CN101328482A - Chrysalid pteromalid antibiotic protein Pp-AP2 and encoding nucleic acid sequence thereof - Google Patents
Chrysalid pteromalid antibiotic protein Pp-AP2 and encoding nucleic acid sequence thereof Download PDFInfo
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- CN101328482A CN101328482A CNA2008101201464A CN200810120146A CN101328482A CN 101328482 A CN101328482 A CN 101328482A CN A2008101201464 A CNA2008101201464 A CN A2008101201464A CN 200810120146 A CN200810120146 A CN 200810120146A CN 101328482 A CN101328482 A CN 101328482A
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Abstract
The invention discloses a nucleic acid sequence for coding a pteromalus puparum antimicrobial protein Pp-AP2. The separated DNA molecule comprises: a nucleotide sequence for coding peptides with pteromalus puparum antimicrobial protein activity, the nucleotide sequence and the nucleotide sequence from the 175th ribonucleotide to the 486th ribonucleotide in the SEQ ID NO:1 have a 70 percent homology; or the nucleotide sequence can be hybridized with the nucleotide sequence from the 175th ribonucleotide to the 486th ribonucleotide in the SEQ ID NO:1 at a temperature of between 40 and 55 DEG C. The invention also discloses a pteromalus puparum antimicrobial protein Pp-AP2 peptide which has an amino acid sequence shown in the SEQ ID NO: 2. The pteromalus puparum antimicrobial protein Pp-AP2 has antibiosis performance.
Description
Technical field
The present invention relates to molecular biology and genetically engineered field.The nucleotide sequence of particularly a kind of antibiotic protein Pp-AP 2 of in the chrysalis tiny golden wasp, expressing and coding thereof.
Background technology
In recent years, because the abuse of medicine, problem such as drug residue and bacterial drug resistance is day by day serious, existing antibiotic medicine is losing original curative effect, thereby caused the concern of people to food safety, more and more countries begins to appeal the forbidding microbiotic, and antibacterial peptide (antibacterial peptides) is active and be different from traditional antibiotic special role mechanism because of its unique biological, cause people's great research interest, become one of the focus in molecular biology and biochemical research field.The researchist of various countries has found hundreds of kind antibacterial peptide from Mammals and insect, Amphibians etc., wherein some separation and Extraction come out, and its structure, anti-microbial activity etc. have been done many research, find that its anti-microbial activity is efficient, wide spectrum, and itself is nontoxic, harmless, and bacterium, virus, some protozoon, fungi and tumour cell etc. are had selective killing effect.
From Boman research group of Stockholm Univ Sweden in 1980, first by intestinal bacteria injections induce purify in the cecropia moth diapause pupa hemolymph of processing since the antibacterial peptide, domestic and international many scholars have done a large amount of screenings with regard to the antibacterial protein/peptide in insect source, determination of activity, work such as gene cloning and expression, and clear and definite insect antibacterial protein/peptide can be divided into cecropin (cecropins), defensin (defensins), the antibacterial peptide of proline rich (prolin-rich antibacterialpeptides) and be rich in antibacterial peptide (glycine-rich antibacterial the peptides) (Brey﹠amp of glycine; Hultmark, 1998).
As the biology of most species on the earth, insect has the characteristics fast, wide adaptability of breeding; Except that the ocean, all ecotopes all have the distribution of insect.From 19th century, insect just is used as the research object of immune defence system.Remain after tellurian insect all is to win the victory in violent struggle for existence and just survive, they have strengthened the ability of resisting various pathogen infections in the process of evolving, this just provides abundant genetic resources storehouse for we screen insect antibacterial protein/peptide and goal gene thereof with anti-microbial effect.Numerous researchists have found hundreds of antibacterial proteins from different insects, but do not have any bibliographical information to separate the antibacterial peptide that obtains as yet from the chrysalis tiny golden wasp.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of have the chrysalis tiny golden wasp antibiotic protein Pp-AP 2 of anti-microbial property and the nucleotide sequence of coding thereof.
In order to solve the problems of the technologies described above, the invention provides a kind of nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 coding, its isolated dna molecular comprises: coding has the active polypeptide nucleotide sequence of chrysalis tiny golden wasp antibacterial protein, shows at least 70% homology from the nucleotides sequence of Nucleotide 175-486 position among this nucleotide sequence and the SEQ ID NO:1; Perhaps this nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO:1 in from the nucleotide sequence hybridization of Nucleotide 175-486 position.
Improvement as the nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 of the present invention coding: this sequence has the nucleotide sequence of 175-486 position among the SEQID NO:1.
Further improvement as the nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 of the present invention coding: comprise 8～312 continuous nucleotides in this sequence.
The present invention also provides above-mentioned chrysalis tiny golden wasp antibiotic protein Pp-AP 2 polypeptide simultaneously, and it has the aminoacid sequence shown in the SEQ ID NO:2.
Improvement as chrysalis tiny golden wasp antibiotic protein Pp-AP 2 polypeptide of the present invention: chrysalis tiny golden wasp antibacterial protein polypeptide is polypeptide, its conservative property variation polypeptide, its active fragments or its reactive derivative.
The nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 provided by the present invention and coding thereof makes it can be applied in the new antibacterial protein of expressing in the chrysalis tiny golden wasp, encoding sequence and at foodstuff additive that are developed to using value and medicine and be applied to a plurality of fields such as agricultural, industry and food sanitation.
The invention has the advantages that: the present invention passes through structure and the high-efficient cloning screening method based on the chrysalis tiny golden wasp antibiotic protein/peptide gene in cDNA expressivity library, is experiment material with the chrysalis tiny golden wasp, screens the antibacterial protein of its expression in vivo.This method is in view of can the cause death characteristics of host bacterium of potential antibacterial protein/peptide purpose representation own, and expression of gene and the representation anti-microbial activity rough determination of the chrysalis tiny golden wasp of potential antibacterial protein/peptide closely is harmonious, and screens.When screening representation can make the clone of host bacterium death the time, also just obtained to come from accordingly the goal gene in the library of insertion of chrysalis tiny golden wasp.
After plasmid with anti-microbial effect gene is transformed into the SOLR bacterium, after genetic expression under the inducing of inductor IPTG (sec.-propyl-b-d-thiogalactoside), produce antimicrobial albumen/peptide, cause the cytolemma of thalline to receive destruction, thereby allow dye molecule to enter bacterial cell, color reaction takes place, and promptly observes and finds blue clone.So these are dyed blue clone is exactly the clone who contains antifungal genes.They are chosen the back order-checking, just can obtain the corresponding antibacterial protein of corresponding gene order and coding thereof.
The present invention is achieved by the following technical solutions: the present invention isolated dna molecular comprise: coding has the nucleotide sequence of the active polypeptide of chrysalis tiny golden wasp antibiotic protein Pp-AP 2, and shows at least 70% homology from the nucleotides sequence of Nucleotide 175-486 position among described nucleotide sequence and the SEQ IDNO:1; Perhaps described nucleotide sequence can be under the 40-55 degrees celsius with SEQ ID NO:1 in from the nucleotide sequence hybridization of Nucleotide 175-486 position.Preferable, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2.More preferably, described sequence has among the SEQ ID NO:1 nucleotide sequence from Nucleotide 175-486 position.
The isolated chrysalis tiny golden wasp of the present invention antibiotic protein Pp-AP 2 comprises: polypeptide or its conservative property variation polypeptide or its active fragments, perhaps its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is to have SEQ ID NO:2 polypeptide of sequence.
Dna molecular of the present invention comprises 8-312 continuous nucleotide in the described dna molecular.
Dna molecular transformed host cells of the present invention is a prokaryotic cell prokaryocyte.
In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, also refer to this dna fragmentation with native state under the component of adjoint kernel thuja acid separate, and separate with the protein of in cell, following it.
In the present invention, the nucleotide sequence of chrysalis tiny golden wasp antibacterial protein (or polypeptide) Pp-AP2 coding refers to: coding has the nucleotide sequence of the active polypeptide of chrysalis tiny golden wasp antibiotic protein Pp-AP 2, as 175-486 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO:1.This degenerate sequence is meant, is arranged in SEQ ID NO:1 sequence encoding frame 175-486 position Nucleotide, has the be encoded degenerate codon of same amino acid of one or more codons to replace the sequence that the back produces.Because the degeneracy of password, thus with SEQ ID NO:1 in 175-486 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO:1 of also encoding out.
Also comprising can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO:1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 175-486 position.Also comprise with SEQ ID NO:1 in from the homology of nucleotide sequence at least 70% of Nucleotide 175-486 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.Also comprising to encode has the variant form of open reading frame sequence among the proteic SEQ ID NO:1 of natural chrysalis tiny golden wasp antibiotic protein Pp-AP 2 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance of Nucleotide, insertion/or replace, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
Chrysalis tiny golden wasp antibiotic protein Pp-AP 2 or polypeptide refer in the present invention: have the active SEQ ID of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 NO:2 polypeptide of sequence.This term also comprises having and variant form natural chrysalis tiny golden wasp antibiotic protein Pp-AP 2 identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion/or replace, and add one or several at C-terminal and/or N end and (be generally in 20, preferably be in 10, more preferably with in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again not as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of chrysalis tiny golden wasp antibiotic protein Pp-AP 2.
The polypeptide of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 conservative property variation in the present invention refers to: compare with the aminoacid sequence of SEQ ID NO:2, there are 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also comprises the analogue of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 or polypeptide.The difference of these analogues and natural antibacterial polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, can also pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its proteolysis performance or optimized solubility property by modifying.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing chrysalis tiny golden wasp antibacterial protein polypeptide Pp-AP2 of the present invention, chrysalis tiny golden wasp antibiotic protein Pp-AP 2 encoding sequence operationally can be connected in expression regulation sequence, thereby form chrysalis tiny golden wasp antibiotic protein Pp-AP 2 expression vector.
As used herein " operationally being connected in " refers to such a case, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can translate, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
Host cell is a prokaryotic cell prokaryocyte in the present invention.Prokaryotic host cell commonly used refers to such an extent that be Bacillus coli cells.
Whether and quantity the expression of also available Northern blotting technical Analysis chrysalis tiny golden wasp antibiotic protein Pp-AP 2 gene product, the existence of rna transcription thing in cell of promptly analyzing chrysalis tiny golden wasp antibiotic protein Pp-AP 2.
In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-312 continuous amino acid of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding chrysalis tiny golden wasp antibiotic protein Pp-AP 2.
The present invention relates to whether exist in the test sample method of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to chrysalis tiny golden wasp antibiotic protein Pp-AP 2 nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In addition, according to chrysalis tiny golden wasp antibacterial protein nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening chrysalis tiny golden wasp antibiotic protein Pp-AP 2 homologous gene or homologous protein.
In order to obtain the dot matrix with the chrysalis tiny golden wasp cDNAs of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 gene-correlation, can screen chrysalis tiny golden wasp cDNA library with dna probe, these probes are under low stringent condition, use
32P chrysalis tiny golden wasp antibiotic protein Pp-AP 2 all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from the chrysalis tiny golden wasp.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, and for example available from Clonetech, Stratagene, Palo Alto......, this screening method also can discern the nucleotide sequence with the gene family of chrysalis tiny golden wasp antibiotic protein Pp-AP 2.
Chrysalis tiny golden wasp antibiotic protein Pp-AP 2 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and obtain relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., SanFrancisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected the molecule that produces total length with chemical process then.Utilize chrysalis tiny golden wasp antibiotic protein Pp-AP 2 of the present invention,, can filter out chrysalis tiny golden wasp antibiotic protein Pp-AP 2 interactional material takes place, perhaps acceptor, inhibitor or anti-antagonistic agent etc. by various conventional screening methods.
The present invention has tangible effect in resistant proof, the growth that suppresses bacterium is had positive effect.China has numerous castes, but seldom finds new antibacterial protein in its body, and chrysalis tiny golden wasp antibiotic protein Pp-AP 2 of the present invention possesses the new albumen of anti-microbial effect just, therefore, has very big using value.
The nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 coding of the present invention can obtain in accordance with the following methods:
(1) cDNA (complementary DNA (cDNA)) of chrysalis tiny golden wasp antibacterial protein/peptide Pp-AP2 gene, the structure in expressivity library:
Separate total RNA from the chrysalis tiny golden wasp, extract poly (A)+RNA, the clone make up the cDNA expressivity library of chrysalis tiny golden wasp antibacterial protein/peptide Pp-AP2 gene;
(2) cDNA expressivity library screening:
A, preparation SOLR bacterium liquid;
B, bite that Jun body library is transformed in the SOLR bacterium liquid for preparing and measure and tire former;
C, preparation LB/Amp+ filter membrane flat board;
D, get the SOLR bacterium liquid bed board that has transformed, coated plate on agarose/penbritin millipore filtration flat board of 0.45 millimeter earlier, being inverted to cultivate for 37 degrees centigrade had less bacterium colony to form to film in about 15 hours; Transfer to agarose/penbritin/sec.-propyl-b-d-thiogalactoside substratum, be inverted for 37 degrees centigrade and cultivated 3～4 hours,, go to the dyeing nutrient agar, dyeed about 10 minutes to colony growth to normal size (1 millimeter); Simultaneously with comparing without sec.-propyl-b-d-thiogalactoside (IPTG) inducing culture; The picking blue colonies contains in the agarose/penbritin nutrient solution of 20% glycerine in 100 microlitres, and subzero 70 degrees centigrade of storages get chrysalis tiny golden wasp antibacterial protein/peptide Pp-AP2 gene.
In the above-mentioned steps (1): total RNA obtains from integral body or the certain organs separation of insect, adopts the purified mRNA test kit to obtain poly (A)+RNA, employing cDNA synthetic agent box,
The synthetic agent box and
Gigapack III clone test kit (buying no Chinese) by U.S. clontech company's T akana Agcy make up cDNA expressivity library.
Among the d of above-mentioned steps (2): the dyeing nutrient agar by * * per-cent (according to the preparation of following per-cent): tongue phenol indigo plant 0.0005% (0.005%), tetrabromophenol sulfonphthalein 0.0005% (0.005%), agar 0.4% (takes by weighing the blue and 0.5g tetrabromophenol sulfonphthalein of 0.5g tongue phenol, be dissolved in respectively in the 9.5 gram water, thereby be made into blue mother liquor of 5% tongue phenol and tetrabromophenol sulfonphthalein mother liquor respectively.Other takes by weighing 4 gram agar and is dissolved in the 1L water, and heating makes its thorough dissolving; Add the blue mother liquor of 1 milliliter of tongue phenol and 1 milliliter of tetrabromophenol sulfonphthalein mother liquor, make that the final concentration of two kinds of dyestuffs is 0.005%, placement gets final product its cooled and solidified half an hour behind the mixing.)。Carry this SOLR bacterium liquid in the SOLR bacterium liquid cDNA synthetic agent box.
The former Jun body library of biting of step (2) is cDNA expressivity library constructed in the step (1).
Can be according to following formulated agarose/penbritin filter membrane flat board: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water.About 120 celsius temperature autoclaving postcooling to 50 degree centigrade, 1 milliliter in penbritin (Amp) mother liquor (autogamy) that adds 100 mg/ml, shaking up the back falls in culture dish, wait after its cooled and solidified and to put the millipore filtration that high-temperature sterilization crosses in media surface (aperture is 0.45 micron, purchase in Baoding, Hebei province laboratory apparatus trade Co., Ltd of Wanke), this is agarose/penbritin filter membrane flat board.
Can be according to following formulated agarose/penbritin/sec.-propyl-b-d-thiogalactoside substratum: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water.About 120 celsius temperature autoclaving postcooling to 50 degree centigrade, 1 milliliter in penbritin (Amp) mother liquor (autogamy) that adds 100 mg/ml, 0.5 1 milliliter in the isopropylthiogalactoside of mol (IPTG) mother liquor (autogamy), shake up the back and fall in culture dish, waiting after its cooled and solidified is agarose/penbritin/sec.-propyl-b-d-thiogalactoside substratum.
Sequence that the present invention relates to and mark are as follows respectively:
(1) sequence signature:
(A) length: 678bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(2) molecule type: Nucleotide
(3) sequence description:
Zhejiang University
Chrysalis tiny golden wasp antibiotic protein Pp-AP 2 encoding sequence
678
DNA
Chrysalis tiny golden wasp (Pteromalus puparum)
GGGAACAAaA AGCTGGAGCT CCACCGCGGT GGCGGCCGCT CTAGAACTAG TGGATCCCCC 60
GGGCTGCAGG AATTCGGCAC GAGGCAACCA TCAACTTGAA TAGTTTCCAA AACTAAAAAC 120
TCAAAAGCCA CAATGAAGTT CGTCCTCAGT TTCGTGTTGC TCGCTGTAGC CCTT ATG GTA 180
Met Val
1
GCC GTA GAT GCT TCA CCA TAC GTA CCA CCA GTT CAA AAG CCT CAC CCG AAC 231
Ala Val Asp Ala Ser Pro Tyr Val Pro Pro Val Gln Lys Pro His Prp Asn
5 10 15
GGA CCC AAA TTC CCG ACT TTC CCC GGT CAA GGA ACT TGG AGT GGC AGA CCT 282
Gly Pro Lys Phe Pro Thr Phe Pro Gly Gln Gly Thr Trp Ser Gly Arg Pro
20 25 30 35
CGT CGC TCA CCT CAG AAG AAC GGT CAA ATC GAA ATC CAC GGC AAG AAG GAA 333
Arg Arg Ser Pro Gln Lys Asn Gly Gln Ile Glu Ile His Gly Lys Lys Glu
40 45 50
GGT GGA AAG ACT TCC TGG AGC GTT GAG GGC CAG CAC AAA GTC TGG GGT AAC 384
Gly Gly Lys Thr Ser Trp Ser Val Glu Gly Gln His Lys Val Trp Gly Asn
55 60 65 70
GAG CAC GGC AGC ATT CAC GTC AGC GGT GGT GCC AAC AAG CAG CCC GGT GGT 435
Glu His Gly Ser Ile His Val Ser Gly Gly Ala Asn Lys Gln Pro Gly Gly
75 80 85
AAG CCC CAA GGA CAG GTC GGA ATC GGC GGA TCC TTC CAC TGG GGA AAA TAA 486
Lys Pro Gln Gly Gln Val Gly Ile Gly Gly Ser Phe His Trp Gly Lys stop
90 95 100
ATTAACACCA ATTTCAGTCA AAAAATTATT ACAAGTTCCT ACACACTAAC AACTGAACAT 546
GTTCCTCGTA AATTTTAGCT TTATTCATAA GAACTCAGTT GTAAGCATCG TTGCCAACAG 606
TACTTAATTT GAAAATATAT TTTTTATCAT AACGTGAAAA AAAAAAAAAA AAAAACTCGA 666
GGGGGGGCCC GG 678
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the high-efficient cloning triage techniques scheme synoptic diagram of the insect source antibiotic protein based on cDNA (complementary DNA) expressivity library of the present invention/peptide Pp-AP2 gene;
The bacterium colony figure of SOLR bacterium when Fig. 2 is of the present invention the screening on the filter membrane;
Annotate: in a large amount of white colonies the less blue colonies of shape being arranged, promptly is the goal gene that comprises the potential anti-microbial activity of tool;
Fig. 3 is of the present invention with the inhibition design sketch of paper disk method test synthetic chrysalis tiny golden wasp antibiotic protein Pp-AP 2 to the SOLR bacterium; " 1 " represents 0.1% acetate among the figure, and " 2 " represent 1m mole sample.
Embodiment
Below in conjunction with the concrete testing data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1:(is with reference to Fig. 1 :)
1, the structure in chrysalis tiny golden wasp cDNA (complementary DNA) expressivity library:
The chrysalis tiny golden wasp derives from insect science institute of Zhejiang University insect Physiology and biochemistry laboratory.
A, from the total RNA of the overall separation of chrysalis tiny golden wasp, obtain poly (A)+RNA with the purified mRNA test kit, adopt again cDNA synthetic agent box,
The synthetic agent box and
Gigapack III clone test kit (buying no Chinese) by U.S. clontech company's T akana Agcy make up cDNA (complementary DNA) expressivity library.
B, basis, reference reagent box operation instructions are carried out each step operation, promptly make up cDNA (complementary DNA) expressivity library.
2, chrysalis tiny golden wasp cDNA (complementary DNA) expressivity library screening:
SOLR is (colibacillary a kind of for a, preparation; no Chinese name) bacterium liquid: it is extremely red to connect collarium calcination on spirit lamp; dip in the intestinal bacteria SOLR glycerol stock (mixture of bacterium liquid and glycerine that takes a morsel after the cooling; the volume ratio of glycerine is about 20%; helping bacterium liquid preserves at low temperatures and does not influence its normal activity) on the agarose substratum that contains kantlex, rule, cultivated 12 hours for 37 degrees centigrade.Picking list bacterium colony is in 50 milliliters agarose rich medium (LB nutrient agar medium), and 37 degrees centigrade, 200 rev/mins (rpm) cultivated about 12 hours.Be transferred in 50 milliliters of centrifuge tubes, 1000 rev/mins, 4 degrees centigrade centrifugal 10 minutes.Remove supernatant liquor, with the Adlerika suspension precipitation of 10 mmoles, reconciling the OD600 value is 1.0 again.
B, preparation agarose/penbritin filter membrane flat board: the agarose plate for preparing the penbritin that contains 100 mcg/ml is some.LB/Amp
+The preparation method of filter membrane flat board is as follows:
According to following formulated LB substratum: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water; Behind the uniform mixing, about 120 ℃ of High Temperature High Pressure (0.4Mpa) sterilizations postcooling to 50 ℃, add 1 milliliter in the penbritin mother liquor of 100 mg/ml; Shake up the back and fall in culture dish, waiting after its cooled and solidified and putting aperture that high-temperature sterilization crosses in media surface is 0.45 micron millipore filtration, promptly gets LB/Amp
+The filter membrane flat board; Promptly get sterilized filter membrane (aperture 0.45 μ m) and be laid on LB/Amp
+On the flat board, note not having bubble between film and the flat board.
C, bite that Jun body library (being the constructed cDNA expressivity library of step 1) is transformed in the SOLR bacterium liquid for preparing and measure and tire: will formerly bite Jun body library and add distilled water and dilute 50 times former, get bacterium liquid 1 microlitre that diluted respectively in 200 microlitre SOLR bacterium liquid of above-mentioned steps a gained, 37 degrees centigrade of incubations 15 minutes, get 100 microlitres coated plate on agarose/penbritin filter membrane flat board, the triangle rod that is used in calcination on the spirit lamp and cooled off is coated with out, and faces up to place after 20 minutes 37 degrees centigrade and be inverted and cultivated 12 hours.
Check the bacterial growth situation, thus the decision phasmid library, conversion reaction system Central Plains extension rate since the dilution 50 times and 100 times after changing effect bad, make growth concentration lower (thousands of clones should grow on the every substratum) thus do not dilute.
The chrysalis tiny golden wasp phasmid (be we oneself the chrysalis tiny golden wasp that makes up cDNA expressivity library) of getting 100 microlitres is in 2 milliliters of SOLR bacterium liquid of above-mentioned steps a gained, 37 degrees centigrade of incubations 15 minutes, get 100 microlitres coated plate on agarose/penbritin filter membrane flat board, be used in calcination on the spirit lamp and the good triangle rod of cooling is coated with out, face up and place after 20 minutes 37 degrees centigrade and be inverted and cultivated 12 hours, check in conjunction with effect.Place 50 milliliters of agaroses to enrich nutrient solution (LB nutritive medium) with 2 milliliters of the products that combine of SOLR the chrysalis tiny golden wasp phasmid of gained of last step, 37 degrees centigrade, 200 rev/mins of shaking culture 4～5 hours, to thick, get 100 microlitres coated plate on agarose/penbritin filter membrane flat board again, be inverted for 37 degrees centigrade and cultivated 12 hours, mensuration is tired.50 milliliters of bacterium liquid of gained of last step are added glycerine in 20% ratio, be distributed into 1 milliliter/pipe after shaking up, standby in subzero 70 degrees centigrade of storages.
Behind the above-mentioned bacterium liquid of dilution proportion by 100 times, 1000 times and 10000 times, respectively get 100 microlitres coated plate on agarose/penbritin filter membrane flat board, face up and place after 20 minutes 37 degrees centigrade and be inverted and cultivated 12 hours.The colony growth situation is to find out best multiple on the observation film.
Tire and optimum diluting multiple by mensuration.It is long-pending to find out the optimum response bacteria liquid, and promptly 100 microlitres/plate has several thousand bacterium colonies on every LB filter membrane flat board after cultivating.
D, bed board, dyeing:
Get SOLR bacterium liquid 100 microlitres that transformed coated plate on agarose/penbritin filter membrane flat board of subzero 70 degrees centigrade of storages, face up and place after 20 minutes, being inverted to cultivate in 37 degrees centigrade had that less (being generally 0.1～0.5mm) bacterium colony forms in about 15 hours to the film.Transfer to agarose/penbritin/sec.-propyl-b-d-thiogalactoside substratum, be inverted for 37 degrees centigrade and cultivated 3～4 hours, (be generally 0.9～1.1mm) to colony growth to normal size, go to the dyeing nutrient agar, dyeed about 10 minutes, referring to Fig. 2, examine back sterilization toothpick picking blue colonies, contain in the agarose/penbritin nutrient solution of 20% glycerine in 100 microlitres, and the bacterium colony size of record picking and dyeing depth situation, be divided into two pipes behind the vortex vibration mixing, on two pipes, put on identical number, as PP1a, subzero 70 degrees centigrade of storages.
The making method of above-mentioned dyeing nutrient agar is as follows: take by weighing the blue and 0.5g tetrabromophenol sulfonphthalein of 0.5g tongue phenol, be dissolved in respectively in the 9.5 gram water, thereby be made into 5% tongue phenol indigo plant mother liquor and tetrabromophenol sulfonphthalein mother liquor respectively; Other takes by weighing 4 gram agar and is dissolved in the 1L water, and heating makes its thorough dissolving, adds the blue mother liquor of 1 milliliter of tongue phenol and 1 milliliter of tetrabromophenol sulfonphthalein mother liquor again, places behind the mixing half an hour its cooled and solidified to be got final product.Be that gained dyeing nutrient agar is: tongue phenol indigo plant 0.0005%, tetrabromophenol sulfonphthalein 0.0005%, agar 0.4%.
Above-mentioned LB/Amp
+/ IPTG
+The preparation method of substratum is as follows:
According to following formulated LB substratum: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water; Behind the uniform mixing, about 120 ℃ of autoclave sterilization postcooling to 50 ℃, 1 milliliter in the penbritin mother liquor that adds 100 mg/ml, 0.5 1 milliliter in the isopropylthiogalactoside mother liquor of mol, shaking up the back falls in culture dish, after waiting its cooled and solidified, promptly get the LB/Amp+/IPTG+ substratum.
E, the checking of coated plate method:
Take out the PP1a bacterium liquid sample that above-mentioned d step is preserved, place on ice.Draw 5 microlitres and be applied to LB/Amp
+/ IPTG
-And LB/Amp
+/ IPTG
+The filter membrane flat board faces up and places after 10 minutes 37 degrees centigrade and be inverted and cultivated 12 hours.
LB/Amp
+/ IPTG
-The preparation method of filter membrane flat board is as follows:
According to following formulated LB substratum: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water; Behind the uniform mixing, about 120 ℃ of high temperature 0.4Mpa sterilization postcooling to 50 ℃, add 1 milliliter in the penbritin mother liquor of 100 mg/ml; Shake up the back and fall in culture dish, waiting after its cooled and solidified and putting aperture that high-temperature sterilization crosses in media surface is 0.45 micron millipore filtration, promptly gets LB/Amp
+/ IPTG
-The filter membrane flat board.
LB/Amp
+/ IPTG
+The preparation method of filter membrane flat board is as follows:
According to following formulated LB substratum: 1 liter of agar 20 gram, sodium-chlor 10 grams, peptone 10 grams, yeast extract 5 grams, distilled water; Behind the uniform mixing, about 120 ℃ of autoclave sterilization postcooling to 50 ℃, 1 milliliter in the penbritin mother liquor that adds 100 mg/ml, 0.5 1 milliliter in the isopropylthiogalactoside mother liquor of mol, shaking up the back falls in culture dish, waiting after its cooled and solidified and putting aperture that high-temperature sterilization crosses in media surface is 0.45 micron millipore filtration, promptly gets LB/Amp
+/ IPTG
+The filter membrane flat board.
Film taken off place dyeing to dye on the nutrient agar, the size of same sample colony growth on the comparative film, density, degree of staining and at LB/Amp
+/ IPTG
+Whether the bacterium colony on the filter membrane flat board presents the situation of unified color, and details is carried out record.The bacterium colony of newly choosing (PP1b) is stored in 100 microlitres to be contained in the agarose/penbritin nutrient solution of 20% glycerine, be divided into two pipes behind the vortex vibration mixing, on two pipes, put on the number identical with raw sample, as PP1b, b represents it is the bacterium liquid sample of picking again after the coated plate method checking, subzero 70 degrees centigrade of storages.According to painted different situations, make different processing:
If 1. the bacterium colony on two films is not all dyed blueness, illustrate that then this sample does not contain the gene of germicidal action, discardable this sample.
If 2. bacterium colony is at LB/Amp
+/ IPTG
+Partly dyed blueness on the filter membrane, and at LB/Amp
+/ IPTG
-Do not dye, illustrate that the sample of picking has the gene of germicidal action, but this sample mix other do not contain the clone of germicidal action gene, picking LB/Amp then
+/ IPTG
+Being dyed blue single bacterium colony on the filter membrane contains in the agarose/penbritin nutrient solution of 20% glycerine in 100 microlitres, be divided into two pipes behind the vortex vibration mixing, on two pipes, put on the number identical with raw sample, as PP1b, b represents it is the bacterium liquid sample of picking again after the method for scoring checking, subzero 70 degrees centigrade of storages.
If 3. bacterium colony is at LB/Amp
+/ IPTG
+All dyed blueness on the filter membrane, and at LB/Amp
+/ IPTG
-On do not dye, illustrate that the sample of picking has the gene of germicidal action, and this sample only contains this kind clone, then picking is at LB/Amp
+/ IPTG
-On colourless single bacterium colony in the 100 microlitres agarose/penbritin nutrient solution that contains 20% glycerine, be divided into two pipes behind the vortex vibration mixing, on two pipes, put on the number identical with raw sample, as PP2b, subzero 70 degrees centigrade of storages.
If 4. bacterium colony is at LB/Amp
+/ IPTG
+Partly dyed blueness on the filter membrane, and at LB/Amp
+/ IPTG
-Also have part to be dyed blueness on the film, illustrate that the sample of picking contains the gene of germicidal action, thereby and this gene produce sterilization effect not having also to express under sec.-propyl-b-d-thiogalactoside inductive situation; But this sample mix other do not contain the clone of germicidal action gene, treatment process is with 2..
If 5. bacterium colony is at LB/Amp
+/ IPTG
+And LB/Amp
+/ IPTG
-All dyed blueness on two filter membranes, illustrate that the sample of picking contains the gene of germicidal action, and thereby this gene is not having to express the generation sterilization effect under sec.-propyl-b-d-thiogalactoside inductive situation yet, and this sample only contains this kind clone, therefore only need to preserve original sample of preserving, the new bacterium colony of picking again.
2. and 4. also need carry out coated plate once more for sample that does not also have purifying such as situation, until at LB/Amp
+/ IPTG
+Present till the unified blueness on the filter membrane flat board.Blueness is that dead cell is coloured to blueness by tongue phenol orchid because the albumen/Toplink of insect source genetic expression makes the death of SOLR bacterium in this bacterium colony, otherwise is white.
3, the preliminary identification of anti-microbial activity
The liquid bacteriostatic experiment
A, the bacterium liquid sample of depositing (the bacterium liquid of preserving after 5. above-mentioned steps is verified) of going bail for are applied to LB/Amp
+/ IPTG
-And LB/Amp
+/ IPTG
+The filter membrane flat board goes to the dyeing of dyeing nutrient agar, if LB/Amp after the overnight incubation
+/ IPTG
+Clone on the plate all dyes, then picking LB/Amp
+/ IPTG
-White clone is 5～10 on the plate; If LB/Amp
+/ IPTG
+Clone on the plate is not whole painted words, illustrate the bacterium sample after preservation through multi-pass operations, may be impure, picking LB/Amp then
+/ IPTG
+5～10 of dyeing clones on the plate.
B, the clone of picking is inserted 5 persons of outstanding talent rises the LB/Amp+ nutrient solutions, about 37 ℃, 200rpm concussion cultivation 10h, the OD600 of bacterium is reached about 1.0.
C, to get above-mentioned bacterium liquid (be OD
600Reaching the bacterium liquid about 1.0) 30 μ l are in 3ml agarose/penbritin nutrient solution (the fresh adding of penbritin), and (each sample connects 4 pipes, 2 pipe LB/Amp
+/ IPTG
+, 2 pipe LB/Amp
+/ IPTG
-).Cultivate respectively and survey OD600 (often observing) after 2,3,4 hours when the OD600 value of the bacterium liquid of agarose/penbritin is surveyed its value during roughly in 0.5 left and right sides.(make discovery from observation, the bacterium liquid of agarose/penbritin/sec.-propyl-b-d-thiogalactoside and the bacterium liquid phase of agarose/penbritin relatively are clarification, perhaps have precipitation to occur, and the bacterium liquid of agarose/penbritin then presents cotton-shaped mycelia; By spectrophotometric mensuration, the OD600 value of the bacterium liquid of agarose/penbritin/sec.-propyl-b-d-thiogalactoside is significantly less than the bacterium liquid of agarose/penbritin, explanation antibacterial peptide gene under the inducing of sec.-propyl-b-d-thiogalactoside is expressed, thereby bacterial growth is produced restraining effect.)
4, order-checking:
The bacterium liquid sample of the plasmid that contains antibacterial protein/peptide of screening gained and process checking is delivered to order-checking company and is checked order; Detailed sequence is seen SEQ ID NO:1 and SEQID NO:2.The length of the chrysalis tiny golden wasp antibiotic protein Pp-AP 2 full-length cDNA that the present invention is new is 678bp, and wherein open reading frame is positioned at 175-486 position Nucleotide.Derive the aminoacid sequence of chrysalis tiny golden wasp antibacterial protein according to full-length cDNA, totally 103 amino-acid residues, molecular weight 10949.21kD, PI value 10.25.
Full length cDNA sequence and coded protein Genebank (http://www.ncbi.nlm.nih.gov/BLAST) and AMP database (http://aps.unmc.edu/AP/main.php with chrysalis tiny golden wasp antibiotic protein Pp-AP 2; Http:// reseach.izr.astar.edu.sg/Templar/DB/ANTIMIC) carries out the retrieval of Nucleotide and protein resemblance and homologue.Judge that by sequence alignment the gene that is screened is new gene, on gene and albumen water product, do not have tangible homology with all kinds of antibacterial protein/polypeptide of having found at present.
5, the synthetic of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 (finishing) by the safe bio tech ltd of last hypo
A, reagent
Fluorenylmethyloxycarbonyl (Fmoc)-amino acid is U.S. SIAM company product; PyBOP, Wang resin are U.S. SIAM company product; Hexahydropyridine, lutidine are Merck company product; Dimethyl formamide (DMF) be Japanese import (before using earlier through triketohydrindene hydrate soak and
Molecular sieve dehydration, and measure no free amine group); Methylene dichloride (DCM) is China Medicine's product (using the Anhydrous potassium carbonate immersion treatment before using); Trifluoroacetic acid (TFA) is a GEELBELGIUM company product; Methyl alcohol is Shanghai development chemical industry one factory's product; HPLC methyl alcohol is Merck company product; Tetrahydrofuran (THF) is Shanghai chemical reagent station centralization factory products.
B, instrument
431A type Peptide synthesizer is an Applied biosystems product, and high performance liquid chromatography is Agilent 1100 chromatographic instruments; Freeze drier (FREEZE DRYER 18) is the LABCONCO product; Mass spectrograph is Finnigan LCQ.
C, method
Composition sequence: Y V P P V Q K P H P N G P K F P T F P
Synthesizing of peptide chain
The synthetic employing Fmoc/PyBOP method of peptide chain.Fmoc raises one's hat with the DMF solution of 30% hexahydropyridine.Peptide chain from the resin cut with cut peptide reagent (trifluoroacetic acid/crystallization phenol/water/dithioglycol/first and second thioethers /=81.5/5/5/5/2.5/1).Take by weighing 100mg Fmoc-Ile-wang resin, successively by various Fmoc-amino acid
Per step connects reactive polypeptide and adds
PyBOP--------Mwt：520.30----------------1123.9[mg/coupling]
HOBt+H2O--Mwt：153.10----------------4320[micro-L/coupling]
NMM----------10g.95[mL/mol]-------------3240[micro-L/coupling]
The reaction of raising one's hat adds:
Hexahydropyridine-----------30%------------------------9000 * 2[micro-L/coupling]
Use during washing
DMF----------------------------------------------9000×5[micro-L/coupling]
Connect peptide and carry out on the 431A automatic DNA synthesizer DNA, the resin that has connect all is transferred in the eggplant-shape bottle, add prepare in advance and precooling cut peptide reagent 5ml.25 ℃ of following stirring reactions 2 hours.Filter, collect filtrate.Resin is washed 3 times with a small amount of trifluoroacetic acid.Washings and filtrate are merged, concentrate postcooling, add the 10ml cold diethyl ether then and make the polypeptide precipitation.Centrifugal collection, vacuum-drying.Get the about 60mg of crude product.
Purifying:
Earlier determine target peptide with analytical column:
Method is: use the anti-phase pillar of C18, condition is: A is 95% water (methyl alcohol proportioning) mutually, and B is 95% methyl alcohol (methyl alcohol proportioning) mutually, respectively adds 0.1% TFA then, normal condition: be 20 minutes gradients mutually from A to B.Detect wavelength: 220 nanometers, flow velocity: 1 ml/min, earlier with A solution equilibria pillar, behind the last sample, from A to B solution gradient wash-out 20 minutes, collect target peptide, do the mass spectrum evaluation then.
Preparation: (use instrument: Waters600E)
Use the anti-phase preparation pillar of C18, condition is: A is 95% water (methyl alcohol proportioning) mutually, and B is 95% methyl alcohol (methyl alcohol proportioning) mutually, respectively adds 0.1% TFA then, normal condition: be 30 minutes gradients mutually from A to B.Detect wavelength: 220 nanometers, flow velocity: 10 ml/min, earlier with A solution equilibria pillar, behind the last sample, from A to B solution gradient wash-out 30 minutes, collect the target peptide elutriant, freeze-drying.
6, the mensuration of synthetic peptide anti-microbial activity
A, the synthetic peptide solution of preparation
The synthetic peptide of 30mg is dissolved in the mother liquor that is made into 100mM in the acetic acid solution of 140ul 0.1%, be distributed into 10 pipes be stored in-20 ℃ standby.
B, preparation test organisms liquid:, should use the SOLR bacterium of band plasmid in order to get rid of the possibility that bacteriostatic action is caused by AMP; Bacterium liquid cultivated be positioned over 4 ℃ of preservations when the OD value is 0.5 left and right sides.
C, preparation filter paper: get No. 1 qualitative filter paper of Xinhua, break into the little scraps of paper of 6mm with tapping and plugging machine, sterilizing, drying is standby.
D, according to agar 1%w/v, peptone 1%w/v, yeast extract 0.5%w/v, sodium-chlor 0.05%w/v configuration agarose substratum, pH is transferred to 7.0 back sterilizations, after its cooling such as a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, take the even coated plate of above-mentioned test organisms liquid 200 microlitres of 100 times of LB liquid nutrient medium dilutions, wait the absorption of bacterium liquid after 10 minutes the three metafiltration scraps of paper to be positioned on the substratum.Put the three-ply paper sheet and can increase the application of sample amount.To synthesize peptide solution 5 microlitres then and be added on the filter paper, and do positive control with kantlex, facing up absorbs back inversion overnight incubation fully.Observe inhibition zone (referring to Fig. 3), survey its size.Observe the inhibition zone result of experiment, finding does not have bacterial growth around the filter paper, therefore presents bright inhibition zone.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ ID NO：1
gggaacaaaa agctggagct ccaccgcggt ggcggccgct ctagaactag tggatccccc 60
gggctgcagg aattcggcac gaggcaacca tcaacttgaa tagtttccaa aactaaaaac 120
tcaaaagcca caatgaagtt cgtcctcagt ttcgtgttgc tcgctgtagc ccttatggta 180
gccgtagatg cttcaccata cgtaccacca gttcaaaagc ctcacccgaa cggacccaaa 240
ttcccgactt tccccggtca aggaacttgg agtggcagac ctcgtcgctc acctcagaag 300
aacggtcaaa tcgaaatcca cggcaagaag gaaggtggaa agacttcctg gagcgttgag 360
ggccagcaca aagtctgggg taacgagcac ggcagcattc acgtcagcgg tggtgccaac 420
aagcagcccg gtggtaagcc ccaaggacag gtcggaatcg gcggatcctt ccactgggga 480
aaataaatta acaccaattt cagtcaaaa aattattacaa gttcctacac actaacaact 540
gaacatgttc ctcgtaaatt ttagctttat tcataagaac tcagttgtaa gcatcgttgc 600
caacagtact taatttgaaa atatattttt tatcataacg tgaaaaaaaa aaaaaaaaaa 660
actcgagggg gggcccgg 678
SEQ ID NO：2
Met Val Ala Val Asp Ala Ser Pro Tyr Val Pro Pro Val Gln Lys
1 5 10 15
Pro His Prp Asn Gly Pro Lys Phe Pro Thr Phe Pro Gly Gln Gly
20 25 30
Thr Trp Ser Gly Arg Pro Arg Arg Ser Pro Gln Lys Asn Gly Gln
35 40 45
Ile Glu Ile His Gly Lys Lys Glu Gly Gly Lys Thr Ser Trp Ser
50 55 60
Val Glu Gly Gln His Lys Val Trp Gly Asn Glu His Gly Ser Ile
65 70 75
His Val Ser Gly Gly Ala Asn Lys Gln Pro Gly Gly Lys Pro Gln
80 85 90
Gly Gln Val Gly Ile Gly Gly Ser Phe His Trp Gly Lys
95 100
Claims (5)
1, a kind of nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 coding, it is characterized in that isolated dna molecular comprises: coding has the active polypeptide nucleotide sequence of chrysalis tiny golden wasp antibacterial protein, shows at least 70% homology from the nucleotides sequence of Nucleotide 175-486 position among described nucleotide sequence and the SEQ IDNO:1; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO:1 in from the nucleotide sequence hybridization of Nucleotide 175-486 position.
2, the nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 coding according to claim 1, it is characterized in that: this sequence has the nucleotide sequence of 175-486 position among the SEQ ID NO:1.
3, the nucleotide sequence of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 coding according to claim 1 is characterized in that: comprise 8～312 continuous nucleotides in this sequence.
4, a kind of chrysalis tiny golden wasp antibiotic protein Pp-AP 2 polypeptide is characterized in that: have the aminoacid sequence shown in the SEQ ID NO:2.
5, chrysalis tiny golden wasp antibiotic protein Pp-AP 2 polypeptide according to claim 4 is characterized in that: described chrysalis tiny golden wasp antibacterial protein polypeptide is polypeptide, its conservative property variation polypeptide, its active fragments or its reactive derivative.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260348A (en) * | 2011-07-19 | 2011-11-30 | 浙江大学 | Pteromalus puparum venom serine protease inhibitor Pp-PI polypeptide and application thereof |
| CN102731646A (en) * | 2012-06-20 | 2012-10-17 | 浙江大学 | Pteromalus puparum venom Kazal serine proteinase inhibitor polypeptide and its application |
-
2008
- 2008-07-24 CN CNA2008101201464A patent/CN101328482A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260348A (en) * | 2011-07-19 | 2011-11-30 | 浙江大学 | Pteromalus puparum venom serine protease inhibitor Pp-PI polypeptide and application thereof |
| CN102731646A (en) * | 2012-06-20 | 2012-10-17 | 浙江大学 | Pteromalus puparum venom Kazal serine proteinase inhibitor polypeptide and its application |
| CN102731646B (en) * | 2012-06-20 | 2014-03-05 | 浙江大学 | Pteromalus puparum venom Kazal serine proteinase inhibitor polypeptide and its application |
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