CN1778920A - Antibiotic peptide gene and its yeast expression carrier - Google Patents
Antibiotic peptide gene and its yeast expression carrier Download PDFInfo
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Abstract
A scophthalmus maximus peptide hepcidin gene and its yeast expression vector, the method is to separate and clone Scophthalmus maximus peptide cDNA and it's gene group's gene, regroup this gene's mature peptides to yeast cell expression vector-pGAPZB, then to form confluent expression vector-pGAPZB-the6his, and conversion Pichia pastoris strain SMD1168, and get the yeast plant that can express mature peptide albumen steadily, this peptide gene can be considered to finish expression by passing the SDS-PAGE and Western blot test. This invention clone Scophthalmus maximums peptide gene and form mature yeast expression vector in the cell at the first time, it can be used as fish and other aquatic animal's feed additive by yeast form directly, and it don't request to separate and purificate the regroup peptide. The invention also can be used to clone many fish's peptidegene and its expression vector, and used to prevention and cure fish and other aquatic animal's disease.
Description
Affiliated technical field:
The invention belongs to the fish genetic engineering technique in the biological technical field, is a kind of fish antibacterial peptide genes that can use in the fish diseases control and the method for production of yeast recombinant expression vector thereof.
Background technology:
Generally use along with antibiotic, the resistance problem of pathogenic bacterium more and more receives publicity, and research and development novel antibacterial medicine is extremely urgent.Fish are specific immunity and non-specific immunity and the vertebrates of depositing.In the defensive raction that fish stimulate to external world and causal organism is attacked, non-specific immunity plays an important role.Antibacterial peptide is the non-specific immunity factor that a class can be resisted multiple pathogenic bacterial infection.Discover: antibacterial peptide plays an important role in fish, shrimp opposing pathogen infection process.There are many kinds of antibacterial peptide genes from different types of fish body, to obtain.As important fish natural immune system forming member, resist play a significant role when outside pathogen stimulates (Chen SL, et al., 2005) fish.Under the situation of present disease-resistant vaccine shortage, such material possesses antibiotic as an alternative great potential.
The liver of fish, skin, the cheek, head-kidney, intestines and skin secretion be the antibacterial peptide material mainly where.The fish antibacterial peptide that has been separated to at present mainly contains: class histone class antibacterial peptide (Fernandes JMO, 2004), Mi sgurnin (Park, CB., 1997), Plerocidin (Pat rzykat A., 2003), Moronecidin (Lauth, X., 2002), Hepcidin (Shike, H., 2002; Douglas, SE., 2003; Chen SL., 2005) etc.But the fish antibacterial peptide genes of finding and cloning is less.
Though the ratio that the fish antibacterial peptide genes engineering is carried out is later, has all obtained sizable achievement.Have and studies confirm that: the ditch catfish parental generation of cad gene cecro pi n B and the ability of filial generation anti-microbial pathogen E.ictaluri and Fla vobacterium columnare obviously improve (DunhamRA., 2002).After antibacterial peptide B and P1 are changed over to salmon embryo CHSE-214 clone, can detect genetically modified existence, express isolating product simultaneously and have very strong anti-microbial activity (Sarmasik A., et al, 2003) with RT-PCR and Southern hybridization.Relevant fish antibacterial peptide genes recombinant expression vector makes up and the expression study in yeast, does not still have report at present.
Summary of the invention:
The objective of the invention is in order to make up fish antibacterial peptide (Hepcidin) recombinant yeast expression vector, foundation is the technology of gene recombination antibacterial peptide yeast as the fish feed additive, for the disease-resistant mechanism research of fish and disease control provide genetic material and technological method.
Its technology contents of the present invention is as follows: one, the clone of antibiotic peptide (hepcidin) gene; Two, the structure of reorganization hepcidin antibacterial peptide gene Yeast expression carrier and the expression in yeast.
One, the clone of antibiotic peptide (hepcidin) gene: comprise acquisition, genomic dna sequence analysis and the comparison of cDNA clone, genomic dna sequence.
1, the amplification of cDNA and clone: utilize the TRIzol test kit from the turbot liver, to extract total RNA, utilize the MMLV reverse transcription to obtain cDNA article one chain.According to the homology design degenerated primer of the aminoacid sequence of different plant species hepcidin, amplification obtains the cDNA total length encoder block of turbot hepcidin gene.Sequential analysis shows turbot hepcidin cDNA total length 286bp, the protein of a long 90aa of coding.This protein precursor peptide includes one 24 amino acid whose signal peptide, one 40 amino acid whose leading peptide and one 26 amino acid whose mature peptide.This mature peptide zone includes 8 half light amino acid residues, meets the characteristic feature of such antibacterial peptide.
2. the acquisition of genomic dna sequence:, obtain including the turbot hepcidin gene order of intron with a pair of genomic dna that comes from the primer amplified turbot of cDNA.
3, dna sequence analysis and comparison: sequencing shows this genomic dna total length 571bp, includes 2 introns and is respectively 114nt and 172nt.The dna sequence dna of having deposited in cDNA and genomic dna sequence and the U.S. GENBANK sequence library is compared, calculate the homology of the dna sequence dna of having deposited in cDNA cloned sequence that we obtain and the GENBANK sequence library with BLASTN and BLASTX program.
Two, the structure of antibiotic peptide gene yeast intracellular fusion expression carrier and expression in yeast: comprise that the structure of expression vector, expression vector express in a small amount to the conversion of yeast cell, the screening that contains the recombination yeast of antibiotic peptide gene, recombinant antibacterial peptide.
1. the structure of Yeast expression carrier:
Sequence according to the antibiotic peptide mature peptide gene that determines, design a pair of special primer that contains suitable restriction enzyme site (EcoRI and Xba I), be respectively pcTBHN1 (5 '-ATCGAATTCATGCAATCCCACATCTCCCTTTGC-3 ') and pcTBHC1 (5 '-GCAAGTCTCTAGAAACTTGCAGCAGAAACCACA-3 ').Amplification antibacterial peptide mature peptide sequence is connected to after enzyme is cut with expression vector pGAPZB in the yeast born of the same parents of same enzymic digestion from the cDNA that liver extracts, and is built into antibiotic peptide gene yeast born of the same parents endomixis type expression vector.
2. expression vector is to the conversion of yeast cell:
The antibiotic peptide Hepcidin expression vector pGAPZB-thep6his of preparation is in a large number cut with the BlnI enzyme, make its linearizing, by electroporation method linearizing antibacterial peptide gene expression plasmid of yeast is transformed in the pichia pastoris SMD1168 cell, cultivates 1-2 days screening positive transformants in 30 ℃.
3. contain the screening of the recombination yeast of antibiotic peptide gene:
By using special primer to carry out PCR, further confirm yeast colony that screened, that integrated the external source antibacterial peptide gene.Used PCR primer is pGAPF (5 '-gtccctatttcaatcaattgaa-3 ') and pGAPR (5 '-gcaaatggcattctgacatcc-3 ').
4. recombinant antibacterial peptide is expressed in a small amount:
The positive single bacterium colony of picking is inoculated in and carries out enlarged culturing in the YPD substratum.After cultivate finishing, with nutrient solution with maximum velocity centrifugation 5-10min.With supernatant and centrifugal after bacterium liquid be put in-80 ℃ standby.Get an amount of thalline, add the SCE digestion solution that contains helicase, 37 ℃ digested 3 hours.The centrifuging and taking supernatant carries out the protein electrophorese analysis.
The present invention and prior art contrast are characterized in: the present invention is that material has made up fish antibacterial peptide (hepcidin) gene fusion type Yeast expression carrier first with the turbot, and realized that born of the same parents' endomixis type expresses the antibacterial peptide material, suitable fodder additives as aquatic animals such as fish is used for the diseases prevention and treatment of aquatic animals such as fish. and technological method of the present invention can be used on other fish.
Description of drawings:
Fig. 1: turbot Hepeidin gene and deduced amino acid;
Fig. 2: antibiotic peptide Hepeidin expression vector;
Embodiment:
Clone and expression vector establishment with antibiotic peptide hepcidin gene is example below, in conjunction with the accompanying drawings, technology contents of the present invention is elaborated.
Its technology contents of the present invention is as follows: one, the clone of antibiotic peptide (hepcidin) gene; Comprise amplification, genomic dna sequence analysis and the comparison of cDNA clone, genomic dna sequence.Two, the structure of reorganization hepcidin antibacterial peptide gene Yeast expression carrier and the expression in yeast: comprise that the structure of expression vector, expression vector express in a small amount to the conversion of yeast cell, the screening that contains the recombination yeast of antibiotic peptide gene, recombinant antibacterial peptide.
One, the clone of antibiotic peptide hepcidin gene:
1.cDNA amplification and clone: utilize the TRIzol test kit from the turbot liver, to extract total RNA, utilize the MMLV reverse transcription to obtain cDNA article one chain.According to the amino acid identity design degenerated primer of the known hepcidin sequence of different sorts fish, amplification obtains the full length coding region of hepcidin.Sequencing is found, cDNA total length 286bp, the protein of a long 90aa of coding.This protein precursor peptide includes one 24 amino acid whose signal peptide, one 40 amino acid whose leading peptide and one 26 amino acid whose mature peptide.This mature peptide zone includes 8 half light amino acid residues, meets the characteristic feature of such antibacterial peptide.
2. the amplification of genomic dna sequence:, obtain including the turbot hepcidin gene order of intron with a pair of genomic dna that comes from the special primer amplification turbot of cDNA.Sequencing is thought and is included 2 introns by this genomic dna (see figure 1) total length 571bp, is respectively 114nt and 172nt.
3.DNA sequential analysis and comparison: the dna sequence dna of having deposited in the sequence of cDNA and genomic dna and the U.S. GENBANK sequence library is compared, identify common sequences and unique sequence with PHRAP cluster program; Calculate the homology of the dna sequence dna of having deposited in cDNA cloned sequence that we obtain and the GENBANK sequence library with BLASTN and BLASTX program.
Two, the structure of reorganization hepcidin antibacterial peptide gene Yeast expression carrier and the expression in yeast:
1. the structure of Yeast expression carrier: according to the sequence of the antibiotic peptide mature peptide gene that determines, design contains the special primer of suitable restriction enzyme site, amplification antibacterial peptide mature peptide sequence from the cDNA that the turbot liver extracts, after enzyme is cut, be connected to Yeast expression carrier pGAPZB, be built into the fish antibacterial peptide genes expression plasmid of yeast with same enzymic digestion.For the turbot hepcidin gene mature peptide sequence that increases, characteristics according to expression vector pGAPZB have designed a pair of specific primer, are respectively pcTBHN1 (5 '-atcgaattcatgcaatcccacatctccctttgc-3 ') and pcTBHC1 (5 '-gcaagtctctagaaacttgcagcagaaaccaca-3 ').Primer sequence is on the basis that does not change hepcidin mature peptide coded amino acid, suddenly change according to the codon of zymic codon preference to partial amino-acid, on upstream primer pcTBHN1 and downstream primer pcTBHC1, introduced simultaneously EcoRI and XbaI enzyme cutting site respectively, on N end primer pcTBHN1, introduced and transcribed atg start codon ATG.The C end primer of design sports TTT to last codon TTC of hepcidin mature peptide, make the sequence in XbaI site of carrier become (ctaga) through after suitable enzyme cuts simultaneously by (tctaga), be connected with pGAPZB carrier, produce antibiotic peptide gene expression vector pGAPZB-thep6his (see figure 2) with same enzymic digestion.
2, expression vector is to the conversion of yeast cell: the antibiotic peptide Hepcidin gene yeast expression vector pGAPZB-thep6his that will prepare in a large number cuts with the BlnI enzyme, make its linearizing, by electroporation method linearizing antibacterial peptide gene expression plasmid of yeast is transformed in the pichia pastoris SMD1168 cell, containing on the antibiotic YPDS flat board of 300 μ g/ml Zeocin, cultivating 1-2 days screening positive transformants for 30 ℃.
3, contain the screening of the recombination yeast of antibiotic peptide gene: carry out PCR by using special primer, further confirm yeast colony that screened, that integrated the external source antibacterial peptide gene.Used PCR primer is pGAPF (5 '-gtccctatttcaatcaattgaa-3 ') and pGAPR (5 '-gcaaatggcattctgacatcc-3 ').
4, recombinant antibacterial peptide is expressed in a small amount: the positive single bacterium colony of picking is inoculated in and carries out enlarged culturing in the YPD substratum.Sampling in per 24 hours.The entire operation process keeps aseptic technique.After cultivate finishing, with nutrient solution with maximum velocity centrifugation 5-10min.With supernatant and centrifugal after bacterium liquid be put in-80 ℃ standby.Get an amount of thalline, add the SCE digestion solution (ph7.0) contain helicase (30mg/ml) (the 0.9M sorbyl alcohol, the 0.1M Trisodium Citrate, 0.06M EDTA, PH8.0), 37 ℃ of digestion 3 hours.The centrifuging and taking supernatant carries out the protein electrophorese analysis.
Sequence table
＜110〉Chinese aquatic science institute Huanghai Sea aquatic products institute
＜120〉antibiotic peptide hepcidin gene and Yeast expression carrier thereof
<160>2
<170>PatentIn?version?3.1
<210>1
<211>286
<212>DNA
＜213〉turbot (Scophthalmus maximus)
<400>1
ctcaaaatga?aggcattcag?cattgcagtt?gcagtgacac?tcgtgctcgc?ctttgtttgc 60
attctggaga?gctctgccgt?cccgttccct?ggggtgcaag?agctggaaga?ggcagggagc 120
aatgacactc?cagccgcggc?acatcaagag?acgtcaatgg?aaccgtggac?ggtgccgagt 180
cacatcaggc?agaagcgaca?gagccacatc?tccctttgcc?gctggtgctg?caactgctgc 240
aaggccaaca?agggctgtgg?cttctgctgc?aagttctgag?gattcc 286
<210>2
<211>90
<212>PRT
＜213〉turbot (Scophthalmus maximus)
<400>2
Met?Lys?Ala?Phe?Ser?Ile?Ala?Val?Ala?Val?Thr?Leu?Val?Leu?Ala?Phe
1 5 10 15
Val?Cys?Ile?Leu?Glu?Ser?Ser?Ala?Val?Pro?Phe?Pro?Gly?Val?Gln?Glu
20 25 30
Leu?Glu?Glu?Ala?Gly?Ser?Asn?Asp?Thr?Pro?Ala?Ala?Ala?His?Gln?Glu
35 40 45
Thr?Ser?Met?Glu?Pro?Trp?Thr?Val?Pro?Ser?His?Ile?Arg?Gln?Lys?Arg
50 55 60
Gln?Ser?His?Ile?Ser?Leu?Cys?Arg?Trp?Cys?Cys?Asn?Cys?Cys?Lys?Ala
65 70 75 80
Asn?Lys?Gly?Cys?Gly?Phe?Cys?Cys?Lys?Phe
85 90
Claims (3)
1, a kind of antibiotic peptide hepcidin gene is characterized in that the clone of antibiotic peptide gene mainly comprising the acquisition of full-length cDNA, genomic dna, sequential analysis and comparison;
The clone of cDNA: extracting total RNA with the TRIzol test kit from the turbot liver, is article one chain of cDNA with the MMLV reverse transcription; According to the homology zone design degenerated primer of the aminoacid sequence of the hepcidin of different plant species, amplification obtains the cDNA total length encoder block of hepcidin gene;
The acquisition of genomic dna:, obtain including the hepcidin gene complete sequence of intron with a pair of primer amplified turbot genomic dna that comes from cDNA;
Dna sequence analysis and comparison: the sequence of the hepcidin gene that will deposit from cDNA sequence and GENBANK sequence library is compared, and identifies common sequences and unique sequence with PHRAP cluster program; Calculate the homology of the hepcidin gene order of having deposited in cDNA that we obtain and the GENBANK sequence library with BLASTN and BLASTX program.
2, antibiotic peptide gene according to claim 1, it is characterized in that described cDNA sequence: the cDNA total length of this gene is 286 bases, the open reading frame that comprises 273 bases, this reads 90 amino acid whose precursors of frame coding, this precursor is made up of 3 districts again, 24 signal peptides that amino acid is formed, 40 leading peptide and 26 mature peptides that amino acid is formed that amino acid is formed, this mature peptide comprises that 8 half light amino acid residue .Hepeidin genes, cDNA and deduced amino acid are:
1 CTCAAA
ATGAAGGCATTCAGCATTGCAGTTGCAGTGACACTCGTGCTCGCCTTTGTTTGCATTCTGGAGAGCTCT
M K A F S I A V A V T L V L A F V C I L E S S
76 GCCGTCCCGTTCCCTGGGgtaaggcgtgaacttttaactcacttcatttgcccatgagctataaaatgttttggt
A V P F P G
151 ttttttgtcggggtgccaagatggcggctcgctaaaacatgcacgatccgttcacagGTGCAAGAGCTGGAAGAG
V Q E L E E
226 GCAGGGAGCAATGACACTCCAGCCGCGGCACATCAAGAGACGTCAATGGAACCGTGGACGgtatgttcaattgac
A G S N D T P A A A H Q E T S M E P W T
301 tggatgaattaggccaattactgtgagcaggttcaaattcaagtggcggcgttttccccacagagcagcctggcg
376 ctgtctccacgcagggggtggggggtgatggaatgctggggagagaaaaacgcctcgttcacgtctcttgtctcc
451 cacacagGTGCCGAGTCACATCAGGCAGAAGCGACAGAGCCACATCTCCCTTTGCCGCTGGTGCTGCAACTGCTG
V P S H I R Q K R Q S H I S L C R W C C N C C
526 CAAGGCCAACAAGGGCTGTGGCTTCTGCTGCAAGTTC
TGAGGATTCC
K A N K G C G F C C K F *
3. a Yeast expression carrier is characterized in that the structure of described born of the same parents' endomixis type expression vector and the expression in yeast thereof, and its method is:
The structure of expression vector: according to the antibiotic peptide gene sequence that determines, design a pair of gene specific primer, pcTBHN1 (5 '-ATCGAATTCATGCAATCCCACATCTCCCTTTGC-3 ') and pcTBHC1 (5 '-GCAAGTCTCTAG AAACTTGCAGCAGAAACCACA-3 '), amplification antibiotic peptide Hepcidin mature peptide sequence, after enzyme is cut, be connected to expression vector pGAPZB in the yeast born of the same parents of same enzymic digestion, be built into antibiotic peptide gene yeast born of the same parents endomixis type expression vector;
Expression vector is to the conversion of yeast cell: the antibiotic peptide Hepcidin expression vector pGAPZB-thep6his that will prepare in a large number cuts with suitable enzyme, make its linearizing, by electroporation method linearizing antibacterial peptide gene expression plasmid of yeast is transformed in the pichia pastoris SMD1168 cell, cultivates 1-2 days screening positive transformants in 30 ℃;
Contain the screening of the recombination yeast of antibiotic peptide gene: carry out PCR by using special primer, the yeast colony of external source antibacterial peptide gene has been integrated in screening,
Used PCR primer is: pGAPF (5 '-gtccctatttcaatcaattgaa-3 ') and pGAPR (5 '-gcaaatggcattctgacat cc-3 ');
Recombinant antibacterial peptide is expressed in a small amount: the positive single bacterium colony of picking is inoculated in and carries out enlarged culturing in the YPD substratum; After cultivate finishing, with nutrient solution with maximum velocity centrifugation 5-10min; With supernatant and centrifugal after bacterium liquid be put in-80 ℃ standby; Get an amount of thalline, add the SCE digestion solution that contains helicase, 37 ℃ digested 3 hours; The centrifuging and taking supernatant carries out the protein electrophorese analysis.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182360B (en) * | 2007-10-08 | 2010-07-07 | 国家海洋局第一海洋研究所 | Fusion protein having antibiotic function and uses 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 |
| CN105732790A (en) * | 2016-05-06 | 2016-07-06 | 青岛农业大学 | Fish-derived antibacterial peptide hepcidin mutant and application thereof |
| CN111825748A (en) * | 2020-07-20 | 2020-10-27 | 大连工业大学 | Antibacterial peptide of turbot and application thereof |
| CN113583103A (en) * | 2021-08-31 | 2021-11-02 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp I (67-92) and application thereof |
| CN116425853A (en) * | 2023-05-15 | 2023-07-14 | 中国海洋大学 | Modified antibacterial peptide and application thereof |
-
2005
- 2005-09-27 CN CN 200510104202 patent/CN1778920A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182360B (en) * | 2007-10-08 | 2010-07-07 | 国家海洋局第一海洋研究所 | Fusion protein having antibiotic function and uses 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 |
| CN105732790A (en) * | 2016-05-06 | 2016-07-06 | 青岛农业大学 | Fish-derived antibacterial peptide hepcidin mutant and application thereof |
| CN111825748A (en) * | 2020-07-20 | 2020-10-27 | 大连工业大学 | Antibacterial peptide of turbot and application thereof |
| CN111825748B (en) * | 2020-07-20 | 2021-09-24 | 大连工业大学 | Antibacterial peptide of turbot and application thereof |
| CN113583103A (en) * | 2021-08-31 | 2021-11-02 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp I (67-92) and application thereof |
| CN113583103B (en) * | 2021-08-31 | 2022-05-06 | 中国科学院南海海洋研究所 | Antibacterial peptide HeHamp I (67-92) and application thereof |
| CN116425853A (en) * | 2023-05-15 | 2023-07-14 | 中国海洋大学 | Modified antibacterial peptide and application thereof |
| CN116425853B (en) * | 2023-05-15 | 2023-12-29 | 中国海洋大学 | Modified antibacterial peptide and application thereof |
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