,TassanakajonA,SittipraneedS.MolecularcloningandcharacterizationofcrustinfrommudcrabScyllaparamamosain.MolBiolRep,2009,36(5):841-850.
[4]ArockiarajJ,GnanamAJ,MuthukrishnanD,GudimellaR,MiltonJ,SinghA,MuthupandianS,KasiM,BhassuS.Crustin,aWAPdomaincontainingantimicrobialpeptidefromfreshwaterprawnMacrobrachiumrosenbergii:immunecharacterization.FishShellfishImmunol,2013,34(1):109-118.
[5]KrusongK,PoolpipatP,SupungulP,TassanakajonA.AcomparativestudyofantimicrobialpropertiesofcrustinPm1andcrustinPm7fromtheblacktigershrimpPenaeusmonodon.DevCompImmunol,2012,36(1):208-215.
[6]LiuHP,ChenRY,ZhangQX,WangQY,LiCR,PengH,CaiL,ZhengCQ,WangKJ.Characterizationoftwoisoformsofantiliopolysacchridefactors(Sp-ALFs)fromthemudcrabScyllaparamamosain.FishShellfishImmunol,2012,33(1):1-10.
[7]LiuY,CuiZ,LiX,SongC,ShiG.Anewlyidentifiedanti-lipopolysaccharidefactorfromtheswimmingcrabPortunustrituberculatuswithbroadspectrumantimicrobialactivity.FishShellfishImmunol,2013,34(2):463-470.
[8]LuoT,YangH,LiF,ZhangX,XuX.Purification,characterizationandcDNAcloningofanovellipopolysaccharide-bindinglectinfromtheshrimpPenaeusmonodon.DevCompImmunol,2006,30(7):607-617.
[9]XuWF,QiaoK,HuangSP,etal.Quantitativegeneexpressionandinsitulocalizationofscygonadinpotentiallyassociatedwithreproductiveimmunityintissuesofmaleandfemalemudcrabs,Scyllaparamamosain.FishShellfishImmunol,2011,31(2):243-251.
[10] Qiao Kun. research [J] .2013. of Scylla paramamosain two kinds of sexual gland high expression level antibacterial peptide Scygonadin and SCY2 Property comparison and reproduction immunologic mechanism thereof
Summary of the invention
The first object of the present invention is the preparation method providing Scylla paramamosain antibacterial peptide SCY2.
The second object of the present invention is to provide Scylla paramamosain antibacterial peptide SCY2 preparing the application in bacterial growth inhibitors.
The preparation method of described Scylla paramamosain antibacterial peptide SCY2, comprises the following steps:
1) structure of Scylla paramamosain antibacterial peptide SCY2 expression vector: design upstream primer, before 5 ' end EcoR I restriction enzyme site, the base sequence corresponding to Kex2 restriction enzyme site aminoacid sequence is added during design, pcr amplification Scylla paramamosain antibacterial peptide SCY2 gene, described Scylla paramamosain antibacterial peptide SCY2 gene is inserted in pPIC9K carrier, obtains pPIC9K-SCY2 recombinant expression vector;
2) by step 1) recombinant expression vector that obtains is transformed into host cell, and abduction delivering is carried out to host cell, obtains expression product;
3) purification procedures 2) expression product of gained, then carry out the order-checking of N end, determine that obtained expression product is have the Scylla paramamosain antibacterial peptide SCY2 having identical N to hold with native protein.
In step 1) in, described primer sequence is as follows:
gggGAATTCGAGAAAAGAGGCCTGGCACTCAACAGACTTATG；
The Genbank accession number of described pcr amplification Scylla paramamosain antibacterial peptide SCY2 gene is EF555444.
Described expression vector is used for the Scylla paramamosain antibacterial peptide SCY2 sequence that expressed sequence is SEQIDNo.1.
The amino acid fragment sequence of described Scylla paramamosain antibacterial peptide SCY2 is SEQIDNo.2.
In step 3) in, the method for described separation and purification is by step 2) expression product of gained carries out centrifugal, and collect fermented supernatant fluid, after fermented supernatant fluid is dialysed, after affinity chromatography, namely obtain the Scylla paramamosain antibacterial peptide SCY2 of purifying.
Antibacterial experiment result shows, Scylla paramamosain antibacterial peptide SCY2 is to gram-positive microorganism, as micrococcus lysodeikticus has stronger anti-microbial activity (MIC=12.5 ~ 25 μM), there is certain anti-microbial activity (MIC=25 ~ 50 μM), to Gram-negative bacteria without anti-microbial activity (MIC>50 μM) to micrococcus luteus, Corynebacterium glutamicum.The SCY2 of visible eukaryotic expression has certain anti-microbial activity, and is better than the anti-microbial activity to Gram-negative bacteria to the anti-microbial activity of gram-positive microorganism.
Described Scylla paramamosain antibacterial peptide SCY2 can apply preparing in bacterial growth inhibitors.Described Scylla paramamosain antibacterial peptide SCY2 also can apply preparing in animal feedstuff additive and anti-pathogenic microorganism medicine.
Described bacterium is mainly gram-positive microorganism, as micrococcus luteus (Micrococcusluteus), Corynebacterium glutamicum (Corynebacteriumglutamicum) and micrococcus lysodeikticus (Micrococcuslysodeikticus).
The gene order of described Scylla paramamosain antibacterial peptide SCY2 is:
The aminoacid sequence of described Scylla paramamosain antibacterial peptide SCY2 is:
The present invention is intended to the eukaryotic expression product obtaining the Scylla paramamosain antibacterial peptide SCY2 with native protein with identical N-terminal, and identifies its anti-microbial activity, to developing the novel drugs of resisting pathogenic microbes.The present invention is according to Scylla paramamosain antibacterial peptide SCY2 gene sequence characteristic, by gene engineering expression technology, success obtains has bioactive Scylla paramamosain antibacterial peptide SCY2, it has the N end identical with native protein, checking Scylla paramamosain antibacterial peptide SCY2 has certain anti-microbial activity, for its exploitation as resisting pathogenic microbes new drug lays the foundation.
Accompanying drawing explanation
Fig. 1 is SCY2PCR electrophoretogram.M is DNAMarkerDL2000, and 1 is SCY2PCR fragment.
Fig. 2 is that pPIC9K expression vector enzyme cuts front and back electrophorogram.1 cuts front fragment for pPIC9K carrier enzyme, and M is DNAMarkerDL2000, and 2 cut rear fragment for pPIC9K carrier enzyme.
Fig. 3 is pPIC9K-SCY2 carrier for expression of eukaryon component diagram.
Fig. 4 is the expression and purification situation that SDS-PAGE analyzes SCY2.M: pre-dyed albumen Maker (BioBasic company); 1: the SCY2 albumen of Pichia anomala expression; 2: flow out component; 3,4: the antibacterial peptide SCY2 of purifying.
Fig. 5 is for expressing the N terminal amino acid residue sequence D01lcd sequencing result of antibacterial peptide SCY2 obtained.
Fig. 6 is for expressing the N terminal amino acid residue sequence D02lcd sequencing result of antibacterial peptide SCY2 obtained.
Fig. 7 is for expressing the N terminal amino acid residue sequence D03lcd sequencing result of antibacterial peptide SCY2 obtained.
Fig. 8 is for expressing the N terminal amino acid residue sequence D04lcd sequencing result of antibacterial peptide SCY2 obtained.
Fig. 9 is for expressing the N terminal amino acid residue sequence D05lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 10 is for expressing the N terminal amino acid residue sequence D06lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 11 is for expressing the N terminal amino acid residue sequence D07lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 12 is for expressing the N terminal amino acid residue sequence D08lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 13 is for expressing the N terminal amino acid residue sequence D09lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 14 is for expressing the N terminal amino acid residue sequence D10lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 15 is for expressing the N terminal amino acid residue sequence D11lcd sequencing result of antibacterial peptide SCY2 obtained.
Figure 16 is for expressing the N terminal amino acid residue sequence D11lcd sequencing result of antibacterial peptide SCY2 obtained.
Embodiment
Be described with reference to the accompanying drawings technical scheme of the present invention by the following examples.
The structure of embodiment 1 recombinant expression vector pPIC9K-SCY2
1) increase Scylla paramamosain SCY2 mature peptide sequence
According to pPIC9K vector multiple cloning site and Scylla paramamosain SCY2 gene order (Genbank accession number: JX228177), the upstream and downstream primer of design amplification SCY2 gene.Upstream primer 5 ' end add Kex2 restriction enzyme site and EcoR I, downstream primer 3 ' end add Not I restriction enzyme site, terminator codon and 6 × His histidine-tagged.The design of primers of SCY2 gene is as follows:
Upstream primer F:ggg
gAATTc
gGCCTGGCACTCAACAGACTTATG
EcoR I Kex2 recognition site
Downstream primer R:cat
gCGGCCGC aTGGTGATGGTGATGATGgTAGGAAGCAAGCCAGTCC
Not I terminator codon 6 × His-Tag
TCGAGGTC
With the total serum IgE reverse transcription of mud crab ejaculatory duct tissue extraction become cDNA for amplification template, respectively with SCY2-F and SCY2-R for upstream and downstream primer, with FastPfu polymeric enzymatic amplification object fragment, reaction system is
Mix, PCR response procedures is as follows:
PCR primer length is 300bp (see accompanying drawing 1).
2) double digestion of SCY2 goal gene fragment:
The two kinds of N getting the above-mentioned recovery of 2 μ g hold the goal gene fragment of SCY2, add EcoR I and Not I Restriction Enzyme respectively, hatch 5h for 37 DEG C, and it is as follows that enzyme cuts system:
After reaction terminates, with 1.5% (W/V) sepharose-TAE electrophoresis detection digesting efficiency; SCY2 goal gene fragment after cutting with Dongsheng gel recovery test kit recovery enzyme.SCY2 goal gene fragment after process has the sticky end (see accompanying drawing 2) of EcoR I and Not I.
5) extraction of pPIC9K plasmid and double digestion
Conservation bacterial strain DH5 α with pPIC9K is lined on the LB flat board containing the ammonia benzyl mycin of 50 μ g/mL, 37 DEG C of overnight incubation.Mono-clonal bacterium colony on next day picking flat board, is inoculated in the LB liquid nutrient medium of 5mL containing the ammonia benzyl mycin of 50 μ g/mL, 37 DEG C, 180r/min cultivates 8h to logarithmic phase, collected by centrifugation thalline, extract pPIC9K in a small amount, concrete operation method is shown in Dongsheng plasmid extraction kit specification sheets.
Get the pPIC9K carrier of the above-mentioned purifying of 2 μ g, add EcoR I and Not I Restriction Enzyme respectively, hatch 5h for 37 DEG C, it is as follows that enzyme cuts system:
After reaction terminates, with 1% (W/V) sepharose-TAE electrophoresis detection digesting efficiency; If carrier enzyme cuts entirely, the carrier after cutting with Dongsheng gel recovery test kit recovery enzyme.PPIC9K carrier after process has the sticky end of EcoR I and Not I.
6) structure of pPIC9K-SCY2 carrier, conversion and qualification
Be connected having EcoR I after a series of process with the gene fragment with identical sticky end SCY2 with the pPIC9K carrier of the sticky end of Not I, reaction system is as follows:
Mixed system is placed in 16 DEG C of overnight incubation; Get 10 μ L ligation liquid next day to be converted in 80 μ LE.coliDH5 α competent cells, be coated on the LB agar plate containing ammonia benzyl mycin (50 μ g/mL), 37 DEG C of overnight incubation.Next day by by bacterium liquid PCR method, picking mono-clonal bacterium colony, identifies that the positive colony bacterium obtained transfers to Invitrogen company to carry out DNA nucleotide sequence mensuration.
Result shows, and connects correct, open reading frame (ORF) continuous programming code of Nucleotide, finally successfully builds pPIC9K-SCY2 fusion expression vector (accompanying drawing 3).The feature of this recombinant eukaryotic expression plasmid adopts AOX1 promotor, and the signal peptide of yeast α-factor factor guides the secreting, expressing of antibacterial peptide SCY2; Before EcoRI restriction enzyme site, Kex2 restriction enzyme site is added during design primer, by adding the recognition site of Kex2 in the foreign protein precursor of yeast expression, can realize the object that foreign protein precursor is processed, thus acquisition and native protein has the antibacterial peptide SCY2 that identical N holds; C holds interpolation 6 × His-Tag to be convenient to affinitive layer purification target protein.
The abduction delivering of embodiment 2pPIC9K-SCY2 recombinant plasmid in Pichia pastoris GS115
1) linearizing of pPIC9K-SCY2
By streak culture for the bacterial strain containing expression vector correct for order-checking, picking mono-clonal shakes bacterium and cultivates, and extract plasmid, to 10 μ g plasmid Sac I linearizings, reaction system is as follows:
Reclaim test kit with Dongsheng gel and reclaim the pPIC9K-SCY2 after linearizing.Plastid transformation after linearizing in Pichia pastoris GS115 competent cell, is coated MD flat board by the conversion of employing electric shocking method, cultivates 48h for 28 DEG C.
2) abduction delivering of antibacterial peptide pPIC9K-SCY2
Picking mono-clonal bacterium colony from MD flat board, be inoculated in 10ml growth medium (BMGY), 28 DEG C of 230rpm/min are cultured to logarithmic phase, centrifugal substratum of outwelling collects thalline, add 10ml and express substratum (BMGY), express 0,24 and 48h with 0.5% methanol induction, utilize the expression of polyacrylamide gel electrophoresis (SDS-PAGE) detection fusion albumen.
Result shows: the Pichia pastoris GS115 methanol induction 24h of pPIC9K-SCY2 recombinant plasmid transformed, compared with before induction, has obvious expressing protein band (accompanying drawing 4) in about 10kDa position.
Embodiment 3 affinity chromatography purifying antibacterial peptide SCY2
1) preparation of sample before upper prop
Describe according to embodiment 2,12,000rpm low-temperature centrifugation 30min after recombinant bacterial strain induction 24h, collect fermented supernatant fluid.4 DEG C, to dialyse pichia spp abduction delivering supernatant liquor 2 ~ 3 times with the PBS dialyzate (50mM phosphoric acid buffer+50mMNaCl, pH8.0) of precooling, 12,000rpm low-temperature centrifugation 30min, collect fermented supernatant fluid again subsequently, through 0.45 μm of membrane filtration, treat column purification.
2) affinity chromatography purifying antibacterial peptide SCY2
With nickel ion chelating affinity column affinity purification, chromatography reagent is:
Solution A: 20mM phosphoric acid buffer+50mMNaCl+10mM imidazoles, pH8.0;
Solution B: 20mM phosphoric acid buffer+500mMNaCl+1M imidazoles, pH8.0;
With 5 ~ 10 column volume MilliQ water cleaning prepacked column (HisTrap
tMfFCrude5mL), then 5 ~ 10 column volume solution C balance HisTrap chromatography columns, by the supernatant liquor after filtration with the whole upper prop of 2mL/min, collect stream simultaneously and wear sample; Then cross post by the solution C of 5 ~ 10 column volumes, wash away unconjugated albumen; Cross post by 30% solution D, elution antimicrobial peptide collects elution peak, takes a morsel and carries out SDS-PAGE electroresis appraisal.
SDS-PAGE electrophoresis result display (see accompanying drawing 4), SCY2 fusion protein expression products can specificly be combined with nickel ion chelating affinity column, after the imidazole solution competition washing of higher concentration, fusion expressed product is by wash-out, and purity is higher.Albumen is dialysed in phosphate buffered saline buffer, finally dialyses in MilliQ water.
The N end order-checking of embodiment 4 antibacterial peptide SCY2
N end order-checking student on commission work bio-engineering corporation completes, and this experiment N that Edman degradation method determines two kinds of SCY2 holds 12 amino acid residue sequences.The N of antibacterial peptide SCY2 holds sequencing result (D01lcd ~ D12lcd) see Fig. 5 ~ 16.
From N hold sequencing result, the sample N terminal sequence that checks order be: YVEFGLALNRLM, i.e. NH2-Tyr-Val-Glu-Phe-Gly-Leu-Ala-Leu-Asn-Arg-Leu-Met.Protein sequence known in this sequence and American National Biotechnology Information central database (NCBI) is compared.Result shows, after this albumen n end, 8 amino acid residue sequence Gly-Leu-Ala-Leu-Asn-Arg-Leu-Met hold front 8 amino acid residue sequences identical with Scylla paramamosain SCY2 mature peptide N in database, and the known polypeptide by the external eukaryotic expression gained of Pichia pastoris GS115 is Scylla paramamosain antibacterial peptide SCY2.
The anti-microbial activity qualification of embodiment 5 antibacterial peptide SCY2
After protein sample filtration sterilization, be that standard substance application Bradford method draws protein concentration typical curve with BSA, the concentration of antibacterial peptide SCY2 can be calculated according to formula.By protein solution doubling dilution to 1.6 ~ 50 μM.Being determined on 96 porocyte culture plates of MIC (MinimumInhibitoryConcentration, minimal inhibitory concentration) is carried out.
(1) preparation of bacteria suspension: get tested bacterium, at the flat lining out of nutrient broth, cultivates 12 ~ 16h; Picking 2 ~ 3 clones are inoculated in MH inclined-plane, continue cultivation 12 ~ 16h; 10mM sodium phosphate buffer (pH7.2) rinses the MH bacterium slant culture of fresh preparation, adjustment OD
600be 0.003.Be inverted cultivation 2 ~ 5 days.
(2) often kind of tested bacterium is according to following operation setting blank group, negative control group and testing sample experimental group, and often group arranges 2 Duplicate Samples, and often kind of bacterium repeats 3 times:
I. positive controls: add 50 μ L sodium phosphate buffer and 50 μ L bacteria suspensions;
Ii. blank group: add 50 μ L testing protein samples and 50 μ L sodium phosphate buffer;
Iii. sample experiments group: add 50 μ L testing protein samples and 50 μ L bacteria suspensions;
(3), after the suitableeest cultivation 24 ~ 48h of bacterium, MIC result is observed.
Antibacterial experiment result shows, SCY2 is to gram-positive microorganism, as micrococcus lysodeikticus has stronger anti-microbial activity (MIC=12.5 ~ 25 μM), there is certain anti-microbial activity (MIC=25 ~ 50 μM), to Gram-negative bacteria without anti-microbial activity (MIC>50 μM) to micrococcus luteus, Corynebacterium glutamicum.The SCY2 of visible eukaryotic expression has certain anti-microbial activity, and is better than the anti-microbial activity to Gram-negative bacteria to the anti-microbial activity of gram-positive microorganism.
The invention provides the structure, expression product and preparation method thereof of the expression vector containing Scylla paramamosain antibacterial peptide SCY2.Inserting in expression vector of the present invention for recombinant expressed sequence is the SCY2 sequence of SEQIDNo.1, in host's pichia spp, carry out abduction delivering, obtains expression product.Before EcoRI restriction enzyme site, Kex2 restriction enzyme site is added due to during design primer, by adding the recognition site of Kex2 in the foreign protein precursor of yeast expression, realize the object that foreign protein precursor is processed, thus acquisition and native protein has the antibacterial peptide SCY2 that identical N holds.This antibacterial peptide has certain anti-microbial activity, and to micrococcus lysodeikticus, micrococcus luteus, Corynebacterium glutamicum etc. all have significant Antibacteria effect, will have important using value at culture fishery, pharmaceutical sector etc.
Claims (10)
1. the preparation method of Scylla paramamosain antibacterial peptide SCY2, is characterized in that comprising the following steps:
1) structure of Scylla paramamosain antibacterial peptide SCY2 expression vector: design upstream primer, before 5 ' end EcoR I restriction enzyme site, the base sequence corresponding to Kex2 restriction enzyme site aminoacid sequence is added during design, pcr amplification Scylla paramamosain antibacterial peptide SCY2 gene, described Scylla paramamosain antibacterial peptide SCY2 gene is inserted in pPIC9K carrier, obtains pPIC9K-SCY2 recombinant expression vector;
2) by step 1) recombinant expression vector that obtains is transformed into host cell, and abduction delivering is carried out to host cell, obtains expression product;
3) purification procedures 2) expression product of gained, then carry out the order-checking of N end, determine that obtained expression product is have the Scylla paramamosain antibacterial peptide SCY2 having identical N to hold with native protein.
2. the preparation method of Scylla paramamosain antibacterial peptide SCY2 as claimed in claim 1, is characterized in that in step 1) in, described primer sequence is as follows:
gggGAATTCGAGAAAAGAGGCCTGGCACTCAACAGACTTATG。
3. the preparation method of Scylla paramamosain antibacterial peptide SCY2 as claimed in claim 1, is characterized in that in step 1) in, described expression vector is used for the Scylla paramamosain antibacterial peptide SCY2 sequence that expressed sequence is SEQIDNo.1.
4. the preparation method of Scylla paramamosain antibacterial peptide SCY2 as claimed in claim 1, is characterized in that in step 1) in, the amino acid fragment sequence of described Scylla paramamosain antibacterial peptide SCY2 is SEQIDNo.2.
5. the preparation method of Scylla paramamosain antibacterial peptide SCY2 as claimed in claim 1, it is characterized in that in step 3) in, the method of described separation and purification is by step 2) expression product of gained carries out centrifugal, collect fermented supernatant fluid, after fermented supernatant fluid is dialysed, after affinity chromatography, namely obtain the Scylla paramamosain antibacterial peptide SCY2 of purifying.
6. the Scylla paramamosain antibacterial peptide SCY2 prepared by claim 1 applies preparing in bacterial growth inhibitors.
7. the Scylla paramamosain antibacterial peptide SCY2 prepared by claim 1 applies preparing in animal feedstuff additive.
8. the Scylla paramamosain antibacterial peptide SCY2 prepared by claim 1 applies preparing in anti-pathogenic microorganism medicine.
9. apply as claimed in claim 6, it is characterized in that described bacterium is gram-positive microorganism; Described gram-positive microorganism includes but not limited to micrococcus luteus (Micrococcusluteus), Corynebacterium glutamicum (Corynebacteriumglutamicum), micrococcus lysodeikticus (Micrococcuslysodeikticus).
10. the gene order of the Scylla paramamosain antibacterial peptide SCY2 prepared by claim 1 is:
The aminoacid sequence of described Scylla paramamosain antibacterial peptide SCY2 is:
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| CN107903319A (en) * | 2017-12-28 | 2018-04-13 | 厦门大学 | Expression and application of a kind of thymosin extrasin PROTEIN C q TRP1 of Anti-infection to WSSV in Pichia pastoris |
| CN108285481A (en) * | 2018-01-30 | 2018-07-17 | 厦门大学 | The gene C q-BAF and preparation method of promotion WSSV infection and application |
| CN111051335B (en) * | 2018-06-20 | 2022-06-21 | 厦门大学 | Scylla paramamosain antibacterial peptide Sparamosin and application thereof |
| WO2019241938A1 (en) * | 2018-06-20 | 2019-12-26 | 厦门大学 | Scylla paramamosain antibacterial peptide sparamosin and application thereof |
| CN111051335A (en) * | 2018-06-20 | 2020-04-21 | 厦门大学 | Scylla paramamosain antibacterial peptide Sparamosin and application thereof |
| US11254717B2 (en) | 2018-06-20 | 2022-02-22 | Xiamen University | Antimicrobial peptide Sparamosin from Scylla paramamosain and application thereof |
| WO2020103511A1 (en) * | 2018-11-21 | 2020-05-28 | 厦门大学 | Scylla paramamosain antibacterial peptide scyreprocin and application thereof |
| US11512120B2 (en) | 2018-11-21 | 2022-11-29 | Xiamen University | Antimicrobial peptide Scyreprocin of Scylla paramamosain and method thereof |
| CN110028568A (en) * | 2019-03-11 | 2019-07-19 | 厦门大学 | A kind of Scylla paramamosain antibacterial polypeptide Sp-NPFin and its application |
| CN110028568B (en) * | 2019-03-11 | 2021-06-01 | 厦门大学 | Scylla paramamosain antibacterial polypeptide Sp-NPFin and application thereof |
| CN112724221A (en) * | 2021-01-26 | 2021-04-30 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamplin58-82And uses thereof |
| CN112724221B (en) * | 2021-01-26 | 2022-03-15 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamplin58-82And uses thereof |
| WO2022268115A1 (en) * | 2021-06-25 | 2022-12-29 | 厦门大学 | Scylla paramamosain antibacterial polypeptide spampcin 56-86 and application thereof |
| CN115058430A (en) * | 2022-06-12 | 2022-09-16 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
| CN115058430B (en) * | 2022-06-12 | 2023-06-09 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
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