CN101948839B

CN101948839B - Antimicrobial peptide gene from pinctada fucata and application

Info

Publication number: CN101948839B
Application number: CN2010102653358A
Authority: CN
Inventors: 江世贵; 张殿昌; 崔淑歌
Original assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Current assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Priority date: 2010-08-26
Filing date: 2010-08-26
Publication date: 2012-06-13
Anticipated expiration: 2030-08-26
Also published as: CN101948839A

Abstract

The invention discloses cDNA of antimicrobial peptide gene AP from pinctada fucata, the nucleotide sequence is as shown in SEQ ID NO. 1, and the amino acid sequence can be deduced to be as shown in SEQ ID NO.2. The invention further discloses an expression vector containing the antimicrobial peptide gene from the pinctada fucata and a recombinant microbe transformed by utilizing the vector, discloses a method for preparing the antimicrobial peptide gene from the pinctada fucata by utilizing the microbe, and simultaneously discloses an application of the antimicrobial peptide gene from the pinctada fucata in the preparation of drugs for treating or preventing sea water pearl oyster type anti-gram-negative bacteria. The invention adopts the genetic engineering technology for producing recombinant antimicrobial peptide protein from the pinctada fucata, and the recombinant antimicrobial peptide protein is utilized for preparing sea water pearl oyster antibacterial agents or fungicides.

Description

A kind of pinctada fucata antibacterial peptide gene and application

Technical field

The invention belongs to gene engineering technology field; What be specifically related to is a kind of pinctada fucata antibacterial peptide gene and carrier and the recombinant bacterial strain that contains this gene; And the application of this gene in preparation antiseptic-germicide or sterilant, also can be used as target gene and be applied to the shellfish transgenic breeding.

Background technology

Antibacterial peptide (Antimicrobial peptide; AP) be through the micromolecule polypeptide of a kind of biologically active of inducing generation in the organism; Molecular weight generally is made up of 20～60 amino-acid residues about 2000～7000, and molecular weight is about 4KDa; Have the common characteristic: 1. the N-end is rich in polare Aminosaeren, is strong basicity; 2. C-holds common amidation, is neutral hydrophobicity; 3. the middle interconnecting piece branch is rich in Pro, and terminal amidation mostly directly influences the fungicidal activity of antibacterial peptide; 4. the amino acid of most of antibacterial peptide on the 2nd is Trp, and the height of antibacterial peptide fungicidal activity is played an important role.1972, Sweden scientist Boman etc. at first found antibacterial peptide and immunologic function thereof in fruit bat.After this, from bacterium, fungi, batrachians, insect, higher plant, Mammals and even the mankind, find in succession and separate and obtain the polypeptide that more than 600 kind of endogenous has anti-microbial activity.According to amino acid The Nomenclature Composition and Structure of Complexes characteristic, can be divided into 4 types to antibacterial peptide: (1) alpha-helix type antibacterial peptide.Intramolecularly has α-Luo Xuanjiegou, like the oozy magainin of skin of Africa xenopus (Xenopus laevis); (2) beta sheet type antibacterial peptide.Have antiparallel beta sheet structure, intramolecularly contains two or above disulfide linkage, like alexin defensin; (3) antibacterial peptide of laminated structure.Lack typical secondary structure, be rich in Pro, Gly, form by 13 amino-acid residues, wherein just contain 5 tryptophanes like isolating indolicidin in the ox neutrophil(e) cell; (4) antibacterial peptide of ring texture.Have the highly variable structure, at the C end disulfide linkage is arranged usually, like thanatin from thorn shoulder stinkbug (Podisu smaculiventris).Therefore, the development and application of antibacterial peptide research has become domestic and international forage science and pharmacological research focus, has broad application prospects at plant-animal transgenic engineering and drug development field.

Pinctada fucata (Pinctada fucata) is subordinate to Mollusca (Mollusca); Lamellibranchiata (Lamellibranchia); Anisomyaria (Anisomyaria), Pteriidae (Pteriidae) is the main kind that China's sea water pearls is cultivated; Have higher economic value, its pearl that produces is " Nan Zhu " world-famous.Yet the pinctada fucata disease takes place frequently in recent years, has caused higher mortality ratio, becomes one of technical bottleneck of restriction pinctada fucata aquaculture industry Sustainable development, but in the pinctada fucata disease control, does not also have a kind of effective antiseptic-germicide at present.

Summary of the invention

The aminoacid sequence that first purpose of the present invention is to provide a kind of cDNA of pinctada fucata antibacterial peptide gene and infers thus.

Second purpose of the present invention is to provide expression vector that contains above-mentioned pinctada fucata antibacterial peptide gene and the recombinant microorganism that utilizes this carrier to transform.

The 3rd purpose of the present invention is to provide a kind of process from described mikrobe preparation reorganization pinctada fucata antibacterial peptide gene.

The 4th purpose of the present invention provides above-mentioned reorganization pinctada fucata antibacterial peptide gene and treats or prevent the application in the anti-gram negative bacterium medicine in preparation.

First purpose of the present invention realizes through following technical scheme: a kind of cDNA of pinctada fucata antibacterial peptide gene, its nucleotide sequence is shown in SEQ ID NO.1.Its aminoacid sequence is shown in SEQ ID:2.

Pinctada fucata antibacterial peptide full-length gene of the present invention is that the cDNA that obtains with the reverse transcription of pinctada fucata soft body total tissue RNA is a template; The construction cDNA library; Obtained the antibacterial peptide Partial cDNA Sequence through extensive EST order-checking screening; Obtain end sequence through the amplification of RACE method then, after sequence assembly obtains pinctada fucata antibacterial peptide cDNA full length gene sequence.

The antibacterial peptide mature peptide gene order that the present invention is used to express pinctada fucata antibacterial peptide recombinant protein is to be template with pinctada fucata antibacterial peptide full-length gene double-stranded DNA; Obtain through the PCR method amplification, it derives from the mature peptide area relative gene fragment of pinctada fucata antibacterial peptide full-length gene.

Mature peptide gene order according to above-mentioned pinctada fucata antibacterial peptide recombinant protein; It just in time is pinctada fucata antibacterial peptide full-length gene 109bp to the 381bp fragment of (being equivalent to the 35th to the 124th amino acids), and its nucleotide sequence and coded aminoacid sequence thereof are as follows.

The cDNA of pinctada fucata antibacterial peptide gene, its full length nucleotide sequence is as follows:

TTCATT

60

TG GTCTAAAACA 120

GCCTCTAGGT GTTTTGACGT CTGGTCTAGA TGTTCTGGAT GGAGCCGTGC TGCTACTGGT 180

TATCTCTGGC TTAGTTGCAA CAAGTGCTGC CAATGTAAGG GAAAATCTGG AGGGCGTTGT 240

GTATTAGTTG CAGCGAGAGG ATGCCCCTTA TCTAAGAATG CATACCAGTG TCAGTGCAAT 300

GGGAGCAGCC TAAGGGGAGG AAAACCGGGG TTTTGTGGTG GCAAAGCAAA TTATCTTTTC 360

TCTTGTGATA ACAAGAACTA AAACAAAATG AAGATGACGC CAAGCGAAAT ACGACTGAAC 420

CGTAGAGTTG ACATTGTCTT TATATTGATG ATAATTTCAA ATACGGCAAA AGTCTGATGT 480

ATTTAAAATA AATAATATCA TTTGAATGGC AAAAAAAAAA AAAAAAAAAA AAAAAA 536

Be the dna sequence dna of the signal peptide of pinctada fucata antibacterial peptide in the skeleton diagram wherein, the bottom underscore partly is the dna sequence dna of the mature peptide of pinctada fucata antibacterial peptide.

The aminoacid sequence of the pinctada fucata antibacterial peptide gene of being inferred by above-mentioned cDNA sequence is as follows:

Met Asn Cys Pro Arg Val Leu Pro Asp Arg Lys Thr Val Gln Ser Lys

-30 -25 -20

Tyr Val Val Val Val Leu Leu Phe Ile Val Met Cys Pro Tyr Val Asn

-15 -10 -5

Ala Trp Trp Ser Lys Thr Ala Ser Arg Cys Phe Asp Val Trp Ser Arg

-1 1 5 10

Cys Ser Gly Trp Ser Arg Ala Ala Thr Gly Tyr Leu Trp Leu Ser Cys

15 20 25 30

Asn Lys Cys Cys Gln Cys Lys Gly Lys Ser Gly Gly Arg Cys Val Leu

35 40 45

Val Ala Ala Arg Gly Cys Pro Leu Ser Lys Asn Ala Tyr Gln Cys Gln

50 55 60

Cys Asn Gly Ser Ser Leu Arg Gly Gly Lys Pro Gly Phe Cys Gly Gly

65 70 75

Lys Ala Asn Tyr Leu Phe Ser Cys Asp Asn Lys Asn

80 85 90

Amino acid sequence analysis of the present invention shows that the pinctada fucata antibacterial peptide is participated in by 124 amino acid and forms; Wherein preceding 34 amino residue are signal peptide sequence; 90 amino-acid residues in back are the mature peptide sequence; Mature peptide has the former excision signal peptide of antibacterial peptide to form, and Gram-negative bacteria is had the intensive bacteriostatic activity.

Second purpose of the present invention realizes through following technical scheme: a kind of expression vector, and it is the expression vector that contains the cDNA of above-mentioned pinctada fucata antibacterial peptide gene; And the recombinant microorganism that utilizes above-mentioned carrier to transform.

The construction process of expression vector of the present invention is by ordinary method; Will through PCR method synthetic pinctada fucata antibacterial peptide gene through enzyme cut with separation and purification after; Be connected between the corresponding restriction enzyme site (being BamH I and Hind III) of existing respective carrier, promptly be built into the required expression vector that contains the pinctada fucata antibacterial peptide gene.

The recombinant expression vector that the above-mentioned coli expression carrier that contains the pinctada fucata antibacterial peptide gene of the present invention preferentially is built into by synthetic pinctada fucata antibacterial peptide gene of the present invention and coli expression carrier pET-32 α, called after pET-32 α-PoAP.

The 3rd purpose of the present invention realizes through following technical scheme: the method for preparing reorganization pinctada fucata antibacterial peptide gene AP provided by the invention; Comprise with above-mentioned expression vector and transform host cell; The culture transformation body obtains the pinctada fucata antibacterial peptide gene of recombinating.

Wherein host cell is constructed a kind of recombinant strain earlier preferably from intestinal bacteria, and it is the intestinal bacteria that contain the pinctada fucata antibacterial peptide gene.Adopt the above-mentioned corresponding expression vectors that contains the pinctada fucata antibacterial peptide gene to make up the intestinal bacteria recombinant bacterial strain that can efficiently express the pinctada fucata antibacterial peptide.

The bacterial strain that the above-mentioned intestinal bacteria recombinant bacterial strain that can express the pinctada fucata antibacterial peptide of the present invention is preferentially obtained by the expression vector pET-32 α-PoAP transformed into escherichia coli BL21 that contains the pinctada fucata antibacterial peptide gene, called after pET-32 α-PoAP-BL21.

The concrete preparation method of above-mentioned pinctada fucata antibacterial peptide gene realizes through following technical scheme: choose intestinal bacteria recombinant bacterial strain pET-32 α-PoAP-BL21 and be seeded in the LB liquid nutrient medium that 10mL contains penbritin; 37 ℃, spend the night, as kind of a daughter bacteria; Be inoculated in the identical substratum by 1: 100 inoculum size next day again; When the reorganization bacteria growing got into logarithmic phase, adding 100mM IPTG was 1.6mM to final concentration, the 220rpm shaking culture;, the reorganization bacteria growing collects thalline after getting into plateau, through the separation and purification pinctada fucata antibacterial peptide product that obtains recombinating.

Last purpose of the present invention is above-mentioned pinctada fucata antibacterial peptide gene is used for treating or preventing the anti-gram negative bacterium medicine of sea water pearls shellfish in preparation application.

The present invention compared with prior art has the following advantages: pinctada fucata has great economic worth; It is the main shellfish kind of producing " Nan Zhu "; Adopt genetic engineering technique; Utilize pinctada fucata antibacterial peptide gene of the present invention and recombinant bacterial strain pET-32 α-PoAP-BL21, produce reorganization pinctada fucata antimicrobial peptide protein, and further as preparation seawater pearl shell antiseptic-germicide or sterilant.

Description of drawings

Fig. 1 a is the electrophorogram that the post-stimulatory pinctada fucata soft tissue of vibrio alginolyticus is extracted total RNA;

Fig. 1 b is to use the isolating single stranded RNA electrophorogram of test kit;

Fig. 2 is the electrophorogram of the amplified production in pcr amplification pinctada fucata cDNA library;

Fig. 3 a and Fig. 3 b are the electrophorograms of twice amplification of PCR pinctada fucata antibacterial peptide, 3 ' terminal amplified production;

Fig. 4 is the electrophorogram of the synthetic pcr amplification product of pinctada fucata antibacterial peptide mature peptide gene;

Fig. 5 is the plasmid enzyme restriction figure that contains the pET-32 α-AP of pinctada fucata antibacterial peptide gene and coli expression carrier;

Fig. 6 is the SDS-PAGE gel electrophoresis figure that shows through inductive pET-32 α-AP-BL21;

Fig. 7 is the immunoblotting figure as a result of mouse-anti 6 * His antibody;

Fig. 8 is the SDS-PAGE gel electrophoresis figure of purified recombinant pinctada fucata antimicrobial peptide protein;

Fig. 9 is the active detected result figure of reorganization pinctada fucata antimicrobial peptide protein.

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is further specified, but do not limit the present invention in any form.

Embodiment 1: pinctada fucata cDNA library construction and EST screening

Getting the post-stimulatory pinctada fucata soft tissue of vibrio alginolyticus extracts total RNA and (Fig. 1 a), uses

Synthesis kit (Stratagene), Gigapack III Gold Cloning kit (Stratagene) and

mRNA isolation system III test kits such as (Promega) structure EST library (Fig. 1 b).The picking mono-clonal is used primer T at random ₃(AATTAACCCTCACTAAAGGG) check order, unload body through the Crossmatch program; Through the Phrap program; Splice cluster; Obtain the UniGene DS, after the Phrap routine processes, the est sequence that can gather together is the Contigs sequence; The est sequence that can not gather together is the Singlets sequence, and two portions sequence obtains the UniGene DS after EST goes redundancy altogether.Respectively Contigs that is obtained and Singletons are carried out BLASTn and BLASTx analysis in DB; The antibacterial peptide that identifies wherein other species in 1 est sequence and the GeneBank DB has higher homology, obtains pinctada fucata antibacterial peptide Partial cDNA Sequence with this.

Embodiment 2:PCR amplification pinctada fucata cDNA library

The pinctada fucata cDNA that obtains with GeneRacer Kit reverse transcription is a template, and primer is respectively 5 ' of GeneRacerKit-Primer and 3 '-Primer, increases to set up pinctada fucata cDNA library through the touchdown PCR method; Reaction conditions is: 94 ℃ of preparatory sex change 5min are 40 circulations then, divide two stages: 1. 94 ℃ of sex change 30S; Each circulation reduces by 0.5 ℃ from 68 ℃ to 54 ℃; Annealing 30S, 72 ℃ are extended 2min, totally 32 circulations; 2. 94 ℃ of sex change 30S, 50 ℃ of annealing 30S, 72 ℃ are extended 2min, totally 8 circulations.Last 72 ℃ are extended 10min.The electrophorogram of pcr amplification product such as Fig. 2.

Embodiment 3:PCR amplification pinctada fucata antibacterial peptide 3 ' end

Operation is undertaken by GeneRacer Kit specification sheets.According to the requirement of test kit, according to pinctada fucata antibacterial peptide est sequence time of having checked order two upstream primer GSP1 and GSP2 to carry out nest-type PRC.Article one, upstream primer (GSP1) is from 20 bases of pinctada fucata antibacterial peptide est sequence the 96th bit base of having checked order (5 '-TGTGAATGCCTGGTGGTCTA-3 '), and second upstream primer (GSP2) is 20 bases (5 '-GCCGTGCTGCTACTGGTTAT-3 ') from the 164th.

PCR is a template with the PCR product cut back (1: 100) that the foregoing description 2 obtains for the first time; Primer is GSP1; Through PCR method the 96th the nucleotide sequence that increase to 3 '-polyA end, use the PCR appearance of Eppendorf to set the pcr amplification program, reaction conditions is: 96 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30S then, 53 ℃ of annealing 30S, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 10min.For the second time PCR with the first time PCR product be that template increases, reaction conditions is 96 ℃ of preparatory sex change 2min; 94 ℃ of sex change 30S then, 55 ℃ of annealing 30S, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 10min.

The electrophorogram of twice PCR amplified production such as Fig. 3 a and Fig. 3 b.

The amplified production of 360bp is connected to obtains recombinant plasmid pMT-18-3AP on the pMT-18 carrier.The universal primer of quality pMT-18-3AP with pMT-18 of recombinating carried out sequencing, and the sequence of sequencing result and GenBank compares, and the result shows that clone's 360bp product is a 3 ' terminal sequence of pinctada fucata antibacterial peptide.

Embodiment 4: the splicing of pinctada fucata antibacterial peptide gene full length sequence

The pinctada fucata antibacterial peptide est sequence that has checked order and 3 '-RACE sequence are spliced; The full length sequence that obtains the pinctada fucata antibacterial peptide gene is 536bp; And finish since the initiator codon at 7bp place to its terminator codon, infer its aminoacid sequence.Pinctada fucata antibacterial peptide gene total length is seen sequence table SEQ ID NO.1, sees sequence table SEQ ID NO.2 by the aminoacid sequence of its supposition.

Embodiment 5: pinctada fucata antibacterial peptide gene synthetic

According to having spliced pinctada fucata antibacterial peptide gene cDNA complete sequence; The synthetic a pair of primer of design; Upstream primer is restriction enzyme site and 3 the protection base that before the 109th bit base plays 23 bases, adds BamH I (5 '-GGTGGATCCTGGTGGTCTAAAACAGCCTCTAG-3 '), 32bp altogether; Downstream primer is restriction enzyme site 2 protection bases (5 '-CGAAGCTTTTAGTTCTTGTTATCACAAGAGAAAAGAT-3 '), the 37bp altogether that adds HindIII in preceding 29 bases that the 381st bit base rises.With pinctada fucata cDNA library is template; Through the 35th of PCR method amplification pinctada fucata antibacterial peptide to the 124th amino acids sequence corresponding DNA sequence; Be pinctada fucata antibacterial peptide mature peptide corresponding sequence, the pcr amplification condition is: 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 40s then, 55 ℃ of annealing 40s, 72 ℃ are extended 45S, totally 35 circulations; Last 72 ℃ of 10min.

The electrophorogram of pcr amplification product such as Fig. 4.Gone out to be about the fragment of 290bp from the visible pcr amplification of Fig. 4.

Embodiment 6: the structure that contains the coli expression carrier of pinctada fucata antibacterial peptide gene

The PCR product that the foregoing description 5 amplification obtains is after BamH I cuts with Hind III enzyme, and enzyme is cut product with the recovery of the PCR of AxyGen company product purification test kit, the pinctada fucata antibacterial peptide gene fragment of the about 290bp of separation and purification; After expression vector pET-32 α cut with BamH I and Hind III enzyme too, enzyme was cut product and is carried out agarose gel electrophoresis, the big fragment of separation and purification 5.9kb; Mix by 1: 3 with the pinctada fucata antibacterial peptide gene fragment of 290bp; Change in the e. coli bl21 with the standard calcium chloride transformation with 16 ℃ of connections of T4 ligase enzyme back of spending the night, screening has the transformant of amicillin resistance, with standard method extraction plasmid; The reorganization quality of the about 6.2kb of screening size; With restriction enzyme BamH I and Hind III double digestion reorganization quality, obtain two fragments of 290bp and 5.9bp, size is identical with expression vector pET-32 α size with the pinctada fucata antibacterial peptide gene respectively; Proof pinctada fucata antibacterial peptide gene has been cloned among the coli expression carrier pET-32 α, reorganization quality called after pET-32 α-AP.Plasmid enzyme restriction figure such as Fig. 5.

Embodiment 7: the structure that efficiently expresses the intestinal bacteria recombinant bacterial strain pET-32 α-AP-BL21 of pinctada fucata antibacterial peptide

With the standard Calcium Chloride Method with pET-32 α-AP transformed into escherichia coli BL21; Screen transformant containing on the LB flat board of penbritin; Detect the sub-pET-32 α-AP-BL21 of recombinant conversion that obtains to contain pET-32 α-AP with restriction analysis through plasmid, at last sequence verification.

Embodiment 8: utilize recombination bacillus coli pET-32 α-AP-BL21 genetic engineering bacterium production reorganization pinctada fucata antibacterial peptide

Picking intestinal bacteria recombinant bacterial strain pET-32 α-AP-BL21; Earlier bacterial classification inoculation is contained in the LB liquid nutrient medium of penbritin at 10ml; 37 ℃, the 220rpm shaking culture is spent the night, as kind of a daughter bacteria; Be inoculated in the identical substratum by 1: 100 inoculum size next day again, (A when the reorganization bacteria growing gets into logarithmic phase ₆₀₀=0.5-0.6), add 100mMIPTG to final concentration be 1.6mM, 37 ℃, receive bacterium behind the 220rpm vibration inducing culture 10h.Synthetic pinctada fucata antibacterial peptide has obtained to efficiently express in bacillus coli gene engineering bacteria pET-32 α-AP-BL21.

The cell that takes a morsel adds 2 * electrophoresis upper reaches damping fluid; Run the SDS-PAGE gel electrophoresis by standard method after boiling 5min; Result such as Fig. 6 show a new fusion rotein band on the position of inductive pET-32 α-AP-BL21 at about 31kDa, to occur, this band do not occur without inductive.Utilize ordinary method to carry out immunoblotting (Western blot) analysis then.One anti-is mouse-anti 6 * His antibody, and two anti-ly are sheep anti-mouse igg-HRP.Result such as Fig. 7, demonstration mouse-anti 6 * His antibody capable is discerned our the fusion antimicrobial peptide protein with escherichia coli expression.The albumen that these result's proofs obtain is reorganization pinctada fucata antibacterial peptide.

Embodiment 9: reorganization pinctada fucata antimicrobial peptide protein purifying detects with active

Press the method for the foregoing description 8, intestinal bacteria recombinant bacterial strain pET-32 α-AP-BL21 genetic engineering bacterium is carried out enlarged culturing, 10; The centrifugal 10min of 000g collects thalline; Behind ultrasonic disruption, utilize His-Bind Purification Kit Protocol purification of Recombinant pinctada fucata antimicrobial peptide protein, through the SDS-PAGE detected through gel electrophoresis; Result such as Fig. 9 show to have obtained the higher pinctada fucata antibacterial peptide recombinant protein of purity.

The outer bacteriostatic activity of agar diffusion method detection bodies: get bacterial strain, intestinal bacteria, streptococcus aureus, vibrios, Bacillus licheniformis preservation liquid, be coated on the nutrient agar, cultivate 16～18h activation for 37 ℃; Again with loop-carrier respectively from the nutrient agars of the four kinds of bacterium bacterium that gets on, rule on the solid nutrient agar with three step subregion dilution methods, cultivate 16～18h for 37 ℃; 3 single bacterium colonies of picking are cultivated 16～18h activation for 37 ℃ and are increased bacterium in the LB substratum respectively, get 0.1mL and place on the LB agar plate; L shaped bent glass rod with sterilization evenly is coated with; Slightly do the back with the sterilization punch tool, scribbling the plate culture medium surface punching of bacterium, aperture diameter is 6mm; Pitch of holes leaves >=24mm; The pinctada fucata antibacterial peptide recombinant protein that adds 30 μ L equivalent purifying in the hole respectively, and contrast TRX albumen, PBS, every kind of bacterial strain do three parallel.After completing flat board is just being put 37 ℃ of cultivation 16-18h in the incubator; Detected result such as Fig. 9; Show that reorganization pinctada fucata antibacterial peptide has the intensive lytic activity to the intestinal bacteria and the vibrio alginolyticus of Gram-negative bacteria, and the streptococcus aureus and the Bacillus licheniformis of gram-positive microorganism do not had bacteriostatic activity.

The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included in protection scope of the present invention.

Claims

1. the cDNA of a pinctada fucata antibacterial peptide gene AP, its nucleotide sequence is shown in SEQ ID NO.1.

2. pinctada fucata antibacterial peptide gene AP, its amino acid sequence coded is shown in SEQ ID NO.2.

3. the expression vector that comprises the cDNA of claim 1.

4. the expression vector pET-32 α-AP that comprises the cDNA of claim 1.

5. the method for preparing reorganization pinctada fucata antibacterial peptide gene AP comprises with the described expression vector of claim 3 transforming host cell that the culture transformation body obtains the pinctada fucata antibacterial peptide gene of recombinating.

6. method according to claim 5, wherein host cell is intestinal bacteria.

7. the described pinctada fucata antibacterial peptide gene of claim 1 AP is used for treating or preventing the application of sea water pearls shellfish Chinese People's Anti-Japanese Military and Political College enterobacteria and vibrio alginolyticus medicine in preparation.