CN103484466A

CN103484466A - Diamondback moth antibacterial peptide moricin, preparation method and application thereof

Info

Publication number: CN103484466A
Application number: CN201310411620.XA
Authority: CN
Inventors: 金丰良; 许小霞; 李鹏
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2013-09-11
Filing date: 2013-09-11
Publication date: 2014-01-01
Anticipated expiration: 2033-09-11
Also published as: CN103484466B

Abstract

The present invention discloses a diamondback moth antibacterial peptide moricin, a preparation method and an application thereof, wherein the nucleotide sequence of the diamondback moth antibacterial peptide moricin gene is represented by SEQIDNO:1-2, the amino acid sequence for encoding the gene is represented by SEQIDNO:3, and sequence analysis results show that the mature peptide of the diamondback moth antibacterial peptide moricin has an amino acid sequence represented by SEQIDNO:4. According to the present invention, the mature peptide gene of the diamondback moth antibacterial peptide moricin is subjected to eukaryotic expression to obtain the recombinant mature peptide, and the recombinant mature peptide provides significant inhibition or killing effects for Aureobasidium pullulans, and can be used for preparing preparations for inhibition or killing of Aureobasidium pullulans.

Description

A kind of small cabbage moth antibacterial peptide moricin and preparation method thereof and application

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of small cabbage moth antibacterial peptide moricin and preparation method thereof and application.

Background technology

Antibacterial peptide (antibacterial peptide) is that in organism, the class through inducing generation has bioactive micromolecule polypeptide; be present in multiple organism; an important component part (Boman and Hultmark, 1987 of host immune system of defense; Bulet et al., 2004).Antibacterial peptide can not only bacteria growing inhibiting, and some antibacterial peptide all has strong lethal effect to part fungi, protozoon, virus and cancer cells etc., and certain immunoloregulation function is even arranged, can accelerating wound healing, and can not produce resistance.And most antibacterial peptides are harmless to the Mammals normal cell, and its bactericidal mechanism is fully different from traditional microbiotic, by scholars, is thought a kind of natural antibiotics and enjoys favor.Along with deepening continuously of research, more and more demonstrated its unique application in fields such as physianthropy, veterinary science and be worth (Boulanger et al., 2006; Bulet et al., 2004).

Moricin separates and obtains first from silkworm hemolymph, called after moricin1 and moricinA1, the antibacterial peptide that a class is new (Hara and Yamakawa, 1995), both list 97.2% similarity at nucleotides sequence, unique the 6th amino acid phenylalanine that is not both the moricin-1 signal peptide changes to Methionin in moricin-2, but their maturation protein aminoacid sequence is identical.Moricin is the antibacterial peptide that a class is comprised of 42 amino-acid residues, and high alkalinity does not structurally have obvious similarity with other antibacterial peptide.Identified from the domestic silkworm gene group successively afterwards and obtained 11 other moricin mutant (Cheng et al., 2006; Xia et al., 2004).At present successively from other insect as prodenia litura (Spodoptera litura) (Oizumi et al., 2005), cigarette angle hawkmoth (Manduca sexta) (Zhu et al., 2003), the moricin analogue is from prodenia litura (S. litura) (Oizumi et al., 2005) and separate to obtain the moricin analogue in greater wax moth (Galleria mellonella) (Brown et al., 2008).At present, for the distinctive antibacterial peptide of lepidopteran, research and comparison is few.The microorganism of bactericidal mechanism and expression system research act on to(for) this class antibacterial peptide are also few.

As everyone knows, insect is kind and the maximum animal of quantity in the world, but the source of natural antibacterial peptide limited still only has by chemosynthesis and genetic engineering technique and could obtain a large amount of antibacterial peptides.The chemosynthesis antibacterial peptide is expensive, cost is higher, so genetic engineering technique is the prefered method that obtains antibacterial peptide, is also the most popular means of research antibacterial peptide, but the several amino meetings of moricin C-terminal form special construction, are difficult to obtain this antibacterial peptide by chemical synthesis.And moricin has very strong toxicity to host itself, directly express antibacterial peptide gene in Host Strains, likely kill host cell and can't obtain expression product.

Aureobasidium pullulans belongs to aureobasidium genus, is a kind of opportunistic fungus in humans and animals, can cause eye disease, as keratitis, conjunctivitis etc.It is a class yeast-like fungus, in close relations with yeast, has two kinds of forms of yeast sample and radicula byssoidea.The initial thickness of bacterium colony, dirty white, change light green very soon into, is finally black.The bacterium colony quality is by thick to hard and leather shape, and colony edge is obvious root shape.The vegetative propagation mode is various, mainly is similar to saccharomycetic polygon budding form to forming obvious fungal filament.This mould also easily grows on paint, and it is had to larger corrosive nature and special destructive force.

Summary of the invention

The object of the present invention is to provide a kind of small cabbage moth moricin gene.

Another purpose of the present invention is to provide a kind of small cabbage moth moricin albumen.

Another purpose of the present invention is to provide a kind of preparation method of small cabbage moth moricin mature peptide albumen.

Another purpose of the present invention is to provide the application of a kind of small cabbage moth moricin and its mature peptide.

Purpose of the present invention is achieved by following technical proposals:

A kind of small cabbage moth antibacterial peptide moricin gene, its nucleotide sequence is as shown in SEQ ID NO:1～2.The complete genome sequence that wherein SEQ ID NO:1 is small cabbage moth antibacterial peptide moricin gene (comprising 5 ' end non-coding region, 3 ' end non-coding region and coding region sequence); The ORF sequence that SEQ ID NO:2 is small cabbage moth antibacterial peptide moricin gene; ORF sequence 65 the amino acid whose albumen of encoding.

A kind of small cabbage moth antibacterial peptide moricin, its aminoacid sequence is as shown in SEQ ID NO:3.SEQ ID NO:3 is small cabbage moth antibacterial peptide moricin coding protein sequence, and this sequence is comprised of 65 amino acid.

The mature peptide of a kind of small cabbage moth antibacterial peptide moricin, the aminoacid sequence of mature peptide is as shown in SEQ ID NO:4; Nucleotide sequence corresponding to mature peptide is as shown in SEQ ID NO:5.

The primer of pair for amplification small cabbage moth antibacterial peptide moricin, the nucleotide sequence of primer is as shown in SEQ ID NO:11～12.

A kind of recombinant expression vector, insert the nucleotide sequence of the as above described small cabbage moth antibacterial peptide in SEQ ID NO:1～2 moricin or the as above nucleotide sequence of the mature peptide of the described small cabbage moth antibacterial peptide of SEQ ID NO:5 moricin by the multiple clone site of the carrier that sets out.

Preferably, described recombinant expression vector is pMTA-PxMor.

A kind of recombinant cell lines, built and formed by recombinant expression vector transformed acceptor cell as above, and preferably, described recipient cell is that drosophila cell is S2.

The preparation method of the mature peptide of a kind of small cabbage moth antibacterial peptide moricin, concrete grammar for to add the copper sulfate that final concentration is 250 mM in recombinant cell lines as above, collect and within 10 days, induce substratum supernatant after 24 hours continuously, the substratum supernatant of collection is mixed, centrifugal, collect supernatant, remove cell debris, at first use balance buffer balance Anti-V5 gel beads, secondly carry out sufficient incubation with the supernatant of collecting, then Anti-V5 gel beads mixed solution is added in pillar, flow velocity is 0.05 ml/min, after flowing through several times by the acellular substratum of all collections by pillar, with balance buffer wash-out pillar, until the effluent liquid of collecting is when measuring protein concentration A280 and approaching zero, with wash-out buffer eluted protein, and add 100 ml1 M Tris-base in advance in the pipe of collecting albumen, pH 8.0 collects effluent liquid, and by the component of collecting purity and the concentration with 15% SDS-PAGE electrophoretic analysis albumen, finally the albumen of collection is carried out to desalination, utilize ultrapure water to carry out balance to desalting column Excellulose GF-5, then the albumen of collection is joined in pillar and goes, collect the albumen flowed out, be mature peptide.

The application of small cabbage moth antibacterial peptide moricin as mentioned above, for the preparation of the preparation that suppresses or kill aureobasidium pullulans.

The application of the mature peptide of small cabbage moth antibacterial peptide moricin as mentioned above, for the preparation of the preparation that suppresses or kill aureobasidium pullulans.

For great expression and fast purifying moricin, and keep great-hearted antibacterial peptide.This patent utilizes eukaryotic expression system to express the distinctive antibacterial peptide moricin of lepidopteran, by the great-hearted little peptide of method acquisition tool of cation exchange purification, for application, produces and lays the foundation.

In order to filter out the great-hearted antibacterial peptide of Aureobasidium pullulans, we have done a large amount of screening operations, found that mature peptide moricin has very strong restraining effect to Aureobasidium pullulans, demonstrates potential using value.In order to control Aureobasidium pullulans in actual life, the mankind are worked the mischief, the present invention, in conjunction with the advantage in this laboratory, utilizes animal nutrition to produce mature peptide moricin albumen, and albumen is carried out to biological activity determination to Aureobasidium pullulans.Utilize scanning electron microscope and saturating look Electronic Speculum can very clearly observe mature peptide moricin Aureobasidium pullulans is had to very strong killing action.By this patent, can utilize drosophila cell is that S2 expresses mature peptide moricin, and has screened stable clone, and energy fast purifying recombinant protein, therefore, guaranteed the source of a large amount of mature peptide moricin albumen; And utilize this patent, can be easy to observe has very strong effect to mature peptide to Aureobasidium pullulans.Apply in the near future antibacterial peptide moricin and control the harm of Aureobasidium pullulans, and will be highly significant and application value.

Compared with prior art, the present invention has following beneficial effect:

The present invention clones and obtains antibacterial peptide moricin gene first from Important Agricultural insect small cabbage moth.And antibacterial peptide moricin gene order or its mature peptide gene order are expressed and obtained activated mature peptide by eukaryotic expression system, the mature peptide obtained has very strong restraining effect to Aureobasidium pullulans.For the harm of controlling Aureobasidium pullulans by antibacterial peptide moricin in actual life is laid a good foundation.

figure of description

Fig. 1 .SDS-PAGE inspection detects the result of recombinant protein PxMor.

Fig. 2. the scanning electron microscopic observation of recombinant protein PxMoricin to the effect of Aureobasidium pullulans mycelia; A:CK, show that mycelium is mellow and full, smooth surface; B: thick leach protein stoste is directly processed mycelia, shows that mycelium is wilted, shrivelled, depression.

Fig. 3. the transmission electron microscope observing of recombinant protein PxMor to the effect of Aureobasidium pullulans spore; A:CK, show that cell walls, cytolemma are complete, and tenuigenin is even, the organoid clear in structure; B:50% recombinant protein PxMor processes spore, the entocyte pyknosis, and the organoid sex change disappears.

Embodiment

Further describe the present invention below in conjunction with the drawings and specific embodiments.Unless stated otherwise, the reagent adopted in embodiment and method are conventional reagent and the method for using in this area.

The clone of embodiment 1 small cabbage moth antibacterial peptide moricin gene

S1. the laboratory rearing small cabbage moth ( plutella xylostella) larva, the use intestinal bacteria ( escherichia coli) and Aureobasidium pullulans a. Pullulansas supplying the examination bacterial classification.

S2. small cabbage moth is transcribed the mensuration of group sequence: collect 1 age of small cabbage moth to 4 instar larvaes, prepupa, pupa, adult, extract after total RNA and utilized the RNA-seq technical measurement it transcribes the group sequence, the extracting method of total RNA operates with reference to Trizol (Invitrogen, USA) test kit specification sheets.Measured altogether 34,522,812 clean reads, the reads mean length is 90bp, obtains 3,107,053,080 base.Finally be assembled into 107710 Unigene, length range is 200 ~ 3000bp.COG, GO, KEGG annotation and analyze after obtain altogether the Unigene of 68984 higher annotation confidence levels of tool, 725 of the Unigenes directly related with Insect immunity, the Unigene1 bar of Moricin wherein, its sequence is 186 bp, shown in SEQ ID NO:6.

S3. according to Unigene design 3 ' end of Moricin and the special primer of 5 ' end RACE end rapid amplifying reaction, 3 ' RACE primer sequence is: 5 '-CCCACGCCGCGCCCAAGGT-3 ', as shown in SEQ ID NO:7,5 ' RACE primer is 5 '-CCGCCGCGCCGATCACACCGA-3 ', as shown in SEQ ID NO:8.3 ' end and 5 ' end RACE matches and carries out the reaction of RACE end rapid amplifying with the UPMs primer respectively, and the UPMs primer sequence is: UPM-L:5 '-CTAATACGACTCACTATAGGGCAAGCAG

As shown in TGGTATCAACGCAGAGT-3 ' (as SEQ ID NO:9) and UPM-S:5 '-CTAATACGAC TCACTATAGGGC-3 ' as shown in SEQ ID NO:10.

S4.cDNA the first chain is synthetic:

At first, extract the total RNA of small cabbage moth: the Escherichia coli bacteria liquid that is 0.8～1.0 by the OD value stimulates small cabbage moth 4 instar larvaes, carries out inducing of peptidoglycan recognition protein.Get the small cabbage moth of 0.5～1g after 6 hours induce, grind in liquid nitrogen, by the Trizol method, extract the total RNA of small cabbage moth; The extracting method of total RNA operates with reference to Trizol (Invitrogen, USA) test kit specification sheets.

According to the RACE of CLONTECH company test kit specification sheets, carry out: the total RNA of 10 μ g adds reaction solution 20 μ L, 42 ℃ of reaction 90min.72 ℃ of reaction 10min, termination reaction.Reaction solution forms: 50 mmole Repone K, 3 mmole magnesium chlorides, 10 mmole Tris-HCL pH8.3,1 mmole DTT, 5 micromole d NTP, 25U RNA enzyme inhibitors, 8U AMV reversed transcriptive enzyme.

S5.3 ' RACE and 5 ' RACE reaction: cDNA the first chain that the step S4 reverse transcription of take obtains is template, and the primer shown in S3 carries out the PCR reaction, and chain polymerization enzyme reaction (PCR) reagent and condition are as follows:

At first by following reagent mix together

3 '-RACE in, forward primer and reverse primer be respectively 3 '-RACE(is as shown in SEQ ID NO:7) and UPMs primer (as shown in SEQ ID NO:9 and SEQ ID NO:10); 5 '-RACE in, forward primer and reverse primer be respectively 5 '-RACE(is as shown in SEQ ID NO:8) and UPMs primer (as shown in SEQ ID NO:9 and SEQ ID NO:10).

The PCR reaction conditions is: then 94 ℃ of sex change 5min at first enter following circulation: 94 ℃ of 30s, and 65 ℃ of 30 s, 72 ℃ of 50s, carry out 35 circulations altogether, and last 72 ℃ are extended 10min.

S6. reaction product purifying: utilize OMEGA BIO-TEK company product (Gel Extraction Kit) operation steps to be undertaken by product description.Get purified product 5 μ L, be connected with pMD18-T carrier (TaKaRa).Be transformed into e.colistraindh5α, grow overnight on the flat board of the isopropylthiogalactoside (IPTG) of the penbritin that contains 100mg/mL and 0.1moL/mL, 6 hickies of picking, in LB liquid nutrient medium (5mL, containing 100 μ g/mL penbritins) overnight incubation.Collect incubated overnight bacterium liquid 1 ~ 2mL, centrifugal (10000rpm, 1min) collecting cell.With minim DNA purification kit (OMEGA BIO-TEK) plasmid purification, the purification step by specification carries out.

Sequence order-checking and homology retrieval: get the bacterium liquid of 500 μ L containing positive plasmid, serve extra large invitrogen company and checked order.By institute's calling sequence and with the gene pool sequence, compare.

S7. the acquisition of small cabbage moth antibacterial peptide moricin full length gene cDNA

According to the RACE result, design pair of primers ORFF and ORFR, primer sequence SEQ ID NO:11 and EQ ID NO:12.

SEQ ID NO：11 ORFF 5′-ACAGTCGCGTTCAACAGTGAA-3′

SEQ ID NO：12 ORFR 5′-ATGGTGTCCACCCCTGGCACT- 3′

The cDNA of small cabbage moth 4 instar larvaes of take is template, and reaction conditions is as follows:

Described mixed solution is composed as follows:

Figure 201310411620X100002DEST_PATH_IMAGE002

Mixed solution, at 94 ℃ of denaturation 5min, is then entered to following circulation: 94 ℃, 40 seconds, 56 ℃, 40 seconds, 72 ℃, 50 seconds, carry out altogether 35 circulations, last 72 ℃ are extended 7 minutes, and complete rearmounted 4 ℃ of termination reactions increase.Reaction product purifying: utilize OMEGA BIO-TEK company product (Gel Extraction Kit) operation steps to be undertaken by product description.

S8. antibacterial peptide gene clone: get purified product 5 μ L, with pMD18-T(TaKaRa) carrier is connected.Be transformed into e.colistraindh5α, in the dull and stereotyped grow overnight that contains penbritin (100mg/mL) and isopropylthiogalactoside (0.1mol/mL), 6 hickies of picking, in LB liquid nutrient medium (5mL, containing 100 μ g/mL penbritins) incubated overnight.

Plasmid purification: collect incubated overnight bacterium liquid 1 ~ 2mL, centrifugal (10000 turn, 1 minute) collecting cell.With minim DNA purification kit (OMEGA BIO-TEK) plasmid purification, the purification step by specification carries out.

Sequence order-checking and homology retrieval: get 500 μ L bacterium liquid, serve extra large invitrogen company and checked order.By institute's calling sequence and with the gene pool sequence, compare.

The sequencing result analysis is found: the full length sequence of the gene of the small cabbage moth moricin that the clone obtains is that 482bp(is as shown in SEQ ID NO:1), the ORF of small cabbage moth moricin gene is 198bp, the ORF sequence is as shown in SEQ ID NO:2; 65 the amino acid whose albumen of encoding, coding protein sequence is as shown in SEQ ID NO:3.

Through sequential analysis, find: the mature peptide sequence is as shown in SEQ ID NO:4; APKVNVNAL

KKGGRVIKKGLGVIGAAGTAHEVYNHVRNRNQG(is totally 42 aa) be the mature peptide sequence; Nucleotide sequence corresponding to mature peptide is as shown in SEQ ID NO:5.

Embodiment 2 has the preparation method of the small cabbage moth recombinant protein moricin of sterilizing function:

S1. design the primer of amplification small cabbage moth moricin mature peptide gene:

MF:5 '-GCGATATGTGCGCCCAAGGTCAACGTCAACGCCC-3 '; Wherein, ATATGT means that the restriction enzyme site of introducing is the Bgl II; As shown in SEQ ID NO:13

MR:5 '-GCCTCGAGCCCCTGGTTCCTGTTCCGCACG-3 '; Wherein, CTCGAG means that the restriction enzyme site of introducing is XhoI; As shown in SEQ ID NO:14.

S2. the encode amplification of small cabbage moth moricin mature peptide gene:

Described mixed solution is composed as follows:

Containing 20 mM Mg ²⁺10 * PCR damping fluid, 5 μ L;

Concentration is respectively the dNTP mixture 4 μ L of 2.5 mM;

10 μ M primer MF 4 μ L;

10 μ M primer MR 4 μ L;

The high-fidelity DNA polymerase 0.5 μ L of 5U/ μ L

Aqua sterilisa 32.5 μ L.

Mixed solution, at 94 ℃ of denaturation 5min, is then entered to following circulation: 94 ℃, 40s, 62 ℃, 40s, 72 ℃, 50s, carry out 35 circulations altogether, and last 72 ℃ are extended 7min, and complete rearmounted 4 ℃ of termination reactions increase.

S3. the expression of small cabbage moth moricin mature peptide in the S2 cell

S31. the acquisition of recombinant plasmid pMTA-PxMor: the mature peptide cDNA sequence of the small cabbage moth moricin that pcr amplification is obtained is carried out Bgl II and Xho I double digestion, after glue purification reclaims, with external connection of plasmid pMT/Bip/V5/HisA cut back to close through same enzyme, construction expression plasmid pMTA-PxMor;

S32. add 10% heat-inactivated fetal bovine serum and 1% penicillin-Streptomycin sulphate water culture Drosophila S 2 cells in the fruit bat special culture media, be placed in 27 ℃ of incubators and cultivate.When transfection DNA, seed cell will be in serum free medium overnight incubation, according to transfection reagent specification sheets transfection DNA, after the cell of bed board reaches 70% fusion rate, start transfection.Order-checking is identified to correct recombinant plasmid pMTA-PxMor transient transfection drosophila cell is the S2 cell, utilize the expression of Tricine-SDS-PAGE electrophoresis detection moricin in the S2 cell.

S33. for purification of recombinant proteins moricin.Utilize DES Inducible/Secreted test kit to screen stable clone pMTA-PxMor in conjunction with pCoBlast.In order to build stable clone, plasmid pCoBlast and plasmid pMTAMor cotransfection are in the S2 cell, after transfection 48 hours, eccentric cell, remove supernatant, with being added with 25 mg/ml Blasticidine S hydrochloride perfect medium re-suspended cells, after cultivating a week, resistance clone just can be out.

S34. the recombinate purifying of moricin

Restructuring moricin purification result is as Fig. 2, and concrete operations are as follows: adopt molecular weight to hold back with cationic exchange and carry out purification of recombinant proteins: for purification of recombinant proteins moricin, at first build S2 stable cell lines pMTA-PxMor.Add the copper sulfate inducible protein that final concentration is 250 mM in the Tissue Culture Flask of 75cm2.Collect and within 10 days, induce substratum supernatant after 24 hours continuously, and add fresh substratum at every turn in the Tissue Culture Flask of 75cm2.For purifying protein, the substratum supernatant of all collections is mixed, centrifugal 10 min of 1000 g under normal temperature, collect supernatant, removes cell debris.Supernatant is respectively with the ultrafiltration centrifugal column 10 of holding back 10 kDa, and centrifugal 10 min of 000g ultrafiltration, collect filtration fraction, and then, with the ultrafiltration centrifugal column 10 of holding back 3 kDa, centrifugal 10 min of 000g ultrafiltration, abandon filtration fraction, collects not filtration fraction.The sodium acetate dialysed overnight of 0.1M for the liquid of collecting through ultrafiltration, directly loading is in advance through the CM-Sepharose CL-6B of sodium acetate (pH5.0,0.1M) balance post, then with the 150mL balance liquid to 150mL, the sodium acetate buffer of pH 5.0 1 M is made gradient development, after gradient finishes, then use 300mL, 1 M NaAc(pH 5.0) wash-out, flow velocity is 0.2mL/min, every 1min collects a pipe, collects and merges each Peak Activity, and carry out western blot detection.The protein frozen drying of collecting ,-80 ℃ of preservations.

The biological activity determination of embodiment 3 PxMor albumen

Utilize scanning electron microscope to observe the effect of PxMor albumen to the Aureobasidium pullulans mycelia, concrete operations are as follows:

S1. recombinant protein Pxmor processes the Aureobasidium pullulans mycelia: be about to recombinant protein Pxmor and add in substratum, the final content that makes recombinant protein Pxmor is 10%, contrast adds 10 mmol/L pH7.2 phosphate buffered saline buffers, and the inoculation Aureobasidium pullulans is cultivated 5 days for 28 ℃;

The mycelia of S2. recombinant protein Pxmor being processed and contrast mycelia piece (0.5 * 0.5cm ²) after, with 10mmol/L pH 7.2 phosphate buffered saline buffers, rinse 2 times, add 2.5% glutaraldehyde fixedly to spend the night.0.1mol/L phosphate buffered saline buffer rinses 3 times, each 15min; 1% osmic acid is fixedly after 1.5h, then rinses 3 times with the 0.1mol/L phosphate buffered saline buffer; Through 50%, 70%, 80%, 90% dehydration of alcohol respectively once, each 15min, then use 100% dehydration of alcohol 2 times, each 15min; Then process 2 times each 20min with isoamyl acetate.Enter critical point drying, fill afterwards platform, the vacuum metal spraying.The scanning electron microscope sample made is observed and is recorded result, takes pictures under scanning electron microscope.

S3. utilizing transmission electron microscope to observe recombinant protein comprises the following steps the electron microscopic observation of fungi Aureobasidium pullulans spore:

S31. the Aureobasidium pullulans spore suspension (10 in vegetative period of taking the logarithm ⁸individual/mL), add recombinant protein Pxmor to process, making its final concentration is 50%, adds sodium phosphate buffer and the logarithmic phase Aureobasidium pullulans spore suspension (10 of 10 mmol/L pH 7.2 in control tube ⁸individual/mL) each 0.5mL, 37 ℃ of water bath with thermostatic control 3h;

S32. the spore suspension that recombinant protein Pxmor processes and contrast spore, in the centrifugal 5min of 3000 g, collecting precipitation, wash twice with the sodium phosphate buffer of 10 mmol/L pH 7.2, after fixedly spending the night by 2.5% glutaraldehyde, adds 2% agar of 45 ℃ of thawings to fix.Rinse 6 times each 20min with the 0.1mol/L phosphate buffered saline buffer; Fixedly after 3h, rinse 6 times each 10min with the 0.1mol/L phosphate buffered saline buffer after 1% osmic acid; Through 50%, 70%, 80%, 90% dehydration of alcohol respectively once, each 10min, then use 100% dehydration of alcohol 2 times, each 10min; With propylene oxide, process 2 times, each 15min, then through propylene oxide: epoxy resin 3:1,1:1, each mixed solution of 1:3 and pure epoxy resin permeate step by step, and the time is respectively 1h, 2h, 2h, 4h.Then embedding, repair piece, ultrathin section(ing), the two dyeing of acetic acid uranium and lead citrate.The transmission electron microscope sample made is observed and is recorded result, takes pictures under transmission electron microscope.

Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.

SEQUENCE LISTING

<110 > Agricultural University Of South China

<120 > a kind of small cabbage moth antibacterial peptide moricin and preparation method thereof and application

<130>

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<170> PatentIn version 3.3

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<400> 1

acatggggat tcaaccacga gcagtcacag ctaacagtcg tgtcctcccg agtacagtcg 60

cgttcaacag tgaatatgag attcttccac ttgctgatgc tggcgctggc ggccatgacg 120

ctgctgctgg gcggctccca cgccgcgccc aaggtcaacg tcaacgccct caagaagggc 180

ggccgtgtca ttaaaaaagg tctcggtgtg atcggcgcgg cggggacggc gcatgaagtg 240

tacaaccacg tgcggaacag gaaccagggg tagacagcac caggagtaga cagtgccagg 300

ggtggacacc atcagggatg gataccgtcg gcaaacgagg tctacttacc gtataccctg 360

acgtgatggt ctttagtatt attaaaggat gatgcgccta gttgtaaata tattgtttaa 420

gatactgtat aatttaaata aatcatgttc acaataaaaa aaaaaaaaaa aaaaaaaaaa 480

gt 482

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aaaggtctcg gtgtgatcgg cgcggcgggg acggcgcatg aagtgtacaa ccacgtgcgg 180

aacaggaacc aggggtag 198

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Met Arg Phe Phe His Leu Leu Met Leu Ala Leu Ala Ala Met Thr Leu

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Leu Leu Gly Gly Ser His Ala Ala Pro Lys Val Asn Val Asn Ala Leu

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Gly

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Ala Pro Lys Val Asn Val Asn Ala Leu Lys Lys Gly Gly Arg Val Ile

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Tyr Asn His Val Arg Asn Arg Asn Gln Gly

35 40

<210> 5

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gcgcccaagg tcaacgtcaa cgccctcaag aagggcggcc gtgtcattaa aaaaggtctc 60

ggtgtgatcg gcgcggcggg gacggcgcat gaagtgtaca accacgtgcg gaacaggaac 120

caggggtag 129

<210> 6

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<213> Moricin unigene

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atgagattct tccacttgct gatgctggcg ctggcggcca tgacgctgct gctgggcggc 60

tcccacgccg cgcccaaggt caacgtcaac gccctcaaga agggcggccg tgtcattaaa 120

aaaggtctcg gtgtgatcgg cgcggcgggg acggcgcatg aagtgtacaa ccacgtgcgg 180

aacagg 186

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cccacgccgc gcccaaggt 19

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ccgccgcgcc gatcacaccg a 21

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ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

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acagtcgcgt tcaacagtga a 21

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atggtgtcca cccctggcac t 21

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gcgatatgtg cgcccaaggt caacgtcaac gccc 34

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gcctcgagcc cctggttcct gttccgcacg 30

Claims

1. a small cabbage moth antibacterial peptide moricin gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1～2.

2. a small cabbage moth antibacterial peptide moricin, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:3.

3. the mature peptide of a small cabbage moth antibacterial peptide defensin, is characterized in that, the aminoacid sequence of mature peptide is as shown in SEQ ID NO:4, and the corresponding nucleotide sequence of mature peptide is as shown in SEQ ID NO:5.

4. the primer of pair for amplification small cabbage moth antibacterial peptide moricin, is characterized in that, the nucleotide sequence of primer is as shown in SEQ ID NO:11～12.

5. a recombinant expression vector, is characterized in that, inserted the nucleotide sequence of the mature peptide of the nucleotide sequence of the described small cabbage moth antibacterial peptide of claim 1 moricin or the described small cabbage moth antibacterial peptide of claim 3 moricin by the multiple clone site of the carrier that sets out.

6. recombinant expression vector according to claim 5, is characterized in that, described recombinant expression vector is pMTA-PxMor.

7. a recombinant cell lines, is characterized in that, is to build gained by the described recombinant expression vector transformed acceptor cell of claim 5 or 6.

8. the preparation method of the mature peptide of a small cabbage moth antibacterial peptide moricin, it is characterized in that, concrete grammar for to add the copper sulfate that final concentration is 250 mM in recombinant cell lines claimed in claim 7, collect and within 10 days, induce substratum supernatant after 24 hours continuously, the substratum supernatant of collection is mixed, centrifugal, collect supernatant, remove cell debris, at first use balance buffer balance Anti-V5 gel beads, secondly carry out sufficient incubation with the supernatant of collecting, then Anti-V5 gel beads mixed solution is added in pillar, flow velocity is 0.05 ml/min, after flowing through several times by the acellular substratum of all collections by pillar, with balance buffer wash-out pillar, be eluted to when measuring protein concentration A280 and approaching zero, change wash-out buffer wash-out target protein into, when collecting albumen, add 100 μ L 1 M Tris-base(pH 8.0 in advance in the pipe of collecting albumen) the collection effluent liquid, utilize purity and the concentration of 15% SDS-PAGE electrophoretic analysis albumen, finally the albumen of collection is carried out to desalination, utilize ultrapure water to carry out balance to desalting column Excellulose GF-5, then the albumen of collection is joined in pillar and goes, collect the albumen flowed out, be mature peptide.

9. the application of the described small cabbage moth antibacterial peptide of claim 2 moricin, is characterized in that, for the preparation of the preparation that suppresses or kill aureobasidium pullulans.

10. the application of the mature peptide of the described small cabbage moth antibacterial peptide of claim 3 moricin, is characterized in that, for the preparation of the preparation that suppresses or kill aureobasidium pullulans.