CN100344758C - Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application - Google Patents
Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application Download PDFInfo
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- CN100344758C CN100344758C CNB2005100445858A CN200510044585A CN100344758C CN 100344758 C CN100344758 C CN 100344758C CN B2005100445858 A CNB2005100445858 A CN B2005100445858A CN 200510044585 A CN200510044585 A CN 200510044585A CN 100344758 C CN100344758 C CN 100344758C
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Abstract
The present invention discloses a single whey acidic protein structural domain containing antibacterial peptide gene of Chinese prawns and a coded antibacterial peptide thereof. The present invention also discloses the application of the single whey acidic protein structural domain containing antibacterial peptide gene of Chinese prawns to the preparation of recombination protein with antibacterial activity. Recombined single whey acidic protein structural domain containing antibacterial peptide obtained by using the present invention can be used for antibacterial feed additives, food preservation, genetic transformation of animals and plants, and medicine development.
Description
Technical field
The present invention relates to a kind of the contain antibacterial peptide gene of single whey acidic protein structure domain and coded antimicrobial polypeptide and application thereof, relate in particular to a kind of antibacterial peptide gene that contains single whey acidic protein structure domain of Chinese prawn and coded antimicrobial polypeptide and application thereof; Belong to gene engineering technology field.
Background technology
Chinese prawn is the important mariculture kind of China, but since the nineties, prawn disease has been broken out on a large scale, and mariculture is already sustained losses severely.Domestic many scholars are doing a large amount of work aspect prevention and cure of shrimp disease, and have obtained a lot of progress, and the cultured output of prawn also picks up year after year; But up to the present, prawn disease is still a difficult problem of puzzlement shrimp culture industry.Address this problem mainly and should start with from aspects such as control cause of disease and raising prawn immunizing power, the defense mechanism of prawn mainly relies on the activity of hemocyte, comprise and engulf foreign matter or form inclusion body that hemocyte synthetic antimicrobial material (antibacterial peptide or polypeptide) also is discharged into hemolymph; The phenol oxidase system causes local melanism and aggegation in addition, also plays a part certain.
Deatoumieux etc. study all antibacterial peptides of receiving prawn, therefrom separate and obtained Ch-penaedin and (be called the prawn element, Penaeidins) and be cloned into its gene, also it has been carried out research (the Destoumieux D of character, genetic expression and location and aspect such as recombinant expressed simultaneously, Bulet P, Loew D et al.Penaeidin, a new familyof antimicrobial peptides isolated from the shrimp Penaeus vannamei.J Biol Chem, 1997,272:28398-28406).We also from Chinese prawn the clone obtained the prawn plain gene, and it has been carried out character, genetic expression and location and aspect such as recombinant expressed is studied, having acquired has the reorganization of broad spectrum antibiotic activity prawn element.
Many studies show that contained multiple albumen or the peptide class that can resist extraneous pathogenic micro-organism in the prawn.Comprise prawn element (penaeidins), chitin element (crustins), coagulogen (ALF) and the negatively charged ion antibacterial peptide of holding from prawn hemocyanin C-.But, in many relevant reports, do not see the report that has Chinese prawn to contain the antibacterial peptide gene of single whey acidic protein structure domain, the present invention clones from Chinese prawn and obtains a kind of antibacterial peptide gene that contains single whey acidic protein structure domain (peptide containing single whey acidic protein (WAP) domain), though also found similar gene all receiving in the prawn abroad, do not had described Chinese prawn to contain the report of the recombinant expressed and functional study of the antibacterial peptide gene of single whey acidic protein structure domain so far.
Summary of the invention
At the deficiency of present present Research, the purpose of this invention is to provide antibacterial peptide gene and nucleotide sequence thereof that a kind of Chinese prawn contains single whey acidic protein structure domain, and its cloning process and carrier are provided.
The antibacterial peptide gene that Chinese prawn of the present invention contains single whey acidic protein structure domain has the nucleotide sequence shown in the SEQ ID NO.1; Information shown in it is:
(a) sequence signature:
* length: 390 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: prawn (Fenneropenaeius chinensis)
(f) sequence description: SEQ ID NO.1
atggtgaaca?tcaaggaagt?tctgatcgtg?tccgtgttgg?tggccgcggt?ggctgtttct 60
cccgccgatg?ctgttccaac?gagacacgct?aggccccgtc?ctcagcccag?gccgaggcca 120
gggacgtgtc?cggacacgag?cgacatcgtc?tccatctgcg?tcgtgacgga?acgcaactgc 180
ttctcggacg?gcgagtgcgg?agccggccag?aagtgctgtc?cgattggctg?cgggagagag 240
tgcctggctg?tgggatctcc?ctacggaaaa?tgaagatcgt?aggaggggga?cgaaatttcc 300
tcgatgggct?cacagtctcc?ctggaaatat?tattgaagcg?tgttgattca?ttgataataa 360
aattgtgttg?tttcaaaaaa?aaaaaaaaaa 390
Another object of the present invention provides and a kind ofly contains the antibacterial peptide of the antibacterial peptide gene coding of single whey acidic protein structure domain by Chinese prawn, and it has the aminoacid sequence shown in the SEQ ID NO.2; Information shown in it is:
(a) sequence signature
* length: 90 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ ID NO.2
Met?Val?Asn?Ile?Lys?Glu?Val?Leu?Ile?Val?Ser?Val?Leu?Val?Ala
5 10 15
Ala?Val?Ala?Val?Ser?Pro?Ala?Asp?Ala?Val?Pro?Thr?Arg?His?Ala
20 25 30
Arg?Pro?Arg?Pro?Gln?Pro?Arg?Pro?Arg?Pro?Gly?Thr?Cys?Pro?Asp
35 40 45
Thr?Ser?Asp?Ile?Val?Ser?Ile?Cys?Val?Val?Thr?Glu?Arg?Asn?Cys
50 55 60
Phe?Ser?Asp?Gly?Glu?Cys?Gly?Ala?Gly?Gln?Lys?Cys?Cys?Pro?Ile
65 70 75
Gly?Cys?Gly?Arg?Glu?Cys?Leu?Ala?Val?Gly?Ser?Pro?Tyr?Gly?Lys
80 85 90
Wherein, also comprise the varient of the aminoacid sequence shown in the SEQ ID NO.2, its coding has the homologous variation albumen that is less than 8 amino acid changes, and amino acid change is that conservative amino acid changes.
The cloning process of the antibacterial peptide cDNA that contains single whey acidic protein structure domain of Chinese prawn of the present invention is:
Adopt single stage method or RNA kit from prawn, to extract total RNA, perhaps use mRNA kit separation and Extraction mRNA; Utilize total RNA or mRNA reverse transcription to synthesize cDNA then;
Wherein: the primer according to the conserved sequence design of the antibacterial peptide gene that contains single whey acidic protein structure domain of prawn is:
Forward primer: F1 5 ' GTG TTG GTG GCC GCG GTG GC 3 '
Reverse primer: R1 5 ' CAC AGC CAG GCA CTC TCT CC 3 '
The PCR reaction conditions is: at first 94 ℃ of sex change are 2 minutes, enter following circulation then: 94 ℃ 30 seconds, 53 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 30 circulations altogether, last 72 ℃ were extended 10 minutes.
Purifying obtains dna fragmentation by chain polymerization enzyme reaction (PCR) amplification;
Get above-mentioned purified product and be cloned into pGEM-T Easy carrier (Promega company product) or pMD 18-T carrier (TaKaRa company product), transform DH5 α cell (common carrier host cell), the dull and stereotyped cultivation;
Extract and plasmid purification amplification plasmid and order-checking;
Through 3 ' and 5 ' terminal rapid amplifying,, promptly get the antibacterial peptide gene full length nucleotide sequence that Chinese prawn shown in the SEQ ID NO.1 contains single whey acidic protein structure domain again with the splicing of 3 ' and 5 ' terminal sequence.
The antibacterial peptide gene that contains single whey acidic protein structure domain of Chinese prawn of the present invention has application in the recombinant protein of anti-microbial activity in preparation.
Wherein, the method for above-mentioned application is by gene recombination technology described gene to be expressed in intestinal bacteria, yeast and insect nuclear polyhedrosis virus, obtains to have the recombinant protein (Fig. 1) of anti-microbial activity.
For example: the antibacterial peptide gene that contains single whey acidic protein structure domain of the Chinese prawn that obtains is cloned into pET-30a (Novagen company) expression vector, and transformed into escherichia coli BL21 DE3 cell carries out abduction delivering, purifying; And utilize the acquisition recombinant protein to carry out bacteriostatic experiment.
Experimental result shows that the antibacterial peptide that contains single whey acidic protein structure domain of reorganization has the activity (Fig. 2) of anti-leather Lan Shi negative bacterium and positive bacteria.
Utilize the antibacterial peptide gene that contain single whey acidic protein structure domain of method of the present invention, also can be used for other researchs and production by existing gene engineering method modification Chinese prawn.
Utilizing the antibacterial peptide that contains single whey acidic protein structure domain of the Chinese prawn of the reorganization that the present invention obtains to can be used for antibacterial feed additive, Food preservation, animal-plant gene transforms and drug development.
Description of drawings
Fig. 1 Chinese prawn contains the recombinant expressed and purifying of the antibacterial peptide of single whey acidic protein structure domain
Wherein: the abduction delivering of A different strains: 1 induces preceding bacterial strain I, and 2 induce preceding bacterial strain II, and 3 induce back bacterial strain I, and 4 induce back bacterial strain II.
The purifying of B recombinant antibacterial peptide: 1 induces the supernatant liquor electrophoresis result after the cytoclasis of back, the precipitation after 2 cytoclasises, and the prawn of 3 purifying contains the antibacterial peptide of single whey acidic protein structure domain.
The bacteriostatic experiment of the antibacterial peptide that contains single whey acidic protein structure domain of the Chinese prawn of Fig. 2 reorganization
Wherein: A is that the antibacterial peptide of different concns is to colibacillary fungistatic effect;
B is the fungistatic effect of the antibacterial peptide of different concns to micrococcus luteus.
Embodiment
Embodiment 1: Chinese prawn contains the clone of the antibacterial peptide cDNA of single whey acidic protein structure domain
1) extraction of total RNA: adopt the prior art single stage method to extract total RNA.
2) cDNA first chain is synthetic: the total RNA of 6 micrograms, add reaction solution 20 microlitres, reaction solution is 50 mmole Repone K and 3 mmole magnesium chloride mixed solutions, 10 mmole tris-HCI buffer (Tris-HCl) pH8.3,1 mmole dithiothreitol (DTT) (DTT), 5 micromole's oligomerization dT acid (Oligod (T)
17), 500 micromole's deoxyribonucleotide mixtures (dNTP), 25 RNA of unit enzyme inhibitorss, 8 AMV of unit reversed transcriptive enzymes, 42 ℃ were reacted 70 ℃ of 10 minutes termination reactions 90 minutes.
3) PCR reaction: chain polymerization enzyme reaction (PCR) reagent and condition:
At first following reagent is mixed:
10xTaq dna polymerase buffer liquid 5 microlitres (μ l)
Forward primer (1.25 μ g/ μ l) 1 μ l
Reverse primer (1.25 μ g/ μ l) 1 μ l
Deoxynucleoside acid mixture (dNTP) 4 μ l
Taq archaeal dna polymerase 0.25 μ l
Aqua sterilisa 37.75 μ l
Cumulative volume 50 μ l
The primer of conserved sequence design that contains the antibacterial peptide of single whey acidic protein structure domain according to the prawn prawn is:
Forward primer: F1 5 ' GTG TTG GTG GCC GCG GTG GC 3 '
Reverse primer: R1 5 ' CAC AGC CAG GCA CTC TCT CC 3 '
The PCR reaction conditions is: at first 94 ℃ of sex change are 2 minutes, enter following circulation then: 94 ℃ 30 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, carry out 30 circulations altogether, last 72 ℃ were extended 10 minutes.
4) reaction product purifying: utilize the product QIAquick Gel Extraction Kit of German Quinn (QIAGEN) company, operation steps is undertaken by product description.
5) prawn contains the antibacterial peptide cDNA clone of single whey acidic protein structure domain: get purified product 3 microlitres, be connected in pGEM-T Easy carrier (Promega company product).Be transformed into e.colistraindh5, in the dull and stereotyped grow overnight that contains penbritin (100 mcg/ml), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal 0.2 mcg/ml) and isopropylthiogalactoside (0.1 mole/milliliter of IPTG), 3 hickies of picking, overnight incubation in LB liquid nutrient medium (5 milliliters contain 100 mcg/ml peace penicillin G).
6) plasmid purification: collect 2 milliliters of incubated overnight bacterium liquid, centrifugal (10000 rev/mins, 1 minute) collecting cell.With minim DNA purification kit (Wizard plus SV Minipreps DNA Purification System, U.S. Pu Luomaige Promega company) plasmid purification, the purification step by specification carries out.
7) sequencing and homology retrieval: get plasmid purification 4 microlitres, with carrier primer T7 automatically check order (originally being operated in Shanghai life worker company finishes).Institute's calling sequence and gene pool sequence are compared.
8) prawn contains antibacterial peptide cDNA 3 ' the end rapid amplifying of single whey acidic protein structure domain
According to the antibacterial peptide gene fragment that obtains, design a specificity forward primer F1 5 ' GTG TTG GTG GCCGCG GTG GC 3 ' again, carry out cDNA 3 ' end rapid amplifying, operation steps is undertaken by precious biological 3 ' RACE test kit specification sheets.
Get the about 20 μ g of the total RNA of hemocyte, the modulation of other reagent and reaction conditions by specification carry out.Carry out 3 ' end pcr amplification with Auele Specific Primer F3 and 3 ' joint, adopt 56 ℃ of annealing, all the other are identical with above-mentioned pcr amplification program, and products therefrom is cloned, verified and check order by preceding method.
9) prawn contains antibacterial peptide cDNA 5 ' the end clone of single whey acidic protein structure domain
Utilize total RNA of above-mentioned acquisition, press the SMART of CLONTECH company (U.S.)
TMIt is synthetic that PCR cDNA library construction test kit specification sheets carries out the first chain cDNA.
With above-mentioned cDNA is template, and 5 ' the PCR primer and reverse primer R1 5 ' the CAC AGC CAGGCA CTC TCT CC 3 ' that provide with test kit are that primer carries out pcr amplification, obtains the 5 ' sequence that prawn contains the antibacterial peptide cDNA of single whey acidic protein structure domain.
With the splicing of 3 ' and 5 ' terminal sequence, promptly obtain the antibacterial peptide cDNA full length gene nucleotide sequence that Chinese prawn shown in the SEQ ID NO.1 contains single whey acidic protein structure domain.
Embodiment 2: recombinant expression vector structure, expression and bacteria resistance function are measured
(1) contain the sequence of antibacterial peptide gene of single whey acidic protein structure domain and the cloning site of expression vector pPET30a (Novagen company) according to Chinese prawn, the design primer:
Wap?F：CGC?GAT?
GAA?TTC?ATG?GTG?AAC?ATC?AAG?GAA?G(EcoR?I)
Wap?R：TAG?ATC?
CTC?GAG?TTT?TCC?GTA?GGG?AGA?TCC?CAC(XhoI)
The present invention has selected the EcoR I and the Xho I restriction enzyme site of pET30a cloning site, therefore, has introduced EcoR I restriction enzyme site at upstream primer during the design primer, has introduced Xho I restriction enzyme site on the downstream primer.
(2) gene amplification, clone and recombinant plasmid screening
With pMD-18T-Wap is template, carries out the PCR reaction with above-mentioned primer, and amplification condition is: 94 ℃, and the pre-sex change of 2min; 94 ℃, 30s, 55 ℃, 45s, 72 ℃, 45s, 35 circulations; 72 ℃ are extended 10min.
2% agarose gel electrophoresis PCR product detects.
The PCR product is made the preparation electrophoresis, with UNIQ-5 Column DNA Gel Extraction Kit (chemical product is given birth in Shanghai) recovery, purified pcr product, through EcoR I and Xho I endonuclease digestion, the cutting-out two ends have the housefly phylaxin mature peptide cDNA fragment of Xho I and EcoR I restriction enzyme site, same expression vector pET30a exposes the Xho I and the EcoR I restriction enzyme site at multiple clone site two ends through EcoR I and XhoI endonuclease digestion.Then, the amplified production after enzyme cut is connected with the T4 dna ligase with expression vector, transforms DH5 α competent cell, the dull and stereotyped PCR screening positive clone of LB+Amp.The bacterium colony that picking PCR is sieved to, 37 ℃ of vibrate amplification cultivation and extracting plasmids after EcoR I and Xho I double digestion and the sequence verification, are recombinant expression plasmid pET30a-Wap.Switching through E.coli expression strain BL21 DE3 competent cell, coating LB+Amp flat board is inverted incubated overnight for 37 ℃.
(3) screening expression strain
8 mono-clonal bacterium colonies of picking from the above-mentioned LB+Amp flat board, 37 ℃ of shaken overnight of 2ml LB+Amp liquid nutrient medium are cultivated, and get 20 μ l incubated overnight liquid and join the transfer of 2ml LB+Amp liquid nutrient medium and cultivate next day, and 37 ℃ of shaking culture 3h are to OD
600Between 0.5～0.7, add then IPTG to final concentration be 1mmol, continue 37 ℃ of shaking culture abduction delivering 4h.Before inducing, from a sample, take out 0.5ml bacterium liquid at random, do not induce contrast during electrophoresis detection.
After having expressed, respectively get 0.5ml bacterium liquid, the centrifugal 5min collecting cell of 5000r/min comprises not inductive sample, is resuspended in the 100 μ l deionized waters, makes 15% SDS-PGAE as the electrophoresis sample.According to electrophoresis result, identify expression strain.
(5) the reorganization prawn contains the peptide expression and the purifying of single whey acidic protein structure domain
Picking expression strain mono-clonal 37 ℃ of overnight shakings in the LB+Amp liquid nutrient medium are cultivated, next day, join the transfer of 100ml LB+Amp substratum at 1: 100 according to volume ratio and cultivate, behind 37 ℃ of shaking culture 3h, adding IPTG again is 0.5mmol to final concentration, 37 ℃ of vibration inducing culture 4h again.Take out the bacterium liquid of 0.5ml before inducing, as inducing preceding control sample.After inducing culture is intact, the centrifugal 10min collecting cell of bacterium liquid 7000r/min in suitable centrifuge tube, cell is resuspended in 1 * PBS of 5ml precooling, adds the Triton X-100 of 50 μ l 20%, fully ice bath 30min behind the mixing.The ultrasonic disruption cell, ultrasonic circulating is: ultrasonic 1s; Interval 1s; Omnidistance 40s.Repeat 4 times, with bacterium liquid mixing in ice bath, avoid local temperature too high during each gap, make protein denaturation.At last, with the centrifugal 15min of bacterium liquid 10000r/min after the fragmentation, collect supernatant liquor and precipitation, supernatant liquor and precipitation keep sample respectively, standby.
Recombinant protein exists with the form of inclusion body, through inclusion body purifying, renaturation and affinity chromatography, has promptly acquired recombinant protein with ordinary method---and the reorganization prawn contains the antibacterial peptide of single whey acidic protein structure domain and (sees Figure 1A, B).
(6) the recombinant protein bacteriostatic activity is measured
Detect bacteriostatic activity with cup-plate method, method is as follows:
Solid plate preparation: lower floor's glue of solid plate is 1.5% agar of one deck 0.5～1cm thickness, upper strata glue is the low bacterial nutrition substratum (1%tryptone of the 8mL solid that contains 5 μ L logarithmic phase bacteriums of 0.5～1cm thickness, 0.5%NaCl, 1.5%Agar, pH7.5).Level leaves standstill, and cooling is standby.
A plurality of Oxford cuvettes (as Fig. 2) through sterilization are placed in design on above-mentioned solid plate, carry out mark in the bottom surface of flat board, get 50 μ L testing samples respectively, add in the cuvette of corresponding Oxford, overnight incubation is left standstill in 28 ℃ of fronts, observes fungistatic effect, measures antibacterial circle diameter.
Experimental result: the antibacterial peptide that contains single whey acidic protein structure domain of the Chinese prawn of reorganization of the present invention is to having tangible fungistatic effect (see figure 2) to intestinal bacteria or micrococcus luteus.
SEQ?ID?NO.1
＜110〉Shandong University
＜120〉Chinese prawn contains the antibacterial peptide and the application of the antibacterial peptide gene and the coding thereof of single whey acidic protein structure domain
<141>2005-9-6
<160>2
<210>1
<211>390
<212>cDNA
＜213〉prawn (Fenneropenaeius chinensis)
＜221〉Chinese prawn contains the antibacterial peptide gene of single whey acidic protein structure domain
<222>(1)…(390)
<400>1
atggtgaaca?tcaaggaagt?tctgatcgtg?tccgtgttgg?tggccgcggt?ggctgtttct 60
cccgccgatg?ctgttccaac?gagacacgct?aggccccgtc?ctcagcccag?gccgaggcca 120
gggacgtgtc?cggacacgag?cgacatcgtc?tccatctgcg?tcgtgacgga?acgcaactgc 180
ttctcggacg?gcgagtgcgg?agccggccag?aagtgctgtc?cgattggctg?cgggagagag 240
tgcctggctg?tgggatctcc?ctacggaaaa?tgaagatcgt?aggaggggga?cgaaatttcc 300
tcgatgggct?cacagtctcc?ctggaaatat?tattgaagcg?tgttgattca?ttgataataa 360
aattgtgttg?tttcaaaaaa?aaaaaaaaaa 390
SEQ?ID?NO.2
<210>2
<211>90
<212>PRT
＜221〉Chinese prawn contains the antibacterial peptide of the antibacterial peptide gene coding of single whey acidic protein structure domain
<222>(1)…(90)
<400>2
Met?Val?Asn?Ile?Lys?Glu?Val?Leu?Ile?Val?Ser?Val?Leu?Val?Ala
5 10 15
Ala?Val?Ala?Val?Ser?Pro?Ala?Asp?Ala?Val?Pro?Thr?Arg?His?Ala
20 25 30
Arg?Pro?Arg?Pro?Gln?Pro?Arg?Pro?Arg?Pro?Gly?Thr?Cys?Pro?Asp
35 40 45
Thr?Ser?Asp?Ile?Val?Ser?Ile?Cys?Val?Val?Thr?Glu?Arg?Asn?Cys
50 55 60
Phe?Ser?Asp?Gly?Glu?Cys?Gly?Ala?Gly?Gln?Lys?Cys?Cys?Pro?Ile
65 70 75
Gly?Cys?Gly?Arg?Glu?Cys?Leu?Ala?Val?Gly?Ser?Pro?Tyr?Gly?Lys
80 85 90
Claims (3)
1. the antibacterial peptide gene that contains single whey acidic protein structure domain of a Chinese prawn, it has the nucleotide sequence shown in the SEQ ID NO.1; Its sequence is:
atggtgaaca?tcaaggaagt?tctgatcgtg?tccgtgttgg?tggccgcggt?ggctgtttct 60
cccgccgatg?ctgttccaac?gagacacgct?aggccccgtc?ctcagcccag?gccgaggcca 120
gggacgtgtc?cggacacgag?cgacatcgtc?tccatctgcg?tcgtgacgga?acgcaactgc 180
ttctcggacg?gcgagtgcgg?agccggccag?aagtgctgtc?cgattggctg?cgggagagag 240
tgcctggctg?tgggatctcc?ctacggaaaa?tgaagatcgt?aggaggggga?cgaaatttcc 300
tcgatgggct?cacagtctcc?ctggaaatat?tattgaagcg?tgttgattca?ttgataataa 360
aattgtgttg?tttcaaaaaa?aaaaaaaaaa 390。
2. the antibacterial peptide of the antibacterial peptide gene coding that contains single whey acidic protein structure domain of the described Chinese prawn of claim 1, it has the aminoacid sequence shown in the SEQ ID N0.2; Its sequence is:
Met?Val?Asn?Ile?Lys?Glu?Val?Leu?Ile?Val?Ser?Val?Leu?Val?Ala
5 10 15
Ala?Val?Ala?Val?Ser?Pro?Ala?Asp?Ala?Val?Pro?Thr?Arg?His?Ala
20 25 30
Arg?Pro?Arg?Pro?Gln?Pro?Arg?Pro?Arg?Pro?Gly?Thr?Cys?Pro?Asp
35 40 45
Thr?Ser?Asp?Ile?Val?Ser?Ile?Cys?Val?Val?Thr?Glu?Arg?Asn?Cys
50 55 60
Phe?Ser?Asp?Gly?Glu?Cys?Gly?Ala?Gly?Gln?Lys?Cys?Cys?Pro?Ile
65 70 75
Gly?Cys?Gly?Arg?Glu?Cys?Leu?Ala?Val?Gly?Ser?Pro?Tyr?Gly?Lys
80 85 90。
3. the antibacterial peptide gene that contains single whey acidic protein structure domain of the described Chinese prawn of claim 1 has application in the recombinant protein of anti-microbial activity in preparation, its method is by gene recombination technology described gene to be expressed in intestinal bacteria, yeast or insect nuclear polyhedrosis virus, obtains to have the recombinant protein of anti-microbial activity.
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| CNB2005100445858A CN100344758C (en) | 2005-09-13 | 2005-09-13 | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101955523B (en) * | 2010-03-10 | 2014-06-25 | 中国科学院海洋研究所 | Preparation and application of Eriocheir sinensis whey acidic protein (WAP) with bacteriostatic activity |
| CN103865933B (en) * | 2013-11-30 | 2016-03-23 | 济南大学 | The application of a kind of WAP gene in Drosophila transgenic |
| CN103981188A (en) * | 2013-12-27 | 2014-08-13 | 内蒙古科技大学 | Pennaus vannamei SWD gene subtype Lv-SWDi, amino acid sequence coded by pennaus vannamei SWD gene subtype Lv-SWDi, and cloning method of pennaus vannamei SWD gene subtype Lv-SWDi |
| CN105385705A (en) * | 2015-10-16 | 2016-03-09 | 济南大学 | Method for preparing soluble penaeus chinensis SWD antibacterial peptide through yeast recombinant expression |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1381587A (en) * | 2002-01-23 | 2002-11-27 | 山东大学 | Antibacterial peptide gene of Chinese prawn and its colon technique |
| US6642203B1 (en) * | 1997-07-21 | 2003-11-04 | Institut Francais De Recherche Pour L'exploitation De La Mer | Crustacean antimicrobial peptides |
| US20040235738A1 (en) * | 2003-05-16 | 2004-11-25 | Academia Sinica | Novel antimicrobial peptide isolated from penaeus monodon |
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2005
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6642203B1 (en) * | 1997-07-21 | 2003-11-04 | Institut Francais De Recherche Pour L'exploitation De La Mer | Crustacean antimicrobial peptides |
| CN1381587A (en) * | 2002-01-23 | 2002-11-27 | 山东大学 | Antibacterial peptide gene of Chinese prawn and its colon technique |
| US20040235738A1 (en) * | 2003-05-16 | 2004-11-25 | Academia Sinica | Novel antimicrobial peptide isolated from penaeus monodon |
Non-Patent Citations (2)
| Title |
|---|
| Molecular cloning and sequencing of shrimp (Penaeusmonodon) penaeidin-5 cDNA Jyh.Yih Chen et al,Fish&Shellfis Immunology,No.16 2004 * |
| 中国对虾抗菌肽成熟肽的cDNA克隆 康翠洁等，山东大学学报(理学版)，第37卷第6期 2002 * |
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