CN1721439A - Scylla serrata antibacterial peptide and its genes and clone method for genes - Google Patents
Scylla serrata antibacterial peptide and its genes and clone method for genes Download PDFInfo
- Publication number
- CN1721439A CN1721439A CN 200510053239 CN200510053239A CN1721439A CN 1721439 A CN1721439 A CN 1721439A CN 200510053239 CN200510053239 CN 200510053239 CN 200510053239 A CN200510053239 A CN 200510053239A CN 1721439 A CN1721439 A CN 1721439A
- Authority
- CN
- China
- Prior art keywords
- val
- leu
- gly
- ala
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention Scylla serrata antibiotic peptide and its gene and genetic cloning method, and relates to one kind of active antibiotic peptide scygonadin and its gene. Antibiotic peptide scygonadin is separated and purified from male sexual gland of Scylla serrata, and degenerate primer is designed and synthesized based on the total RNA of the male sexual gland as template and the detected 20 amino acid sequence in N end of antibiotic peptide scygonadin and cloned to the whole length cDNA gene sequence one the antibiotic peptide scygonadin by means of RT-PCR, RACE or other molecular biological process. Genetic engineering strain with high efficiency expression of the antibiotic peptide may be constituted via constituting eukaryotic expression vector of the gene so as to externally produce antibiotic peptide as feed additive for aquatic animal to resist drug resisting bacteria. In addition, the present invention may be used also in transgenic blue crab breeding.
Description
Technical field
The present invention relates to from the male gland of Young Crab (Scylla serrata (forskal)) separation and purification to a kind of Antimicrobially active polypeptides scygonadin, and the gene of this antibacterial peptide of coding that clones in view of the above.
Background technology
Young Crab, English name mud crab, blue crab latin name Scylla serrata (forskal), be commonly called as that mud crab, lock crab, worm seek, decoy, NATURAL DISTRIBUTION is wider, in the temperate zone, the subtropics, all there is its trace the tropical seas, is the important economic kind of sea farming.In recent years, along with the breakthrough of mud crab artificial breeding technique, the improvement of cultural technique, blue crab cultivation is changed to intensive culture by extensive breed gradually.But increase along with cultivation density, aquaculture water eutrophication phenomenon is more and more serious, causes breeding environment to worsen, and diseases such as virus disease, bacteriosis, mycosis and parasitosis are constantly broken out, both caused tremendous economic loss, again serious contusion people's breed enthusiasm.
(Antibacterial Peptides is the natural small peptide material that a class has very strong wide spectrum antibacterial activity ABP) to antibacterial peptide, is considered to one of main component of animal non-specific immunity systems of defense such as fish, crustaceans, shellfish.When organism sustains damage or pathogenic micro-organism when invasion and attack, can produce antibacterial peptide rapidly, to prevent and to kill and wound the invasion of pathogenic micro-organism, its resultant velocity is fast, it is not available to spread characteristics such as rapid, flexible in vivo and be other macro-molecular proteins (as antibody etc.) and immunocyte, is playing an important role aspect defence seawater bacterium and the poisoning intrusion.
The antibacterial peptide research of crustacean is less, in the crab class, up to (Schnapp D et al. such as Schnapp in 1996, Eur.J.Biochem., 1996,240:532-539) with ion exchange chromatography and high performance liquid chromatography (HPLC), divide offshore crab (Carcinus maenas) hemocytolysis thing to obtain antibacterial peptide (6.5KDa), and measure its N-terminal portions sequence, found that closely, be the positively charged ion resisting gram-positive bacterium and the gram negative bacterium active polypeptide of proline rich with mammiferous bactenecin 7.(RelfJ.M.et al. such as Relf in 1999, Eur.J.Biochem., 1999,264:350-357) granulosa cell of branch offshore crab receives the antibacterial peptide of a 11.5kDa, this albumen is to thermally-stabilised, halophilic ampholytic cation polypeptide, but only the ocean gram positive bacterium is played toxic action, its aminoacid sequence is different with known antibacterial peptide sequence, but with the mankind's antileukoprotease height homology.(Jayasankar V et al. such as V.Jayasankar in 1999, Journal ofExperimental Marine Biology and Ecology.1999,236:253-259) separate Young Crab [Scylla serrata (Forskal)] seminal fluid and obtain the antibacterial protein of a kind of 20KDa, at first proved in the invertebrates seminal fluid to have antibacterial substance, but not further further investigation.The same year, (Khoo LH et al. such as Khoo, Mar Biotechnol., 1999,1:44-51) reported from the C.sapidus hemocyte and be separated to 3.7kDa, the active antibacterial peptide of anti-E.coli---Callinectin, and measure its partial amino-acid series, find the terminal proline rich of its N-, do not form Pro-Arg-Pro motif, and do not have significant homology with known antibacterial peptide.
The basic antibiotic mechanism of antibacterial peptide is: the antibacterial peptide molecule destroys the membrane structure of bacterium, causes that water-soluble substances oozes out in a large number in the born of the same parents, causes bacterium to stop growing or death.Its antibiotic mechanism is different from traditional microbiotic, because molecular weight is little, very easily is diffused into infection site, does not exist after the use to produce the resistance problem, and does not have hazard residue after entering body.Therefore, antibacterial peptide will be a kind of antibacterials that have a extensive future in the diseases prevention and treatment of sea farming crab class; But directly extract or utilize the natural antibacterial peptide of chemical process synthetic by the crab class, be subjected to the restriction of animal-origin and chemical synthesis process, be difficult to obtain the antibacterial peptide of q.s, and expense costliness, thereby separating clone antibacterial peptide gene, utilizing the external mass production antibacterial peptide of gene engineering method, is to obtain the antibacterial peptide of high yield and guarantee its prerequisite in the culture fishery large scale application.
Summary of the invention
Purpose of the present invention aims to provide a kind of antibacterial peptide scygonadin of separation and purification from the male gland of Young Crab and this antibacterial peptide gene of clone.This gene can make up carrier for expression of eukaryon and set up the engineering strain that efficiently expresses Scylla serrata Antibacterial Peptides, and external mass production Scylla serrata Antibacterial Peptides is as some microbiotic in the alternative conventional feed of feed immunity additive.
The present invention said from the male gland of Young Crab the Scylla serrata Antibacterial Peptides scygonadin molecular weight of separation and purification be about 10.8KDa, terminal 20 aminoacid sequences of its N-are " Gly Gln Ala Leu Asn Lys Leu Met Pro LysIle Val Ser Ala Ile Ile Tyr Met Val Gly ".
The said Young Crab scygonadin of the present invention antibacterial peptide gene cDNA has 564nt, wherein contain 5 ' the end long 56nt of non-coding region (5 ' UTR), read the long 378nt of frame for 1,3 ' the non-coding translation head of district 130nt (3 ' UTR), its GenBank series number is AY864802, and its sequence is as follows:
Sequence table one: the scygonadin antibacterial peptide full length cDNA sequence of Young Crab (Scylla serrata):
acacacccgc?aacctctatc?accaccacaa?catccactcg?cctccagacc?ctcaca
c 60
gttcatctct?cctactcggc?cttacagtgg?tggtgctgct?gggcgtcatc?gtgcctccat 120
gcatggcagg?ccaggcactc?aacaaactta?tgcctaaaat?cgtcagcgcc?ataatttata 180
tggtcgggca?acccaatgca?ggtgtcactt?ttctgggcca?ccaatgtctg?gtggagtcaa 240
cgaggcaacc?agacgggttt?tacaccgcaa?agatgtcgtg?tgcttcctgg?actcatgata 300
atcctattgt?tggggaagga?agaagccggg?ttgaacttga?ggcgcttaaa?ggttccatca 360
caaactttgt?ccagacagca?tccaattaca?agaagttcac?catagatgag?gtcgaggact 420
ggattgcttc?ttac
gac?gctcacctga?cgtgctctcc?gagtccacca?aagctgtctt 480
tgctggagcc?actaagagtg?gtttggttat?tgaggacaat?aaataacttt?ctgaatttta 540
aaaaaaaaaa?aaaaaaaaaa?aaaa 564
Sequence table two: Young Crab scygonadin antibacterial peptide cDNA predictive coding Argine Monohydrochloride sequence:
Met?Arg?Ser?Ser?Leu?Leu?Leu?Gly?Leu?Thr?Val?Val?Val?Leu?Leu?Gly
-20 -15 -10
Val?Ile?Val?Pro?Pro?Cys?Met?Ala?Gly?Gln?Ala?Leu?Asn?Lys?Leu?Met
-5 -1 1 5
Pro?Lys?Ile?Val?Ser?Ala?Ile?Ile?Tyr?Met?Val?Gly?Gln?Pro?Asn?Ala
10 15 20
Gly?Val?Thr?Phe?Leu?Gly?His?Gln?Cys?Leu?Val?Glu?Ser?Thr?Arg?Gln
25 30 35 40
Pro?Asp?Gly?Phe?Tyr?Thr?Ala?Lys?Met?Ser?Cys?Ala?Ser?Trp?Thr?His
45 50 55
Asp?Asn?Pro?Ile?Val?Gly?Glu?Gly?Arg?Ser?Arg?Val?Glu?Leu?Glu?Ala
60 65 70
Leu?Lys?Gly?Ser?Ile?Thr?Asn?Phe?Val?Gln?Thr?Ala?Ser?Asn?Tyr?Lys
75 80 85
Lys?Phe?Thr?Ile?Asp?Glu?Val?Glu?Asp?Trp?Ile?Ala?Ser?Tyr
90 95 100
Sequence table three: Young Crab scygonadin antibacterial peptide cDNA reading frame and predicted protein aminoacid sequence:
acacacccgc?aacctctatc?accaccacaa?catccactcg?cctccagacc?ctcaca?atg 59
Met
cgt?tca?tct?ctc?cta?ctc?ggc?ctt?aca?gtg?gtg?gtg?ctg?ctg?ggc?gtc 107
Arg?Ser?Ser?Leu?Leu?Leu?Gly?Leu?Thr?Val?Val?Val?Leu?Leu?Gly?Val
5 10 15
atc?gtg?cct?cca?tgc?atg?gca?ggc?cag?gca?ctc?aac?aaa?ctt?atg?cct 155
Ile?Val?Pro?Pro?Cys?Met?Ala?Gly?Gln?Ala?Leu?Asn?Lys?Leu?Met?Pro
20 25 30
aaa?atc?gtc?agc?gcc?ata?att?tat?atg?gtc?ggg?caa?ccc?aat?gca?ggt 203
Lys?Ile?Val?Ser?Ala?Ile?Ile?Tyr?Met?Val?Gly?Gln?Pro?Asn?Ala?Gly
35 40 45
gtc?act?ttt?ctg?ggc?cac?caa?tgt?ctg?gtg?gag?tca?acg?agg?caa?cca 251
Val?Thr?Phe?Leu?Gly?His?Gln?Cys?Leu?Val?Glu?Ser?Thr?Arg?Gln?Pro
50 55 60 65
gac?ggg?ttt?tac?acc?gca?aag?atg?tcg?tgt?gct?tcc?tgg?act?cat?gat 299
Asp?Gly?Phe?Tyr?Thr?Ala?Lys?Met?Ser?Cys?Ala?Ser?Trp?Thr?His?Asp
70 75 80
aat?cct?att?gtt?ggg?gaa?gga?aga?agc?cgg?gtt?gaa?ctt?gag?gcg?ctt 347
Asn?Pro?Ile?Val?Gly?Glu?Gly?Arg?Ser?Arg?Val?Glu?Leu?Glu?Ala?Leu
85 90 95
aaa?ggt?tcc?atc?aca?aac?ttt?gtc?cag?aca?gca?tcc?aat?tac?aag?aag 395
Lys?Gly?Ser?Ile?Thr?Asn?Phe?Val?Gln?Thr?Ala?Ser?Asn?Tyr?Lys?Lys
100 105 110
ttc?acc?ata?gat?gag?gtc?gag?gac?tgg?att?gct?tct?tac?taagacgctc 444
Phe?Thr?Ile?Asp?Glu?Val?Glu?Asp?Trp?Ile?Ala?Ser?Tyr
115 120 125
acctgacgtg?ctctccgagt?ccaccaaagc?tgtctttgct?ggagccacta?agagtggttt 504
ggttattgag?gacaataaat?aactttctga?attttaaaaa?aaaaaaaaaa?aaaaaaaaaa 564
The separation and purification and the cDNA gene clone process thereof of Young Crab scygonadin antibacterial peptide are as follows:
1) separation and purification of Young Crab scygonadin antibacterial peptide: adopt technological methods such as ion exchange chromatography, reversed phase chromatography and polyacrylamide gel electrophoresis; the deferential acid extract liquid of separation and purification Young Crab; obtain a molecular weight and be about the active polypeptide scygonadin that 10.8KDa has the resisting gram-positive bacterium, and measure terminal 20 aminoacid sequences of its N-by Jikang Biotechnology Co Ltd, Shanghai.
2) mensuration of Young Crab scygonadin antibacterial peptide anti-microbial activity: adopt micrococcus lysodeikticus (G+) and Aeromonas hydrophila (G-) for detecting bacterium, measure its anti-microbial activity with the micro liquid culture method.The result shows, the Scygonadin antibacterial peptide has the activity of very strong resisting gram-positive bacterium and stronger anti-gram negative bacterium.
3) Young Crab scygonadin antibacterial peptide cDNA gene clone process
According to 20 aminoacid sequence designs of Young Crab scygonadin antibacterial peptide N end degenerated primer: F1 5 '-AAYAAR YTN ATG CCN AAR ATH GT-3 ': F2 5 '-GCN ATH ATH TAY ATG the GT-3 ' (R=A+G that has measured; Y=T+C; H=A+C+T; N=T+C+A+G), and the 3 site Adaptor Primer of 3 '-Full RACE Core Set (TaKaRa) of the precious biotech firm in downstream primer employing Dalian (5`-CTGATCTAGAGGTACCGGATCC-3`, S2).With the total RNA of the sexual gland of healthy Young Crab is template, adopt oligodT-3sites Adaptor Primer (S1) reagent in the biological 3 '-Full RACE Core Set test kit of Dalian treasured to carry out reverse transcription, carry out pcr amplification with F1 and S2 primer, get 1 μ L product as template, carry out PCR with F2 and S2 again and obtain about 400bp specific amplified cDNA band, obtain the Young Crab scygonadin antibacterial peptide gene fragment of 394bp through order-checking; (http://www.ncbi.nlm.nih.gov/) analyzes by Blast and confirms that the gene fragment that obtains is new gene in the NCBI website.
3) according to the synthetic special primer GSP (codon ATG downstream 201bp-227bp) of gene fragment design that obtains as downstream primer: GSP:5`-GCACACGACATCTTTGCGGTGTAGAAC-3`, utilize 5 '-CDS primer (Clontech.): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1=A, G or C and SMARTIIA oligo (SMART RACE cDNAAmplification Kit, Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG is as upstream primer, with the total RNA of Young Crab sexual gland is template, carries out 5`race.
4) be the amount that further obtains 5 ' race product, utilize UPM (Clontech) .:(5 ') CTA ATA CGA CTC ACTATA GGG CAA CGC AGA GT, NUPM (Clontech): (5 ') AAG CAG TGG TAT CAA CGC AGA GT carries out nested pcr amplification with GSP1 and obtains about 300bp cDNA band, checks order to such an extent that 283bp Young Crab scygonadin antibacterial peptide contains the 5` end cDNA segment.Sequencing result through 5 ' race and 3 ' race splices and acquisition Young Crab scygonadin antibacterial peptide full-length cDNA.
Separation and purification is to a kind of novel antimicrobial peptide scygonadin from the male gland of Young Crab with technology such as ion-exchange, reverse chromatographies in the present invention, and this peptide has obvious suppression or kills micrococcus lysodeikticus gram positive bacteriums such as (Micrococcus lysoleiksicus) and inhibition fish-pathogenic bacteria---Aeromonas hydrophila effects such as gram negative bacterium such as (Aeromonas hydrophila) external.And be template with the total RNA of male gland of Young Crab, according to 20 aminoacid sequences of scygonadin antibacterial peptide N end of measuring, the synthetic degenerated primer of design, utilize RT-PCR, RACE equimolecular biology techniques means, successfully be cloned into Scylla serrata Antibacterial Peptides scygonadin full-length cDNA gene order, this gene belongs to a kind of new gene.With the 6.5KDa, the 11.5KDa that from bank crab (Carcinus maenas) hemocyte, separate acquisition abroad, and being separated to the antibacterial peptide of a kind of 20KDa from Young Crab [Scylla serrata (Forskal)] seminal fluid, the primary structure (aminoacid sequence) of its isolating tissue site, molecular weight size, peptide chain and nucleotide sequence are all different.Gene can be set up the engineering strain that efficiently expresses this antibacterial peptide by making up carrier for expression of eukaryon, external mass production goes out antibacterial peptide, as some microbiotic in the alternative conventional feed of feed immunity additive, be used for culture fishery, realize green cultivation, reduce and even elimination microbiotic accumulating in fishery products, improve aquatic product quality; Also can be used as the antibiotic effective substitute of some resistance, be used for the control medicine of culture fishery drug tolerant bacteria.In addition, this gene can also be used for the breeding research of transgenosis mud crab.
Description of drawings
Fig. 1 is reversed phase chromatography and the anti-microbial activity figure of antibacterial peptide Scygonadin.
Fig. 2 is the SDS-PAGE electrophorogram of antibacterial peptide scygonadin.
The Young Crab sexual gland total RNA electrophorogram of Fig. 3 for extracting.
Fig. 4 is the RT-PCR electrophorogram.
Fig. 5 is the 5`race electrophorogram.
Embodiment
Below by embodiment the present invention is elaborated.
Embodiment:
1. the separation and purification of Young Crab scygonadin antibacterial peptide:
The vas deferens of anatomical isolation Young Crab adds isopyknic acid extract liquid [15% acetate, 0.1% trifluoroacetic acid and 1mM phenylformic acid sulfonic acid fluoride (PMSF)], homogenate 20min (6000rmin
-1), homogenate is got supernatant in 4 ℃ of centrifugal 45min of 15000g.With the resuspended precipitation of equal-volume extraction liquid, behind the ultrasonic disruption (broken 15 times of 400w 10s), 4 ℃ of centrifugal 45min of 15000g get supernatant, merge supernatant liquor twice, lyophilize.With 0.05M sodium acetate buffer (pH5.0) sample dissolution, 4 ℃ of centrifugal 10min of 10000g, get supernatant, cross SP-Sepharose Fast Flow ion exchange column, with 1.5M ammonium acetate gradient elution, 5 column volumes of 0-50% wash-out (CV), 50%-80% wash-out 2CV, 80-100% wash-out 5CV.The component of wash-out is that the 350-410ml anti-microbial activity is the strongest, with anti-phase Source 5R RPC (Amersham Sweden) purifying.Use A liquid (0.065% trifluoroacetic acid, 2% acetonitrile) balance 2CV in advance, carry out gradient elution (0-15%, 1.5CV with B liquid (0.05% trifluoroacetic acid, 80% acetonitrile); 15-30%, 10CV; 30-50%, 8CV; 80-1001CV); detect A280; the collection peak value is that the component at 74.89mL place is scygonadin antibacterial peptide sample (see figure 1); carry out the TricineSDS-PAGE gel electrophoresis and identify (see figure 2); order-checking (Jikang Biotechnology Co Ltd, Shanghai) obtains its N-terminal portions sequence and is " Gly Gln Ala Leu Asn Lys Leu Met Pro Lys Ile Val Ser Ala Ile Ile Tyr MetVal Gly ".
2. anti-microbial activity detects: get detected bacterium (Aeromonas hydrophila, G
-Micrococcus lysodeikticus, G
+) inclined-plane one ring, line on the MHA flat board, be inverted for 28 ℃ and cultivate 16～18h.1 single colony inoculation of each picking is in the MHA slant medium from above-mentioned flat board, 28 ℃ of shaking culture 24h.Wash slant culture with 0.05M HEPES (pH6.8), adjust and be diluted to OD
620=0.1, standby.Experiment is carried out on the trace Tissue Culture Plate of 96 holes of standard, and each sample is equipped with two experiments and control wells respectively.In experimental port, add 25 μ L detected samples and 25 μ L bacterial suspensions respectively according to experimental design, in negative control hole, add 25 μ L 50mM HEPES and 25 μ L bacterial suspensions, in control wells, add 25 μ L detected samples and 25 μ L 50mM HEPES (blank) in addition.Then Tissue Culture Plate is placed 28 ℃ to cultivate 3h, every Kong Zhongzai adds the aseptic MHB substratum of 50 μ L, cultivates 2h for 28 ℃.After adding 10 μ L MTS-PMS reagent (Promega company) in every hole, cultivate 1h for 28 ℃.On microplate reader, survey A
492Value.Kill and wound index (KI) by following formula calculating.
Kill and wound index (%)=[1-(experimental group A
492-blank A
492)/(negative control A
492-blank A
492)] * 100
The result judges: the KI value is big more, and the expression anti-microbial activity is strong more.
The extraction of the total RNA of 3 Young Crab sexual glands
Take by weighing the male glandular tissue of 100mg Young Crab, powdered with liquid nitrogen grinding, be added in the nuclease free centrifuge tube that contains 1mL Trizol.2～8 ℃ of centrifugal 10min of following 12000g, sucking-off pink supernatant, incubation 5min under the room temperature thoroughly removes nucleoprotein complex, adds 200 μ L chloroforms then, covers tight lid.Thermal agitation 15sec, incubation 2～3min under the room temperature, 2～8 ℃ of centrifugal 15min of following 12000g.Get supernatant to another nuclease free centrifuge tube, add 500 μ L Virahols again, mixing is with precipitated rna gently.Room temperature incubation 10min, 2～8 ℃ of centrifugal 10min of following 12000g, the visible glue sample precipitation in centrifuge tube bottom is RNA.Abandon supernatant, with 1mL 75% washing with alcohol RNA precipitation, 2～8 ℃ of centrifugal 5min of following 7500g.Airing RNA precipitation, and with 100 μ L nuclease free water dissolution.Survey OD
260nm/ OD
280nmAnd OD
260nm/ OD
230nm, on 1% agarose gel electrophoresis, analyze (see figure 3).
4.RT-PCR amplification purpose fragment scygonadin antibacterial peptide gene
4.1 reverse transcription reaction and 3 ' RACE
According to terminal 20 aminoacid sequences measuring of the Young Crab scygonadin antibacterial peptide N-of separation and purification, (degeneracy is: R=A+G for design upstream primer F1 5 '-AAY AAR YTN ATG CCN AAR ATH GT-3 ' and F2 5`-GCN ATH ATH TAY ATG GT-3 '; Y=T+C; H=A+C+T; N=T+C+A+G), and the 3 site Adaptor Primer of downstream primer employing 3 '-Full RACE CoreSet (TaKaRa) (5`-CTGATCTAGAGGTACCGGATCC-3`, S2).With the total RNA of the sexual gland of healthy Young Crab is template, adopt oligodT-3sites Adaptor Primer (S1) reagent of (TaKaRa) in 3 '-Full RACE Core Set test kit to carry out reverse transcription, carry out pcr amplification with F1 and S2 primer, get 1 μ L as template, carry out PCR with F2 and S2 again and obtain about 400bp specific amplified cDNA band, obtain the Young Crab scygonadin antibacterial peptide gene fragment of 394bp through order-checking;
In the 0.2mL centrifuge tube, add following material: 2 μ L, 10 * RNA PCR buffer, 4 μ L MgCl
2(25mM), 2 μ L dNTP Mixture (10mM each), 1 μ L AMV Reverse Transcriptase XL (5U μ L
-1), 0.5 μ L RNase inhibitor (40U μ L
-1), the total RNA of 1 μ L oligo dT-3sites Adaptor Primer (2.5 μ M) and 1 μ L Young Crab sexual gland replenishes no RNase distilled water and makes cumulative volume reach 20 μ L.After the mixing, centrifugal slightly being placed on the PCR instrument, move as follow procedure: 30 ℃, 10min; 48 ℃, 30min; 95 ℃, 5min; 5 ℃, 5min.
4.2PCR reaction
Add 10 * Mg respectively
2+Free buffer 10.0 μ L, MgCl
2(25mM) 8.0 μ L, dNTPs (10mM) 1.0 μ L, F1 (10 μ M) 2.0 μ L, S2 (10 μ M) 2.0 μ L, cDNA 4.0 μ L, Taq archaeal dna polymerase (1.0U/ μ L) 0.5 μ L use nuclease free water polishing to cumulative volume 100 μ L.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 40sec, 60 ℃ of renaturation 45sec, 72 ℃ are extended 60sec, 30 circulations; 72 ℃ are extended 5min and increase.After product dilutes 100 times with distilled water, get 4.0 μ L and in following reaction system, afterwards add 10 * Mg
2+Free buffer 10.0 μ L, MgCl
2(25mM) 8.0 μ L, dNTPs (10mM) 1.0 μ L, F2 (10 μ M) 2.0 μ L, S2 (10 μ M) 2.0 μ L, Taq archaeal dna polymerase (1.0U/ μ L) 0.5 μ L use the distilled water polishing to cumulative volume 100 μ L.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 40sec, 60 ℃ of renaturation 45sec, 72 ℃ are extended 60sec, 30 circulations; 72 ℃ are extended 5min.Amplification obtains about 400bp specific amplified cDNA band (see figure 4) obtains 394bp through order-checking Young Crab scygonadin antibacterial peptide gene fragment; (http://www.ncbi.nlm.nih.gov/) analyzes by Blast and confirms that the gene fragment that obtains is new gene in the NCBI website.For obtaining this full length gene cDNA sequence, carry out 5 ' race.
5.5′RACE
5.1 reverse transcription reaction
According to the synthetic special primer GSP (codon ATG downstream 201bp-227bp) of gene fragment design that obtains as downstream primer: GSP:5`-GCACACGACATCTTTGCGGTGTAGAAC-3`, utilize 5 '-CDS primer (Clontech.): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1(SMART RACE cDNA AmplificationKit, Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG is as upstream primer for=A, G or C and SMARTIIA oligo.Get the total RNA of 3.0 μ L, 1.0 μ L 5 '-CDS primer and 1.0 μ L SMART II A oligo add in the centrifuge tube of 0.2mL, 70 ℃, 10min, place immediately on ice, add 5 * First-Strand Buffer, 2.0 μ L, DTT (20mM) 1.0 μ L, dNTP (10mM) 1.0 μ L, PowerScript Reverse Transcriptase1.0 μ L again to cumulative volume 10 μ L, 42 ℃ are extended 90min; 72 ℃ of incubation 7min inactivators; 5 ℃, 5min.
5.2PCR reaction
Add 10 * Advantage PCR, 5.0 μ L, dNTP (10mM) 1.0 μ L, 50 * Advantage Polymerase mix1.0 μ L, cDNA 5.0 μ L, UPM (2.0 μ M) 1.0 μ L, GSP (10 μ M) 1.0 μ L, with nuclease free water polishing to 50 μ L.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec, 68 ℃ of annealing 30sec, 72 ℃ are extended 2min, 28 circulations; 72 ℃ are extended 5min.
5.3 nested PCR
Utilize UPM (Clontech.): (5 ') CTA ATA CGA CTC ACT ATA GGG CAA CGC AGA GT, NUPM (Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GT carries out nested PCR with GSP.Get step reaction product 5.0 μ L, add 10 * Advantage PCR, 5.0 μ L, dNTP (10mM) 1.0 μ L, 50 * Advantage Polymerasemix1.0 μ L, NUMP (2.0 μ M) 5.0 μ L, GSP 10 μ M) 1.0 μ L, with nuclease free water polishing to 50 μ L.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec, 68 ℃ of renaturation 30sec, 72 ℃ are extended 2min, 25 circulations; 72 ℃ are extended 5min.Amplification obtains about 300bp cDNA band, checks order to such an extent that 283bp Young Crab scygonadin antibacterial peptide contains 5` end cDNA segment (see figure 5).
6. sequential analysis and gene are determined
The cDNA product that amplification obtains carries out dna nucleotide sequence by Shanghai Bo Ya Bioisystech Co., Ltd and measures.Sequence homology comparison and similarity searching carry out with the online software of NCBI (http://www.ncbi.nlm.nih.gov) BLAST; Multisequencing is relatively used DNAssist2.0 software; With the auxiliary sequence assembly that carries out of BLAST2 software; Signal peptide prediction search with SIGNALP is online (
Http:// www.cbs.dtu.dk/services/SignalP).
The Scylla serrata Antibacterial Peptides full length gene cDNA sequence that obtains that increases submits to international GenBank (http://www.ncbi.nlm.nih.gov) to carry out new genetic comparison evaluation.
Claims (3)
1. Scylla serrata Antibacterial Peptides, it is characterized in that said from the male gland of Young Crab the Scylla serrata Antibacterial Peptides scygonadin molecular weight of separation and purification be about 10.8KDa, terminal 20 aminoacid sequences of its N-are " Gly Gln Ala Leu Asn LysLeu Met Pro Lys Ile Val Ser Ala Ile Ile Tyr Met Val Gly ".
2. Scylla serrata Antibacterial Peptides gene, it is characterized in that said Young Crab scygonadin antibacterial peptide gene cDNA has 564nt, wherein contain 5 ' the end long 56nt of non-coding region (5 ' UTR), read the long 378nt of frame for 1,3 ' the non-coding translation head of district 130nt (3 ' UTR), its GenBank series number is AY864802, and its sequence is as follows:
Sequence table one: the scygonadin antibacterial peptide full length cDNA sequence of Young Crab (Scylla serrata):
acacacccgc?aacctctatc?accaccacaa?catccactcg?cctccagacc?ctcaca
c 60
gttcatctct?cctactcggc?cttacagtgg?tggtgctgct?gggcgtcatc?gtgcctccat 120
gcatggcagg?ccaggcactc?aacaaactta?tgcctaaaat?cgtcagcgcc?ataatttata 180
tggtcgggca?acccaatgca?ggtgtcactt?ttctgggcca?ccaatgtctg?gtggagtcaa 240
cgaggcaacc?agacgggttt?tacaccgcaa?agatgtcgtg?tgcttcctgg?actcatgata 300
atcctattgt?tggggaagga?agaagccggg?ttgaacttga?ggcgcttaaa?ggttccatca 360
caaactttgt?ccagacagca?tccaattaca?agaagttcac?catagatgag?gtcgaggact 420
ggattgcttc?ttac
gac?gctcacctga?cgtgctctcc?gagtccacca?aagctgtctt?480
tgctggagcc?actaagagtg?gtttggttat?tgaggacaat?aaataacttt?ctgaatttta 540
aaaaaaaaaa?aaaaaaaaaa?aaaa 564
Sequence table two: Young Crab scygonadin antibacterial peptide cDNA predictive coding Argine Monohydrochloride sequence:
Met?Arg?Ser?Ser?Leu?Leu?Leu?Gly?Leu?Thr?Val?Val?Val?Leu?Leu?Gly
-20 -15 -10
Val?Ile?Val?Pro?Pro?Cys?Met?Ala?Gly?Gln?Ala?Leu?Asn?Lys?Leu?Met
-5 -1 1 5
Pro?Lys?Ile?Val?Ser?Ala?Ile?Ile?Tyr?Met?Val?Gly?Gln?Pro?Asn?Ala
10 15 20
Gly?Val?Thr?Phe?Leu?Gly?His?Gln?Cys?Leu?Val?Glu?Ser?Thr?Arg?Gln
25 30 35 40
Pro?Asp?Gly?Phe?Tyr?Thr?Ala?Lys?Met?Ser?Cys?Ala?Ser?Trp?Thr?His
45 50 55
Asp?Asn?Pro?Ile?Val?Gly?Glu?Gly?Arg?Ser?Arg?Val?Glu?Leu?Glu?Ala
60 65 70
Leu?Lys?Gly?Ser?Ile?Thr?Asn?Phe?Val?Gln?Thr?Ala?Ser?Asn?Tyr?Lys
75 80 85
Lys?Phe?Thr?Ile?Asp?Glu?Val?Glu?Asp?Trp?Ile?Ala?Ser?Tyr
90 95 100
Sequence table three: Young Crab scygonadin antibacterial peptide cDNA reading frame and predicted protein aminoacid sequence:
acacacccgc?aacctctatc?accaccacaa?catccactcg?cctccagacc?ctcaca?atg 59
Met
cgt?tca?tct?ctc?cta?ctc?ggc?ctt?aca?gtg?gtg?gtg?ctg?ctg?ggc?gtc 107
Arg?Ser?Ser?Leu?Leu?Leu?Gly?Leu?Thr?Val?Val?Val?Leu?Leu?Gly?Val
5 10 15
atc?gtg?cct?cca?tgc?atg?gca?ggc?cag?gca?ctc?aac?aaa?ctt?atg?cct 155
Ile?Val?Pro?Pro?Cys?Met?Ala?Gly?Gln?Ala?Leu?Asn?Lys?Leu?Met?Pro
20 25 30
aaa?atc?gtc?agc?gcc?ata?att?tat?atg?gtc?ggg?caa?ccc?aat?gca?ggt 203
Lys?Ile?Val?Ser?Ala?Ile?Ile?Tyr?Met?Val?Gly?Gln?Pro?Asn?Ala?Gly
35 40 45
gtc?act?ttt?ctg?ggc?cac?caa?tgt?ctg?gtg?gag?tca?acg?agg?caa?cca 251
Val?Thr?Phe?Leu?Gly?His?Gln?Cys?Leu?Val?Glu?Ser?Thr?Arg?Gln?Pro
50 55 60 65
gac?ggg?ttt?tac?acc?gca?aag?atg?tcg?tgt?gct?tcc?tgg?act?cat?gat 299
Asp?Gly?Phe?Tyr?Thr?Ala?Lys?Met?Ser?Cys?Ala?Ser?Trp?Thr?His?Asp
70 75 80
aat?cct?att?gtt?ggg?gaa?gga?aga?agc?cgg?gtt?gaa?ctt?gag?gcg?ctt 347
Asn?Pro?Ile?Val?Gly?Glu?Gly?Arg?Ser?Arg?Val?Glu?Leu?Glu?Ala?Leu
85 90 95
aaa?ggt?tcc?atc?aca?aac?ttt?gtc?cag?aca?gca?tcc?aat?tac?aag?aag 395
Lys?Gly?Ser?Ile?Thr?Asn?Phe?Val?Gln?Thr?Ala?Ser?Asn?Tyr?Lys?Lys
100 105 110
ttc?acc?ata?gat?gag?gtc?gag?gac?tgg?att?gct?tct?tac?taagacgctc 444
Phe?Thr?Ile?Asp?Glu?Val?Glu?Asp?Trp?Ile?Ala?Ser?Tyr
115 120 125
acctgacgtg?ctctccgagt?ccaccaaagc?tgtctttgct?ggagccacta?agagtggttt 504
ggttattgag?gacaataaat?aactttctga?attttaaaaa?aaaaaaaaaa?aaaaaaaaaa 564。
3. the cloning process of Scylla serrata Antibacterial Peptides gene is characterized in that clone's process is as follows:
1) separation and purification of Young Crab scygonadin antibacterial peptide: adopt ion exchange chromatography, reversed phase chromatography and polyacrylamide gel electrophoresis, the deferential acid extract liquid of separation and purification Young Crab, obtain a molecular weight and be about the active polypeptide scygonadin that 10.8KDa has the resisting gram-positive bacterium, its N-terminal portions sequence is " Gly Gln Ala Leu Asn LysLeu Met Pro Lys Ile Val Ser Ala Ile Ile Tyr Met Val Gly ";
2) Young Crab scygonadin antibacterial peptide cDNA gene clone process:
According to 20 aminoacid sequence designs of Young Crab scygonadin antibacterial peptide N end degenerated primer: the F1 5 '-AAYAAR YTN ATG CCN AAR ATH GT-3 ' that has measured; F25 '-GCN ATH ATH TAY ATG GT-3 ' (R=A+G; Y=T+C; H=A+C+T; N=T+C+A+G), downstream primer adopts the 3site Adaptor Primer (5`-CTGATCTAGAGGTACCGGATCC-3` of 3 '-Full RACE Core Set (TaKaRa) of the precious biotech firm in Dalian, S2), with the total RNA of the sexual gland of Young Crab is template, adopt oligodT-3sites Adaptor Primer (S1) reagent in the biological 3 '-Full RACE Core Set test kit of Dalian treasured to carry out reverse transcription, carry out pcr amplification with F1 and S2 primer, get pcr amplification product as template, carry out PCR with F2 and S2 again and obtain about 400bp specific amplified cDNA band, obtain the Young Crab scygonadin antibacterial peptide gene fragment of 394bp through order-checking;
3) according to the synthetic special primer GSP (codon ATG downstream 201bp-227bp) of gene fragment design that obtains as downstream primer: GSP:5`-GCACACGACATCTTTGCGGTGTAGAAC-3`, utilize 5 '-CDS primer (Clontech.): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1=A, G or C and SMARTIIA oligo (SMART RACE cDNAAmplification Kit, Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG is as upstream primer, with the total RNA of Young Crab sexual gland is template, carries out 5`race;
4) utilize UPM (Clontech) .:(5 ') CTA ATA CGA CTC ACT ATA GGG CAA CGC AGA GTNUPM (Clontech): (5 ') AAG CAG TGG TAT CAA CGC AGA GT carries out nested pcr amplification with GSP1 and obtains about 300bp cDNA band, checks order to such an extent that 283bp Young Crab scygonadin antibacterial peptide contains the 5` end cDNA segment; Sequencing result through 5 ' race and 3 ' race splices and acquisition Young Crab scygonadin antibacterial peptide full-length cDNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100532396A CN1325515C (en) | 2005-02-17 | 2005-02-17 | Scylla serrata antibacterial peptide and its genes and clone method for genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100532396A CN1325515C (en) | 2005-02-17 | 2005-02-17 | Scylla serrata antibacterial peptide and its genes and clone method for genes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1721439A true CN1721439A (en) | 2006-01-18 |
| CN1325515C CN1325515C (en) | 2007-07-11 |
Family
ID=35912090
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100532396A Expired - Fee Related CN1325515C (en) | 2005-02-17 | 2005-02-17 | Scylla serrata antibacterial peptide and its genes and clone method for genes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1325515C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061303A (en) * | 2010-11-26 | 2011-05-18 | 厦门大学 | Fusion expression product of antimicrobial peptide genes of two marine animals and preparation method of fusion expression product |
| CN102167736A (en) * | 2011-05-17 | 2011-08-31 | 厦门大学 | Green mud crab antibacterial peptide Sphistin and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1171998C (en) * | 2002-01-23 | 2004-10-20 | 山东大学 | Antibacterial peptide gene of Chinese prawn and its colon technique |
| CN1459506A (en) * | 2003-05-30 | 2003-12-03 | 山东大学 | Recombination expression and application of Chinese prawn antibacterial peptide gene |
-
2005
- 2005-02-17 CN CNB2005100532396A patent/CN1325515C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061303A (en) * | 2010-11-26 | 2011-05-18 | 厦门大学 | Fusion expression product of antimicrobial peptide genes of two marine animals and preparation method of fusion expression product |
| CN102061303B (en) * | 2010-11-26 | 2012-07-04 | 厦门大学 | Fusion expression product of antimicrobial peptide genes of two marine animals and preparation method of fusion expression product |
| CN102167736A (en) * | 2011-05-17 | 2011-08-31 | 厦门大学 | Green mud crab antibacterial peptide Sphistin and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1325515C (en) | 2007-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: cloning, distribution and expression | |
| Zhou et al. | Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress | |
| Zhang et al. | Molecular characterization and expression analysis of lipopolysaccharide and β-1, 3-glucan-binding protein (LGBP) from pearl oyster Pinctada fucata | |
| Liu et al. | Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1β in roughskin sculpin (Trachidermus fasciatus) | |
| Bathige et al. | Evidences for the involvement of an invertebrate goose-type lysozyme in disk abalone immunity: Cloning, expression analysis and antimicrobial activity | |
| Zhang et al. | Molecular characterization and expression analysis of a putative LPS-induced TNF-α factor (LITAF) from pearl oyster Pinctada fucata | |
| Mai et al. | Molecular cloning, characterization, expression and antibacterial analysis of a lysozyme homologue from Fenneropenaeus merguiensis | |
| Mai et al. | cDNA cloning, expression and antibacterial activity of lysozyme C in the blue shrimp (Litopenaeus stylirostris) | |
| Maiti et al. | Recombinant ferritin protein protects Penaeus monodon infected by pathogenic Vibrio harveyi | |
| CN109134635B (en) | Antibacterial peptide with composite bioactivity and preparation method and application thereof | |
| CN1721439A (en) | Scylla serrata antibacterial peptide and its genes and clone method for genes | |
| Li et al. | Expression and antimicrobial activity of c-type lysozyme in taimen (Hucho taimen, Pallas) | |
| Duan et al. | The responsive expression of a chitinase gene in the ridgetail white prawn Exopalaemon carinicauda against Vibrio anguillarum and WSSV challenge | |
| Liu et al. | Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in rainbow trout (Oncorhynchus mykiss) with implications for GILT in innate immune response | |
| CN109021088A (en) | A kind of Penaeus monodon cecropin A LFpm10 and preparation method thereof | |
| Sun et al. | Sequence and expression analysis of cathepsin S gene in the miiuy croaker Miichthys miiuy | |
| CN116098233A (en) | Functional feed additive for preventing and treating acute hepatopancreatic necrosis of prawns and application thereof | |
| CN101921788B (en) | Meretrix lysozyme gene and coding protein and application thereof | |
| Dong et al. | cDNA cloning and gene expression pattern following bacterial challenge of peroxinectin in Chinese shrimp Fenneropenaeusáchinensis | |
| Xu et al. | Molecular characterization and expression analysis of IL-10 and IL-20L genes in swamp eel (Monopterus albus) against Aeromonas veronii infection | |
| Han et al. | Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress | |
| KR101121077B1 (en) | Novel genes encoding defensin of olive flounder and uses thereof | |
| CN1238378C (en) | An antibiotic peptide and its coded sequence and use | |
| Jiang et al. | Identification of a trypsin gene from Scylla paramamosain and its expression profiling during larval development | |
| CN102337273A (en) | Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Fujian Hualong Feed Co., Ltd. Assignor: Xiamen University Contract fulfillment period: 2008.8.16 to 2014.8.15 Contract record no.: 2008350000037 Denomination of invention: Scylla serrata antibacterial peptide and its genes and clone method for genes Granted publication date: 20070711 License type: Exclusive license Record date: 2008.9.22 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2008.8.16 TO 2014.8.15 Name of requester: HUALONG FEED CO., LTD., FUJIAN PROVINCE Effective date: 20080922 |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070711 Termination date: 20180217 |