CN1624127A - Hepcidin antibacterial peptide gene of genuine porgy cultured in sea water - Google Patents
Hepcidin antibacterial peptide gene of genuine porgy cultured in sea water Download PDFInfo
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- CN1624127A CN1624127A CN 200410090296 CN200410090296A CN1624127A CN 1624127 A CN1624127 A CN 1624127A CN 200410090296 CN200410090296 CN 200410090296 CN 200410090296 A CN200410090296 A CN 200410090296A CN 1624127 A CN1624127 A CN 1624127A
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Abstract
An antibacterial peptide gene hepcidin of the Pagrosomus major cultured in seawater is cloned through using the general DNA of the Pagrosomus major's liver as template, using the gene sequence of the hepcidin of crossed spotted weever as reference, designing and synthesizing specific primer, and performing RT-PCR, 5'race and 3'race. It has high activity to resist gram-positive and -negative bacteria, and is a iron regulation hormone and related to iron metabolize.
Description
Technical field
The present invention relates to a kind of hepcidin antibacterial peptide gene of from sea farming porgy (Pagrus major), cloning.
Background technology
The sea farming porgy, English name Red Seabream, latin name Pagrus major belongs to Actinopterygii, Perciformes, porgy section, porgy belongs to, and is commonly called as red squama Red Snapper; Increasing at the area of propagating artificially of maritime provinces such as China Guangdong, Fujian to porgy in recent years, become main sea farming economic fish, is one of main famous and precious edible economic fish of outlet Japan, Korea S and country in Southeast Asia.Over nearly 20 years, along with the porgy aquaculture development is swift and violent, the high-density intensive culture has brought multiple negative factor influence, and its disease species, frequency of disease development and hazardness thereof also increase year by year, particularly bacteriosis constantly takes place, and has caused enormous economic loss for whole porgy aquaculture.Especially mariculture industry is widely applied microbiotic, causes and contains excessive violated antibiotic medicine in the fishery products, and quality product is descended, and breeding environment worsens, and outlet is obstructed, and has badly influenced the sound development of water industry.
Antibacterial peptide (Antibacterial Peptides, ABP) be the natural small peptide material that a class has very strong wide spectrum antibacterial activity, be considered to fish, shrimp, one of main component of animal non-specific immunity systems of defense such as shellfish, when the fish body 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, diffusion rapidly in vivo, flexible characteristic is that other macro-molecular proteins (as antibody etc.) and immunocyte are not available, is playing an important role aspect defence seawater bacterium and the poisoning intrusion.The Hepcidin antibacterial peptide is the another kind of antibacterial peptide with peculiar property of discovered in recent years, (Krause A such as Krause A in 2000, et al., FEBS Lett., 2000,480 (2-3): 147-150) the blood filtrate from the people is separated to LEAP-1 (liver-expressed antimicrobial peptide, the antibacterial peptide of liver expression), calendar year 2001 Park etc. isolates a peptide that is rich in halfcystine from human urine, because it derives from liver and antimicrobial characteristic is arranged, so called after hepcidin (Park CH, et al. at first, Biol, Chem., 2001,276 (11): 7806-7810), that continues is separated to 2 hepcidin precursor peptide (PigeonC with family from mouse (Mus musculus), et al., J.Biol.Chem, 276:7811-7819; IlyinG, et al., FEBS Lett., 2003,542:22-26).Shiker equals reported first in 2002, and (Morone chrysops * M.saxatilis) is separated to a hepcidin mature peptide (Shike H from hybridization speckle perch, et al., Eur.J.Biochem., 2002,269:2232-2237), preliminary study finds that the hepcidin antibacterial peptide is the same with the antibacterial peptide of other report, has anti-microbial effect.Studies have shown that the conserved sequence of eight cysteine residues of characteristic feature is contained in the hepcidin antibacterial peptide gene family in different genera source on the same site of mature peptide, the activity of anti-various fungies and Gram-positive, negative bacterium is arranged.Fish, isolate (Shike H the hepcidin gene segment except that reporting from hybridization speckle perch, zebra fish (zebrafish), winter flounder (winter flounder), salmon (salmon) etc., et al., Dvelopment and Comparatvive Immunology., 2004,28:747-745; Douglas SE, et al., Dvelopment and Comparatvive Immunology., 2001,25:137-147; Douglas SE, et al., Dvelopment and Comparatvive Immunology., 2003,27:587-601), report that also analyzing discovery by expressed sequence tag (EST) from the gene library of other fish such as Japanese flatfish (Paralichthys olivaceus), Atlantic salmon (Salmo salar) and goby (Gillichthys mirabilis) etc. has the hepcidin gene order.
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 marine fish; But directly extract or utilize the natural antibacterial peptide of chemical process synthetic by fish, be subjected to the limitation 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 hepcidin antibacterial peptide gene of cloning from sea farming porgy (Pagrus major).This gene can be set up the engineering strain that efficiently expresses hepcidin with structure carrier for expression of eukaryon such as pichia spp, and external mass production porgy hepcidin antibacterial peptide is as some microbiotic in the alternative conventional feed of feed immunity additive; Antibiotic effective antibacterials of some resistance as an alternative.
The said sea farming porgy of the present invention hepcidin antibacterial peptide gene cDNA has 586nt (not comprising poly (A)), wherein contain 5 ' the end long 94nt of non-coding region (5 ' UTR), read the long 264nt of frame for 1,3 ' the non-coding translation head of district 243nt (3 ' UTR).Its GenBank series number is AY557619.Its sequence is as follows:
Sequence table one: the hepcidin antibacterial peptide full length cDNA sequence of sea farming porgy (Pagrus major):
10 20 30 40 50 60
aaccatcaga?caggagaaga?agtgaaagga?gctgacgaga?gtcaccaaaa?gaatcaaagg
70 80 90 100 110 120
actgaacaag?ttaaagcagt?caaagcctcc?taag
atgaag?acattcagtg?ttgcagttgc
130 140 150 160 170 180
agtggccgtc?gtgctcacct?ttatttgcct?tcaggagagc?tctgctgcct?cagttactga
190 200 210 220 230 240
ggtgcaagag?ctggaggagc?caatgagcaa?tggcagtcca?gttgctgcac?atgaagagat
250 260 270 280 290 300
gccagaggag?tcctggaaga?tgccgtataa?caacagacac?aagcgcagcc?ctgctggctg
310 320 330 340 350 360
tcgcttttgc?tgcggttgct?gtcctaacat?gattggatgt?ggtgtctgct?gcaggttc
370 380 390 400 410 420
430 440 450 460 470 480
ttttacttta?aaagcagttt?tacgtctttc?agttgtggct?gttaatatct?gaggatgttt
490 500 510 520 530 540
ttgaatttgg?taatgctgca?gagaatgtgt?gctcactgat?gtgactgtat?catctacaca
550 560 570 580 590 600
cactactaca?gtgtattgtc?ac
aataaact?ttaggtttac?tcatgcaaaa?aaaaaaaaaa
601
a
Sequence table two: the hepcidin antibacterial peptide cDNA predictive coding Argine Monohydrochloride sequence of sea farming porgy:
10 20 30 40 50 60
MKTFSVAVAV?AVVLTFICLQ?ESSAASVTEV?QELEEPMSNG?SPVAAHEEMP?EESWKMPYNN
70 80 88
RHKRSPAGCR?FCCGCCPNMI?GCGVCCRF*
Sequence table three: antibiotic cDNA reading frame of the hepcidin of sea farming porgy and predicted protein aminoacid sequence:
1 A?ACC?ATC?AGA?CAG?GAG?AAG?AAG?TGA?AAG?GAG?CTG?ACG?AGA?GTC?ACC?AAA?AGA?ATC?AAA 58
59 GGA?CTG?AAC?AAG?TTA?AAG?CAG?TCA?AAG?CCT?CCT?AAG?
ATG?AAG?ACA?TTC?AGT?GTT?GCA?GTT 118
1
Met?Lys?Thr?Phe?Ser?Val?Ala?Val 8
119?GCA?GTG?GCC?GTC?GTG?CTC?ACC?TTT?ATT?TGC?CTT?CAG?GAG?AGC?TCT?GCT?GCC?TCA?GTT?ACT 178
9?Ala?Val?Ala?Val?Val?Leu?Thr?Phe?Ile?Cys?Leu?Gln?Glu?Ser?Ser?Ala?Ala?Ser?Val?Thr 28
179?GAG?GTG?CAA?GAG?CTG?GAG?GAG?CCA?ATG?AGC?AAT?GGC?AGT?CCA?GTT?GCT?GCA?CAT?GAA?GAG 238
29?Glu?Val?Gln?Glu?Leu?Glu?Glu?Pro?Met?Ser?Asn?Gly?Ser?Pro?Val?Ala?Ala?His?Glu?Glu 48
239?ATG?CCA?GAG?GAG?TCC?TGG?AAG?ATG?CCG?TAT?AAC?AAC?AGA?CAC?AAG?CGC?AGC?CCT?GCT?GGC 298
49?Met?Pro?Glu?Glu?Ser?Trp?Lys?Met?Pro?Tyr?Asn?Asn?Arg?His?Lys?Arg?Ser?Pro?Ala?Gly 68
299?TGT?CGC?TTT?TGC?TGC?GGT?TGC?TGT?CCT?AAC?ATG?ATT?GGA?TGT?GGT?GTC?TGC?TGC?AGG?TTC 358
69?Cys?Arg?Phe?Cys?Cys?Gly?Cys?Cys?Pro?Asn?Met?Ile?Gly?Cys?Gly?Val?Cys?Cys?Arg?Phe 88
359
TGA?TTC?CTG?CTG?CAG?CCT?GGG?ATT?TAC?ACA?ACA?ACC?ACT?AAA?TAT?ATC?TTT?GTC?AAG 418
89?*** --- 89
419?ATT?TTT?ACT?TTA?AAA?GCA?GTT?TTA?CGT?CTT?TCA?GTT?GTG?GCT?GTT?AAT?ATC?TGA?GGA?TGT 478
479?TTT?TGA?ATT?TGG?TAA?TGC?TGC?AGA?GAA?TGT?GTG?CTC?ACT?GAT?GTG?ACT?GTA?TCA?TCT?ACA 538
539?CAC?ACT?ACT?ACA?GTG?TAT?TGT?CAC?
AAT?AAA?CTT?TAG?GTT?TAC?TCA?TGC?AAA?AAA?AAA?AAA 598
599?AAA
Sea farming porgy hepcidin cDNA gene clone process is as follows:
1) with reference to gene order (the Shike H of hybridizing speckle perch hepcidin, et al., Eur.J.Biochem., 2002,269:2232-2237), by synthetic special primer: the S1 (upstream primer: an initiator codon ATG leading portion sequence 24 bp (containing ATG)+2 a modified base CG): CGA AGC AGT CAA ACC CTC CTA AGA TG of optimized choice design, Al (downstream primer: 25 bp of terminator codon TGA leading portion sequence): GAA CCT GCA GCA GAC ACC ACA TCC G, with sea farming porgy liver total RNA behind the infectation of bacteria is that template is carried out the RT-PCR amplification, obtain about 300bp specific amplified cDNA band, obtain the gene fragment hepc of 287bp through order-checking; (http://www.ncbi.nlm.nih.gov/) analyzes by Blast and confirms that the 287bp gene hepc that obtains has the coding region that comprises initiator codon in the NCBI website, with the antibacterial peptide hepcidin of hybridization speckle perch higher homology (＞85%) is arranged, and this gene deduced amino acid C-terminal also is rich in the conserved sequence of distinctive eight halfcystines of hepcidin gene family.
2) according to the synthetic special primer A2 (codon ATG downstream 105bp-82bp) of hepc fragment design that obtains as downstream primer: TGG CTC CTC CAG CTC TTG CAC CTC, utilize 5 '-CDS primer (Clontech.): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1=A, G or C and SMART II A oligo (SMART
TMRACE cDNA AmplificationKit, Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG is template as upstream primer with porgy liver total RNA behind the infectation of bacteria, carries out 5`race; Amplify a treaty 200bp cDNA band from the porgy hepatic tissue, order-checking obtains one and comprises 5 ' UTR and a part of coding region at the hepc5` of interior 202bp end cDNA segment.
3) utilize specificity upstream primer S1 and Oligo dT-3sites Adaptor Primer (3 '-Full RACE CoreSet, TaKaRa Bio), with porgy liver total RNA behind the infectation of bacteria is template, carry out 3`race and pcr amplification and go out the cDNA band of about 500bp size, it is 530bp 3` end cDNA segment that order-checking obtains liver hepcidin sequence, comprises initiator codon at interior complete hepcidin coding region and 3 ' UTR.The sequencing result of 5 ' race and 3 ' race is proofreaded through sequence assembly and base, finally obtained the porgy total length hepcidin cDNA of 601bp.
The present invention is a template to attack malicious porgy liver total RNA, gene order with reference to hybridization speckle perch hepcidin, Auele Specific Primer by the optimized choice design, utilize RT-PCR, 5`race and 3`race equimolecular biology techniques means, successfully be cloned into sea farming porgy hapcidin cDNA full length sequence, this gene belongs to the new group member of hepcidin gene family.The hepcidin gene order homology of the hybridization speckle perch that its precursor sequence of this gene and Shike H etc. deliver is higher than 85%, and deduced amino acid all is rich in the conserved sequence of eight halfcystines on C-terminal mature peptide same position, the signal amino acid sequence peptide point of contact of both predictions, structural domain point of contact are all consistent, respectively at " SSA ", " RHKR " sequence place.But from deduced amino acid (as follows: Hepc is the sea farming porgy, and Bass is hybridization speckle perch)
Hepc：MKTFSVAVAVAVVLTFICLQESSAASVTEVQELEEPMSNGSPVAAHEEMPEESWKMPYNN?60
Bass：MKTFSVAVAVAVVLAFICLQESSAVPVTEVQELEEPMSNEYQEMPVE-----SWKMPYNN?55
Hepc：61?RHKRSPAG--CRFCCGCCPNMIGCGVCCRF?88
Bass：56?RHKRHSSPGGCRFCCNCCPNMSGCGVCCRF?85
Relatively find with the hybridization speckle perch aminoacid sequence of external report, the present invention clone's porgy precursor protein sequence contains 88 amino acid, 85 amino acid of hybridization speckle perch, both mainly show the mature peptide sequence at amino acid difference, have 8 amino acid differences; Compare with the antibacterial peptide of other kind, the every other kind antibacterial peptide of finding before the Hepc sequence is different from, has particular structure, this antibacterial peptide not only has antimycotic and Gram-positive, the activity of negative bacterium, but also be a kind of and the closely-related important iron adjusting of iron metabolism hormone, can set up the engineering strain that efficiently expresses hepcidin with structure carrier for expression of eukaryon such as pichia spp, external mass production porgy hepcidin antibacterial peptide, as some microbiotic in the alternative conventional feed of feed immunity additive, be used for mariculture industry, 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 genetically engineered fish.
Description of drawings
Fig. 1 is for extracting the liver total rna electrophorogram of attacking malicious porgy.
Fig. 2 is the RT-PCR electrophorogram.
Fig. 3 is the 5`race electrophorogram.
Fig. 4 is the 3`race electrophorogram.
Embodiment
Embodiment:
1. attack the extraction of malicious porgy liver RNA
The extraction of the total RNA of porgy hepatic tissue
Get the porgy hepatic tissue of 50mg and put into mortar respectively, add liquid nitrogen and pulverize, go in the eppendorf pipe of 1.5mL nuclease free (DEPC processing), add 1mLTrizol, abundant mixing, room temperature leaves standstill 5min; 4 ℃ of centrifugal 10min of 12000g discard insoluble agglomerate; Add the 0.2mL chloroform, concussion mixing, the static 2min of room temperature; 4 ℃ of centrifugal 15min of 12000g get the upper strata water in new eppendorf pipe; Add the 0.5mL Virahol, room temperature leaves standstill 10min behind the mixing; 4 ℃ of centrifugal 10min of 12000g abandon supernatant; Add 1mL 75% washing with alcohol precipitation, 4 ℃ of centrifugal 10min of 7500g; Abandon clear liquid, drying at room temperature 10min; Promptly get total RNA extracting solution with nuclease free water (DEPC treating water) dissolution precipitation.Survey OD
260nm/ OD
280nmAnd OD
260nm/ OD
230nm, on 1% agarose gel electrophoresis, analyze (see figure 1).
2.RT-PCR amplification purpose fragment hepc
2.1 cDNA first chain is synthetic: RT-PCR
Gene order (Shike H with reference to hybridization speckle perch hepcidin, et al., Eur.J.Biochem., 2002,269:2232-2237), by synthetic special primer: the S1 (upstream primer: an initiator codon ATG leading portion sequence 24 bp (containing ATG)+2 a modified base CG): CGA AGC AGT CAA ACC CTC CTA AGA TG of optimized choice design, A1 (downstream primer: 25 bp of terminator codon TGA leading portion sequence): GAA CCT GCA GCA GAC ACC ACA TCC G, get total RNA 1～3 μ g, add downstream primer A1 10pmol, add DEPC water to 10 μ L, hatch 5min for 70 ℃, place 5min on ice immediately; Add following reagent: 5 * AMV buffer5.0 μ L, _, 10mM each dNTPs 1 μ L, RNasin (40U/ μ L) 0.5 μ L, AMV ThermoScript II (10U/ μ L) 3.0 μ L, adding DEPC treating water to total reaction volume is 25 μ L; Hatch the 60min reverse transcription for 42 ℃; Hatch 5min for 95 ℃, deactivation AMV ThermoScript II places on ice.
2.2 PCR reaction
The 1 μ L RT-PCR reaction solution of getting 2.1 reactions adds 10 * Mg respectively as template
2+Free buffer2.5 μ L, MgCl
2(25mM) 2.0 μ L, 10mM each dNTPs 0.25 μ L, each 10pmol of upstream and downstream primer (S1, A1), Taq archaeal dna polymerase 0.7U use nuclease free water polishing to cumulative volume 25 μ L.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec, 60 ℃ of renaturation 30sec, 72 ℃ are extended 60sec, 30 circulations; 72 ℃ are continued to extend 5min and finish reaction.Obtain about 300bp specific amplified cDNA band (see figure 2), obtain the gene fragment hepc of 287bp through order-checking; In the NCBI website (http://www.ncbi.nlm.nih.gov/) but analyze to confirm the 287bp gene hepc continuous programming code that obtains by Blast, be higher than 85% with the antibacterial peptide hepcidin homology of hybridization speckle perch, and this gene deduced amino acid C-terminal also is rich in the conserved sequence of distinctive eight halfcystines of hepcidin gene family; For obtaining this full length gene cDNA sequence, carry out 3 ', 5 ' race.
3.5′RACE
3.1 reverse transcription reaction
According to the synthetic special primer A2 (codon ATG downstream 105bp-82bp) of hepc fragment design that obtains as downstream primer: TGG CTC CTC CAG CTC TTG CAC CTC, utilize 5 '-CDS primer ((Clontech.)): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1=A, G or C and SMART II A oligo (SMART RACE cDNA AmplificationKit, Clontech.): (5 ') AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG is as upstream primer, get total RNA～1 μ g, 1.0 μ L5 '-CDS primer and 1.0 μ L SMART II A oligo, add the DEPC treating water to final volume 25 μ L, hatch 2min for 70 ℃, place immediately on ice; Add following reagent more successively: 5 * First-StrandBuffer2.0 μ L, DTT (20mM) 1.0 μ L, dNTP (10mM) 1.0 μ L, PowerScript ReverseTranscriptasel.0 μ L are to cumulative volume 10 μ L.Hatch 1.5hr for 42 ℃ and carry out reverse transcription reaction, add Tricine-EDTA Buffer 20 μ L again and dilute the first chain reaction product, hatch the 7min inactivator for 72 ℃, 5 ℃, 5min places on ice.
3.2 PCR reaction
Utilizing UPM (Clontech.): (5 ') CTA ATA CGA CTC ACT ATA GGG CAA CGC AGA GT and A2 advance PCR.Get 3.1 reaction product, 2.5 μ L, the first chain reaction product as template, add 10 * Advantage PCR, 5.0 μ L, dNTP (10mM) 1.0 μ L, 50 * Advantage Polymerase mix, 1.0 μ L, UMP (2.0 μ M) 1.0 μ L, A2 10pmol, with nuclease free water polishing to 50 μ L.94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30sec, 72 ℃ of 2min, 5 circulations; 94 ℃ of 30s, 70 ℃ of 30s, 72 ℃ of 2min, 5 circulations; 94 ℃ of 30s, 68 ℃ of 30s, 72 ℃ of 2min, 25 circulations; 72 ℃ are continued to extend 5min.Amplification obtains about 200bp cDNA band (see figure 3), check order the hepc5` end cDNA segment of 202bp.
4.3′-RACE
4.1 reverse transcription reaction
Get total RNA 1～3 μ g, add in the centrifuge tube of 0.2mL, 70 ℃ of incubation 10min place on ice immediately.In last pipe, add 10 * RNA PCR Buffer2.0 μ L, MgCl
2(25mM) 4.0 μ L, dNTPs (10mM) 2.0 μ L, RNasin (40U/ μ L) 0.5 μ L, Oligo dT-3sites Adaptor Primer (2.5 μ M) 1.5 μ L, AMV ThermoScript II (10U/ μ L) 1.0 μ L are 20 μ L with water polishing to the cumulative volume of nuclease free.30 ℃ of annealing 10min; 42 ℃ are extended 50min; 95 ℃, the 5min inactivator; 5 ℃, 5min places on ice.
4.2 PCR reaction
Get the step reaction product 1 μ L first chain reaction liquid as template, add 10 * Mg respectively
2+Free buffer2.5 μ L, MgCl
2(25mmol/L) 2 μ L, 10mM each dNTPs 0.25 μ L, 3sites Adaptor Primer 5pmol, S1 (CGA AGC AGT CAA ACC CTC CTA AGA TG) 5pmol, Taq archaeal dna polymerase (1.0U/ μ L) 0.7U is with water polishing to the 25 μ L of nuclease free.94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are continued to extend 5min.Amplification obtains the cDNA band (see figure 4) about 500bp; It is 530bp 3` end cDNA segment that order-checking obtains liver hepcidin sequence.
5. 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).
Claims (7)
1. the hepcidin antibacterial peptide gene of sea farming porgy, it is characterized in that said sea farming porgy hepcidin antibacterial peptide gene cDNA has 586nt (not comprising poly (A)), wherein contain 5 ' the end long 94nt of non-coding region (5 ' UTR), read the long 264nt of frame for 1,3 ' the non-coding translation head of district 243nt (3 ' UTR), its GenBank series number is AY557619, and full length cDNA sequence is as follows:
10 20 30 40 50 60
aaccatcaga?caggagaaga?agtgaaagga?gctgacgaga?gtcaccaaaa?gaatcaaagg
70 80 90 100 110 120
actgaacaag?ttaaagcagt?caaagcctcc?taag
atgaag?acattcagtg?ttgcagttgc
130 140 150 160 170 180
agtggccgtc?gtgctcacct?ttatttgcct?tcaggagagc?tctgctgcct?cagttactga
190 200 210 220 230 240
ggtgcaagag?ctggaggagc?caatgagcaa?tggcagtcca?gttgctgcac?atgaagagat
250 260 270 280 290 300
gccagaggag?tcctggaaga?tgccgtataa?caacagacac?aagcgcagcc?ctgctggctg
310 320 330 340 350 360
tcgcttttgc?tgcggttgct?gtcctaacat?gattggatgt?ggtgtctgct
370 380 390 400 410 420
ctgcagcctg?ggatttacac?aacaaccact?aaatatatct?ttgtcaagat
430 440 450 460 470 480
ttttacttta?aaagcagttt?tacgtctttc?agttgtggct?gttaatatct?gaggatgttt
490 500 510 520 530 540
ttgaatttgg?taatgctgca?gagaatgtgt?gctcactgat?gtgactgtat?catctacaca
550 560 570 580 590 600
cactactaca?gtgtattgtc?ac
aataaact?ttaggtttac?tcatgcaaaa?aaaaaaaaaa
601
a
2. the hepcidin antibacterial peptide gene of sea farming porgy as claimed in claim 1 is characterized in that the hepcidin antibacterial peptide cDNA predictive coding Argine Monohydrochloride sequence of said sea farming porgy is as follows:
10 20 30 40 50 60
MKTFSVAVAV?AVVLTFICLQ?ESSAASVTEV?QELEEPMSNG?SPVAAHEEMP?EESWKMPYNN
70 80 88
RHKRSPAGCR?FCCGCCPNMI?GCGVCCRF*
3. the hepcidin antibacterial peptide gene of sea farming porgy as claimed in claim 1 is characterized in that antibiotic cDNA reading frame of hepcidin and the predicted protein aminoacid sequence of said sea farming porgy is as follows:
1 A?ACC?ATC?AGA?CAG?GAG?AAG?AAG?TGA?AAG?GAG?CTG?ACG?AGA?GTC?ACC?AAA?AGA?ATC?AAA 58
59?GGA?CTG?AAC?AAG?TTA?AAG?CAG?TCA?AAG?CCT?CCT?AAG?
ATG?AAG?ACA?TTC?AGT?GTT?GCA?GTT 118
1 Met?Lys?Thr?Phe?Ser?Val?Ala?Val 8
119?GCA?GTG?GCC?GTC?GTG?CTC?ACC?TTT?ATT?TGC?CTT?CAG?GAG?AGC?TCT?GCT?GCC?TCA?GTT?ACT 178
9?Ala?Val?Ala?Val?Val?Leu?Thr?Phe?Ile?Cys?Leu?Gln?Glu?Ser?Ser?Ala?Ala?Ser?Val?Thr 28
179?GAG?GTG?CAA?GAG?CTG?GAG?GAG?CCA?ATG?AGC?AAT?GGC?AGT?CCA?GTT?GCT?GCA?CAT?GAA?GAG 238
29?Glu?Val?Gln?Glu?Leu?Glu?Glu?Pro?Met?Ser?Asn?Gly?Ser?Pro?Val?Ala?Ala?His?Glu?Glu 48
239?ATG?CCA?GAG?GAG?TCC?TGG?AAG?ATG?CCG?TAT?AAC?AAC?AGA?CAC?AAG?CGC?AGC?CCT?GCT?GGC 298
49?Met?Pro?Glu?Glu?Ser?Trp?Lys?Met?Pro?Tyr?Asn?Asn?Arg?His?Lys?Arg?Ser?Pro?Ala?Gly 68
299?TGT?CGC?TTT?TGC?TGC?GGT?TGC?TGT?CCT?AAC?ATG?ATT?GGA?TGT?GGT?GTC?TGC?TGC?AGG?TTC 358
69?Cys?Arg?Phe?Cys?Cys?Gly?Cys?Cys?Pro?Asn?Met?Ile?Gly?Cys?Gly?Val?Cys?Cys?Arg?Phe 88
359
TGA?TTC?CTG?CTG?CAG?CCT?GGG?ATT?TAC?ACA?ACA?ACC?ACT?AAA?TAT?ATC?TTT?GTC?AAG 418
89?*** 89
419?ATT?TTT?ACT?TTA?AAA?GCA?GTT?TTA?CGT?CTT?TCA?GTT?GTG?GCT?GTT?AAT?ATC?TGA?GGA?TGT 478
479?TTT?TGA?ATT?TGG?TAA?TGC?TGC?AGA?GAA?TGT?GTG?CTC?ACT?GAT?GTG?ACT?GTA?TCA?TCT?ACA 538
539?CAC?ACT?ACT?ACA?GTG?TAT?TGT?CAC?
AAT?AAA?CTT?TAG?GTT?TAC?TCA?TGC?AAA?AAA?AAA?AAA 598
599?AAA
4. the hepcidin antibacterial peptide gene of sea farming porgy as claimed in claim 1 is characterized in that said special upstream primer S1 is an initiator codon ATG leading portion sequence 24 bp (containing ATG)+2 a modified base CG.
5. the hepcidin antibacterial peptide gene of sea farming porgy as claimed in claim 1 is characterized in that said special downstream primer A1 is 25 bp of terminator codon TGA leading portion sequence.
6. the hepcidin antibacterial peptide gene of sea farming porgy as claimed in claim 1 is characterized in that said special primer A2 is codon ATG downstream 105bp-82bp.
7, the cloning process of the hepcidin antibacterial peptide gene of sea farming porgy is characterized in that the steps include:
1) by synthetic special primer: the S1 (upstream primer) of optimized choice design: CGA AGC AGT CAA ACC CTC CTA AGATG, A1 (downstream primer): GAA CCT GCA GCA GAC ACC ACA TCC G, with sea farming porgy liver total RNA behind the infectation of bacteria is that template is carried out the RT-PCR amplification, obtains the specific amplified cDNA band hepc of 287bp;
2) according to the synthetic special primer A2 of hepc fragment design that obtains as downstream primer: TGG CTC CTC CAG CTC TTGCAC CTC, utilize 5 '-CDS primer (Clontech.): 5 '-(T
25) N
-1N-3 ', N=A, C, G or T; N
-1=A, G or C and SMART II A oligo (SMART
TMRACE cDNA Amplification Kit, Clontech.): (5 ') AAG CAGTGG TAT CAA CGC AGA GTA CGC GGG is template as upstream primer with porgy liver total RNA behind the infectation of bacteria, carries out 5`race; From the porgy hepatic tissue, amplify one and comprise 5 ' UTR and a part of coding region at the hepc5` of interior 202bp end cDNA segment;
3) utilize specificity upstream primer S1 and Oligo dT-3sites Adaptor Primer (3 '-Full RACE CoreSet, TaKaRa Bio), with porgy liver total RNA behind the infectation of bacteria is template, carry out 3`race and pcr amplification and go out 530bp 3` end cDNA segment, comprise initiator codon at interior complete hepcidin coding region and 3 ' UTR, the sequencing result of 5 ' race and 3 ' race is proofreaded through sequence assembly and base, finally obtained the porgy total length hepcidin cDNA of 601bp.
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| CN 200410090296 CN1624127A (en) | 2004-11-03 | 2004-11-03 | Hepcidin antibacterial peptide gene of genuine porgy cultured in sea water |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410090296 CN1624127A (en) | 2004-11-03 | 2004-11-03 | Hepcidin antibacterial peptide gene of genuine porgy cultured in sea water |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100402552C (en) * | 2005-12-29 | 2008-07-16 | 中山大学 | Antibiotic peptide and its code sequence and use |
| CN101063145B (en) * | 2007-04-20 | 2010-04-14 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
| CN103224893A (en) * | 2013-05-10 | 2013-07-31 | 国家海洋局第三海洋研究所 | Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof |
| CN103755795A (en) * | 2013-03-18 | 2014-04-30 | 华南师范大学 | Saddletail grouper antimicrobial peptide LEAP-2 gene, vector, recombinant strain and protein, and application thereof |
| CN105985443A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Morone saxatilis-like antimicrobial peptide sb-Ml-7 |
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2004
- 2004-11-03 CN CN 200410090296 patent/CN1624127A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100402552C (en) * | 2005-12-29 | 2008-07-16 | 中山大学 | Antibiotic peptide and its code sequence and use |
| CN101063145B (en) * | 2007-04-20 | 2010-04-14 | 厦门大学 | Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method |
| CN103755795A (en) * | 2013-03-18 | 2014-04-30 | 华南师范大学 | Saddletail grouper antimicrobial peptide LEAP-2 gene, vector, recombinant strain and protein, and application thereof |
| CN103755795B (en) * | 2013-03-18 | 2015-10-28 | 华南师范大学 | Epinephelus coioides antimicrobial petide LEAP-2 gene, carrier, recombinant bacterial strain and albumen and application thereof |
| CN103224893A (en) * | 2013-05-10 | 2013-07-31 | 国家海洋局第三海洋研究所 | Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof |
| CN103224893B (en) * | 2013-05-10 | 2015-09-23 | 国家海洋局第三海洋研究所 | A kind of large yellow croaker antibacterial peptide hepcidin gene yeast expression product and preparation method thereof and application |
| CN105985443A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Morone saxatilis-like antimicrobial peptide sb-Ml-7 |
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