CN103789317A - Scylla paramamosain antibacterial peptide hyastatin gene clone, encoding protein recombination and application - Google Patents
Scylla paramamosain antibacterial peptide hyastatin gene clone, encoding protein recombination and application Download PDFInfo
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Abstract
The invention relates to scylla paramamosain antibacterial peptide hyastatin gene clone, encoding protein recombination and an application. According to the invention, the gene is cloned from a scylla paramamosain haemolymph library to an antibacterial peptide hyastatin gene full-length sequence by using a cDNA (complementary Desoxvribose Nucleic Acid) library building technology and rapid amplification technology of 3'and 5'ends; the gene plays an important role in immune defence of the scylla paramamosain; due to adoption of an in-vitro recombinant expression technology, mature peptide expression protein of the scylla paramamosain antibacterial peptide hyastatin gene is obtained; a molecular weight of the mature peptide expression protein is 29358.0; and an isoelectric point is 5.05. The recombinant protein has some effects of inhibiting gram-positive bacterium, gram-negative bacterium and fungus. Through the scylla paramamosain antibacterial peptide hyastatin gene, a basis of prevention and treatment of disease of scylla paramamosain, development of medicine products and application of feed additives can be established.
Description
Technical field
What the present invention relates to is the hyastatin gene clone of Scylla paramamosain antibacterial peptide and proteins encoded restructuring and purposes, belongs to technical field of molecular biology.
Background technology
Scylla paramamosain (
scylla paramamosain) be under the jurisdiction of Arthropoda (
arthropoda), Crustachia (
crustacea), Decapoda (
decapoda), Brachyura (
brachyura), Portumidae (Portunidae), mud crab belong to (
scylla), there is very high economic worth and nutritive value, be one of Chinese important sea farming kind.Because Scylla paramamosain tool feeding habits are wide, strong adaptability, fast growth, short, resistance to temperature of exposure and dry of culture-cycle be long, easily transportation and culture benefit advantages of higher, liked by raiser, is one of fisherman's Main Economic source.
Along with the breeding environment that cultivation intensity increases and goes from bad to worse, Scylla paramamosain breeding process is constantly subject to comprising the infringement (Wang Rongzhi etc., 2008) of the multiple pathogens such as bacterium, fungi and virus.Early stage at pathogen infection, can only rely on congenital nonspecific immunity as the Scylla paramamosain of invertebrates, identification and removing invading micro-organism, maintain body health (Begum et al, 2000).Wherein, antibacterial peptide is owing to having spectrum antibacterium, the antimycotic and unique mechanism of action, at crab class defence pathogenic micro-organism invasion important role (Sperstad et al, 2009).
At present, expert in crab class different tissues isolation identification multiple antibacterial peptide.1988-2000 multidigit scholar in succession in fiddler crab (
fiddler crab) in isolate
tachyplesin family antibacterial peptide, comprises
tachyplesin Iwith
tachyplesinII, sequential analysis shows that its C end is containing formamido-arginine, N end, containing Pyrrolidonecarboxylic acid, forms two disulfide linkage by four halfcystines, and to Gram-positive and negative bacterium and parasite exist certain inhibition (
nakamura T, et al.1988; Miyata T, et al, 1989; Kawano K, et al, 1990; Kawabata S, et al, 1996; Silva P I, et al, 2000);
schanpp Ddeng (
schnapp D, et al, 1996) from bank crab (
carcinus Maenas) be purified into the antibacterial peptide that a kind of proline rich molecular weight is 6.5 KDa, the similar Mammalian Antimicrobial Peptides of its sequence in hemocyte lysate
bactenecin-7, and all toxic to Gram-positive and negative bacteria; 2008
stensv gdeng (Stensv g K, et al, 2008.) in the crab of loving each other (
hys araneus) isolate in hemocyte comprise 37 amino acid whose antibacterial peptides-
arasin1, its N end proline rich and arginine, protein expression in vitro has obvious anti-microbial activity; Next year
sperstad etc. (
sperstad S V, et al, 2009) from the crab hemocyte of loving each other, be purified into the antibacterial peptide that a kind of molecular weight is 11.7 kDa-
hyastatin, its aminoacid sequence comprises 3 kinds of different zones: N end and is rich in glycine, six cysteine residues of C end tool, and centre is proline(Pro)/arginine rich region, and Gram-positive and negative bacterium and yeast are had to remarkable restraining effect; Peng H etc. from Young Crab (
scylla serrata) being separated to the antibacterial peptide scygonadin that molecular weight is 11 kDa in seminal fluid, its recombinant protein has resistance to gram-positive microorganism and negative bacterium, but to yeast and fungi non-resistant (Peng et al, 2010); Imjongjirak etc. and (Imjongjirak C et al, 2009 such as Yue; Yue F et al, 2010) respectively from Scylla paramamosain (
scylla paramamosain) and mitten crab (
portunus trituberculatus) in hemocyte separation and purification to antibacterial peptide crustin(Crus
spand PtCrustin), two kinds of antibacterial peptides all have at C end the typical conservative property cysteine residues that comprises whey acidic protein (whey acidic protein, WAP) structure, and gram-positive microorganism is had to certain restraining effect.
Antibacterial peptide
hyastatin be (2009) such as Sperstad from spider shape love each other crab (
hyas araneus) Purification a kind of all has the novel antimicrobial peptide that suppresses active to G+/G-bacterium, fungi in hemocyte, its C end structure territory and Ch-penaedin Penaeidins homology, there are 6 conservative Cys residues, form 3 pairs of intramolecular disulfide bonds, belong to Penaeidins family antibacterial peptide.Because antibacterial peptide is the natural small peptide material that a class has very strong spectrum anti-microbial activity, be very easily diffused into infection site, after use, do not exist and produce resistance problem, and enter after body without hazard residue.Therefore, the research and development of antibacterial peptide will be the important guarantee of realizing marine cultured animal healthy aquaculture, reducing microbiotic pollution with application.In marine animal, containing abundant in the world antibacterial substance, the effective substitute of microbiotic that research and development have anti-drug resistance bacterium from marine animal is by the inevitable direction that is the development such as future medicine, agricultural, pharmacy.Therefore, make full use of and excavate the antibacterial peptide resource of Chinese Sea animal, develop the antibacterial peptide gene engineering product of high-efficiency antimicrobial, as additive or the environmental friendly biological pesticide of research and development high-quality feed, to Substitute For Partial microbiotic, the Sustainable Healthy Development of China's culture fishery is had to important scientific value and application prospect.
Summary of the invention
The object of the invention is to carry out the hyastatin gene clone of Scylla paramamosain antibacterial peptide and proteins encoded restructuring from Scylla paramamosain hemolymph, another object of the present invention is open Scylla paramamosain antibacterial peptide hyastatin gene clone and proteins encoded restructuring purposes thereof, for research Scylla paramamosain innate immunity mechanism provides science and technology support, for further understanding Hyastatin gene and the effect in immunne response thereof, for immunity and the control and prevention of disease of Scylla paramamosain are provided fundamental basis, and lay the foundation for developing pharmaceutical prod and fodder additives.
Foregoing invention object is realized by following technical proposals: the hyastatin gene clone of Scylla paramamosain antibacterial peptide and proteins encoded restructuring and purposes, described Scylla paramamosain antibacterial peptide Hyastatin gene, there is base sequence in sequence table SEQ ID NO.1, this gene is the expressed sequence tag screening from Scylla paramamosain hemolymph cDNA library, this sequence total length 837 bp, comprise the open reading frame (ORF) of 396 bp, 131 amino acid of encoding, the 5' non-coding head of district 105 bp, the 3' non-coding head of district 336 bp, there are polyadenylic acid tailing signal and polyadenylic acid tail,
Described Scylla paramamosain antibacterial peptide Hyastatin gene coded protein, there is the aminoacid sequence shown in SEQ ID NO.2, this molecular weight of albumen is 70547.7KDa, iso-electric point is 5.04, wherein the 106-153 of encoding sequence is signal peptide sequence, mature peptide molecular weight is 29358.0, and iso-electric point is 5.05.Amino acid has the peculiar region of Penaeidins family: N end for Pro/Arg district, and C end contains 6 Cys, forms 3 intramolecular disulfide bonds;
The expression product of described Scylla paramamosain antibacterial peptide hyastatin gene clone and proteins encoded restructuring is to the micrococcus luteus in gram-positive microorganism and subtilis, cereuisiae fermentum, Candida albicans in intestinal bacteria, streptococcus aureus and fungi in Gram-negative bacteria have fungicidal activity, have pointed out the application of described expression product at preparation antibacterials, immunostimulant, fodder additives.
The present invention utilizes expressed sequence tag (EST) technology, cDNA end rapid amplifying (RACE) technology from Scylla paramamosain hemolymph, to amplify the full length sequence of Hyastatin gene.As the increase mature peptide fragment of this gene of template design primer, be connected with carrier by double digestion and build pET28a-Hyastatin fusion expression vector, utilize kalamycin resistance to realize in-vitro recombination expression in e. coli bl21 (DE3).Recombinant products after His Trap HP affinity column purifying and dialysis renaturation, to gram-positive microorganism micrococcus luteus (
miccrococcus tetragenus) and subtilis (
bacillus subtilis), Gram-negative bacteria intestinal bacteria (
escherichia coli), streptococcus aureus (
staphyloccocus aureus) and fungi in cereuisiae fermentum, Candida albicans there is obvious restraining effect.Concrete outcome is as shown in table 1, the minimal inhibitory concentration mensuration table of the Hyastatin antibacterial peptide that table 1 provides for example of the present invention;
Wherein-: asepsis growth; +: there is bacteria growing.
Table 1
The present invention can provide science and technology support for research Scylla paramamosain innate immunity mechanism, for further understanding Hyastatin gene and the effect in immunne response thereof, for immunity and the control and prevention of disease of Scylla paramamosain are provided fundamental basis, and lay the foundation for developing pharmaceutical prod and fodder additives.
Accompanying drawing explanation
Fig. 1 is Scylla paramamosain Hyastatin mature peptide gene order amplification agarose electrophoresis figure;
Fig. 2 is the Scylla paramamosain Hyastatin recombinant protein SDS-Page electrophorogram of induction and purifying.
Embodiment
By the following examples technical scheme of the present invention is further described, but the invention is not restricted to this.
As shown in Figure 1, Fig. 1 provides Scylla paramamosain Hyastatin mature peptide gene order amplification agarose electrophoresis figure, wherein, and M:DNA marker, the gene amplification fragment of 1:Hyastatin mature peptide.
As shown in Figure 2, Fig. 2 provides the Scylla paramamosain Hyastatin recombinant protein SDS-Page electrophorogram of induction and purifying, wherein M: protein marker; 1: do not induce whole bacterial protein; Whole bacterial protein after 2:IPTG induction; 3: recombinant protein after purifying.
Scylla paramamosain Hyastatin gene order has sequence shown in SEQ ID NO.1.This gene order clone comprise the following steps:
A) Scylla paramamosain artificial challenge Vibrio parahaemolyticus and hemolymph extract;
B) the total RNA of Scylla paramamosain hemolymph extracts and mRNA purifying;
C) Scylla paramamosain cDNA library construction and expression sequence label order-checking;
D) expressed sequence tag homology analysis and the screening of Hyastatin gene fragment; Adopt the amplification of RACE technology to obtain Hyastatin full length sequence.
Concrete operation step is as follows:
1) Scylla paramamosain artificial challenge Vibrio parahaemolyticus and hemolymph extract: using 1 ml asepsis injector to inject 0.3 ml concentration in Scylla paramamosain the 3rd pereiopoda base pitch membrana articulata place is 1.0 × 10
7cFU/ml Vibrio parahaemolyticus.Attacking 6 h, 12 h, 18 h, 24 h after poison respectively, use containing ACD antithrombotics (82 mmol/l glucose, 23 mmol/l citric acids, 45 mmol/l Trisodium Citrates) asepsis injector in Scylla paramamosain base pitch membrana articulata place extract hemolymph, centrifugal (800 g immediately, 5 min), obtain blood lymphocyte, extract for total RNA.
2) the total RNA of Scylla paramamosain hemolymph extracts and mRNA purifying: extract total RNA according to the operation of TRIzol Reagent specification sheets, adopt nucleic acid-protein determinator (Eppendorf, Germany) measure content and
oD 260/
oD 280value, analyzes its concentration and purity, detects integrity with 1.2% agarose gel electrophoresis.After the RNA balanced mix that different time sections is collected, use PolyATtractmRNA Isolation Systems III test kit separation and purification mRNA, detection method is with total RNA.
3) Scylla paramamosain cDNA library construction and expression sequence label order-checking: according to SMARTer
tMpCR cDNA Synthesis Kit specification sheets carries out.Take the polyA+mRNA after purifying as template, 3 ' SMART CDS Primer II A (5 '-ATTCTAGAGGCCGA-GGCGGCCGACATG-d (30) N-3 ') and SMARTer II A Oligonucleotide
(5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 ') be primer, at ThermoScript II SMARTScribe
tMunder Reverse Transcriptase effect, transcribe synthetic the first chain cDNA.According to Advantage 2 PCR Kit, take the first chain cDNA as template, 5 ' PCR Primer II A (5 '-AAGCAGTGGTATCAACGCAGAGT-3 ') be primer, carry out respectively 15,18,21,24 and 27 circulations, synthetic double chain cDNA (ds cDNA) is also determined optimum cycle number.Ds cDNA, through Proteinase K (0.8 mg/ml) digestion 20 min, by Chroma SPIN-400 fractional separation, collects cDNA fragment more than 500bp.CDNA through above-mentioned processing spends the night and is connected in 4 ℃ with carrier pGEM-T easy, be converted into the competent cell DH5 α of the fresh preparation of 100 μ l, add 1 ml LB substratum, 37 ℃ of 200 rpm/min cultivates 1.5 h, be the original library of full-length cDNA ,-80 ℃ of preservations.
4) expressed sequence tag order-checking: random 300 positive colonies of picking carry out bacterium colony PCR checking, and primer is respectively M13F (5 '-GTAAAACGACGGCCAG-3 ') and M13R (5 '-CAGGAAACAGCTATGAC-3 ').Reaction system: bacterium liquid 1 μ l, 10 × PCR buffer, 1.0 μ l,
taqarchaeal dna polymerase is 1U, Mg
2+concentration is 2.0 mmol/L, and dNTP concentration is 0.2 mmol/L, and primer concentration is 0.2 μ mol/L, adds water to 10 μ l.Reaction conditions: 94 ℃ of denaturation 4 min; 94 ℃ of sex change 30 s, 55 ℃ of annealing 30 s, 72 ℃ of extension 3 min, 30 circulations; 72 ℃ are extended 10 min.PCR reaction is carried out on AG 22331 type pcr amplification instrument (Eppendorf, Germany).Pcr amplification product detects with 1.2% agarose gel electrophoresis (0.5 × TBE), and gel imaging instrument (Alpha innotech, USA) is observed and taken pictures automatically.
300 of more than random choose 500bp positive colony are delivered to the order-checking of Shanghai Sheng Gong biotechnology company limited, adopt for the first time 5 ' hold unidirectional order-checking, and after manual synchronizing, partial sequence is supplemented the order-checking of 3 ' end.
5) expressed sequence tag homology analysis and the screening of Hyastatin gene fragment: application VecScreen (http://www.ncbi.nlm. nih.gov/VecScreen/VecScreen.html), DNAstar5.0, ClustalX1.8 software are removed carrier, inferior quality, tumor-necrosis factor glycoproteins, use DNAMAN5.2.2 software splicing sequence, after hand inspection and correction, obtain contig (Contigs) and unique sequence (Singletons).Use the Blastx program of NCBI website, the single-gene group being made up of Contigs and Singletons bunch (UniGenes) obtaining is carried out to protein homology comparison and functional analysis, E-value is lower than 10
-10or there is sequence in continuous 25 amino acid and database to have 50% to be all mutually homologous gene, otherwise be agnoprotein.Result shows in est sequence has found the sequence higher with Portunus trituberculatus Miers gene similarity, has determined the est sequence of Scylla paramamosain Hyastatin gene according to similarity analysis result.
6) Hyastatin full length sequence is obtained in RACE amplification: 1. 3'RACE amplification: according to the expressed sequence tag design upstream Auele Specific Primer Primer P3-1 (5'-CTTCTCCTCTTGGTGTCCCTC-3') and the Primer P3-2(5'-AAGACAACTGACCGCAGACGA-3' that filter out in cDNA), downstream primer adopts specific primer anchor3(5'-AAGCAGTGGTATCAACGCAGAGT-3').Take the total RNA of Scylla paramamosain hemolymph as template, adopt specific primer oligodT
30-anchor(5'-AAGCAGTGGTATCAACGCAGAGTACT
(30)vN-3'), use synthetic cDNA the first chain of ThermoScript II.As template, use primer Primer P3-1 and anchor3 to carry out pcr amplification, get PCR product 1 μ l as template, then carry out PCR with primer Primer P3-2 and anchor3 and obtain 3' and hold object band.2. 5'RACE amplification: according to the expressed sequence tag design upstream Auele Specific Primer PrimerP5-1(5'-GAGGGAGACCAAGAGGAGAAGG-3' filtering out in cDNA), P5-2 (5'-TCGTGGAGCCTGTGAAGC-3') and P5-3(5'-CGTCTGCGGTCAGTTGTCTT-3'), downstream primer adopts specific primer oligodT
16-anchor(5'-GACCACGCGTATCGATGTCGACT
(16)v-3') and anchor5(5'-GACCACGCGTATCGATGTCGAC-3').After total Scylla paramamosain RNA is reacted with primer PrimerP5-1, utilize ThermoScript II to synthesize cDNA the first chain.After utilizing QIAquick PCR Puification test kit purifying, add A reaction.As template, adopt primer P5-2 and P5-3 and primer anchor5 to carry out nest-type PRC, obtain 5' end object fragment.
After PCR product detects by 1.0% agarose electrophoresis, use UNIQ-10 pillar DNA glue to reclaim test kit and reclaim purified product, be connected with pMD-18T carrier again, be transformed in competent escherichia coli cell, coat on penbritin flat board, select the positive by colony polymerase chain reaction (PCR) method and deliver to the order-checking of Shanghai Sheng Gong biotechnology company limited, acquired results obtains full length sequence through the splicing of DNAMAN software analysis.
'
rACE amplification reaction system and reaction conditions:
50 μ l reaction systems, comprise:
10×PCR buffer 5 μl
MgCl
2(25mM) 2 μl
dNTP Mix（2.5mM） 4 μl
Primer P3(10μM) 1 μl
Primer anchor3(10μM) 1 μl
(5U/ μ is 0.5 μ l l) for Taq enzyme
5×M-MLV buffer 2 μl
Rnase free H
2O 34.5 μl
Reaction conditions: 94 ℃ of denaturation 3 min; 94 ℃ of sex change 30 s, 58 ℃ of annealing 45 s, 72 ℃ are extended 45 s, 30 circulations; Last 72 ℃ are extended 10 min.
'
rACE amplification reaction system and reaction conditions:
50 μ l reaction systems:
10×PCR buffer 5 μl
MgCl
2(25mM) 2 μl
dNTP Mix（2.5mM） 4 μl
Oligo dT
16-anchor primer (10 μ M) 1 μ l
PrimerP5(10μM) 1 μl
(5U/ μ is 0.5 μ l l) for Taq enzyme
DA-tailed cDNA or PCR product 5 μ l
Rnase free H
2O 33.5 μl
Reaction conditions: 94 ℃ of denaturation 3 min; 94 ℃ of sex change 30 s, 58 ℃ of annealing 45 s, 72 ℃ are extended 45 s, 34 circulations; Last 72 ℃ are extended 10 min.
The sequence table of the Scylla paramamosain Hyastatin gene finally obtaining is as shown in SEQ ID NO.1, total length 837 bp, comprise the open reading frame (ORF) of 396 bp, 131 amino acid of encoding, the 5' non-coding head of district 105 bp, the 3' non-coding head of district 336 bp, have polyadenylic acid tailing signal and polyadenylic acid tail.
embodiment 2
Scylla paramamosain Hyastatin coded amino acid mature peptide in-vitro recombination expression, comprises the following steps:
A) Hyastatin gene sequencing
B) Auele Specific Primer design and mature peptide fragment amplification
C) mature peptide fragment transformed clone and abduction delivering
Concrete operation step is as follows:
1) according to obtaining a pair of Auele Specific Primer that contains restriction enzyme site of Hyastatin gene cDNA sequences Design, upstream primer HYBDf is: 5'-ccaa catatg tac aac gcg aag gtt ccg atc-3'(ccaa is protection base; Catatg is
ndei restriction enzyme site), downstream primer HYBDr is: 5'-ccg ctcgag gcc ttt gta ggg tac tgg ata g-3'(ccg is protection base; Ctcgag is
xhoi restriction enzyme site).Take cDNA the first chain as template, carry out PCR amplification.
50 μ l reaction systems:
10×PCR buffer 5 μl
MgCl
2(25mM) 2 μl
dNTP Mix（2.5mM） 4 μl
HYBDf primer (10 μ M) 1 μ l
HYBDr primer (10 μ M) 1 μ l
Pfu Taq enzyme (5U/ μ L) 0.5 μ l
CDNA the first chain 1 μ l
Rnase free H
2O 41.5 μl
Reaction conditions: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 30 s, 58 ℃ of annealing 45 s, 72 ℃ are extended 1 min, 34 circulations; Last 72 ℃ are extended 10 min.
2) mature peptide fragment transformed clone, expression and checking
Pcr amplification product and expression vector pET28a warp respectively
ndei and
xhoi double digestion, more than using 16 ℃ of connection 4 h of T4 ligase enzyme.Connecting product is transformed into large intestine and infects BL21(DE3) in competence, select positive bacterium colony extracting plasmid, further cut with bacterium colony PCR and identify and select positive colony order-checking to determine that expression cassette is correct by enzyme.Be inoculated into containing in kantlex LB substratum in 1:50 ratio expressing correct bacterium colony, 37 ℃ of shaking culture are to OD=0.4-0.6, and adding IPTG is to collect thalline after 0.2 mM induces 3 h to final concentration.Thalline is processed each 4 s of 45 min(with ultrasonic wave 130W under condition of ice bath, and s), the centrifugal supernatant that removes of 12000 rpm/min, uses washing buffer(0.5% Triton-10 successively at interval 4; 50 mmol/l Tris-HCl, pH8.0; 300 mmol/l NaCl; 10 mmol/l EDTA; 10 mmol/l DTT) and resupersion buffer (50 mmol/l Tris-HCl, pH8.0; 100 mmol/l NaCl; 10 mmol/l EDTA; 10 mmol/l DTT) suspension washing precipitation, finally go supernatant to weigh and use 4 ℃ of dissolution precipitations that spend the night of 8 mol/l urea by 30 mg/ml concentration.Use the imidazoles (30 mM, 60 mM, 90 mM, 120 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM) of different concns by the HisTrap HP of GE company 1 ml prepacked column purification of recombinant proteins.Be transferred to afterwards in dialysis tubing, containing 1 mM EDTA, 20% glycerine, 1 × PBS(pH7.4) and the dialysis renaturation liquid of the urea (6M, 4.5M, 3.5M, 2.5M, 1.5M, 0.5M, 0M) that reduces of gradient in dialysis renaturation, finally at 50 mM Tris-HCl(pH=8.0) dialyse 2 times in damping fluid.Refolded protein is concentrated through vacuum lyophilization, and using the BCA determination of protein concentration kit measurement Scylla paramamosain Hyastatin recombinant protein concentration of green skies company is 7.2 mg/ml.
To induce the expression mycoprotein containing empty carrier and Hyastatin gene of front and back after SDS-PAGE electrophoresis, using Mini Trans-Blot to coordinate Mini Protean III electrophoresis chamber (Bio-Rad company) that electricity consumption is turned to immersion steeps the blob of viscose of 30 min and is transferred to (200 mA on nitrocellulose filter, 80 min), then use mouse-anti His-tag monoclonal antibody to react with it, two resist the goat family dependents of military personel in the liberated areas IgG for horseradish peroxidase-labeled, between each step, use containing the phosphoric acid buffer (TBS) of volume fraction 0.1% Teween20 and wash film 4 times, each 10 min, finally use diaminobenzidine (DAB) colour developing to there being clear target protein band to occur, aseptic water washing 10 min termination reactions, Western blot result shows that the rear band occurring of band and induction coincide.Finally obtain the protein as described in SEQ NO.2 amino acid series table, its length is 131 amino acid, chain is strand, topological framework is linear, proteins encoded molecular weight is 70547.7KDa, and iso-electric point is 5.04, and wherein the 1-16 of encoding sequence is signal peptide sequence, mature peptide molecular weight is 29358.0, and iso-electric point is 5.05.Amino acid has the peculiar region of Penaeidins family: N end for Pro/Arg district, and C end contains 6 Cys, forms 3 intramolecular disulfide bonds, and secondary structure presents typical alpha-helix and beta sheet.
embodiment 3
The external bacteriostatic test of Scylla paramamosain Hyastatin recombinant protein:
1) microorganism culturing: streptococcus aureus (
staphylococcus aureus), micrococcus luteus (
micrococcus luteus), Pseudomonas aeruginosa (
pseudomonas aeruginosa) and intestinal bacteria (
escherichia coli) 37 ℃ of cultivations of use ordinary nutrient agar substratum, cereuisiae fermentum (
saccharomyces cerevisiae) and Candida albicans (
candida albicans) use 28 ℃ of cultivations of Sharpe dextrose peptone medium, when above-mentioned bacterial strains cultivation concentration reaches logarithmic phase, use 50 mM Tris-HCl(pH=8.0) damping fluid dilution thalline, make its concentration reach 1 × 10
3cFU/ml.
Antibacterial Activity: use 50 mMTris-HCl(pH=8.0) the concentrated recombinant protein of gained is made into the solution of a series of mass concentration gradients in gradient dilution embodiment 2, is respectively 7.2,3.6,1.8,0.9,0.45,0.225,0.112 5,0.056 25,0.028 125 mg/ml.By each concentration antibacterial peptide and concentration 1 × 10
3cFU/ml bacterium liquid equal-volume mixes, and makes control group.After 37 ℃ of insulation 60 min, this mixture is taken out to 10 μ l dibblings on substratum, static placement 20 min, are inverted for 37 ℃ and cultivate 24 h.The final concentration that does not grow the corresponding antibacterial peptide of bacterium colony is the minimal inhibitory concentration of this bacterium.Find that above-described embodiment recombinant protein all has certain restraining effect to gram-positive microorganism streptococcus aureus and micrococcus luteus, Gram-negative bacteria Pseudomonas aeruginosa and intestinal bacteria and fungi cereuisiae fermentum and Candida albicans, minimal inhibitory concentration is respectively 0.45,0.9,0.9,0.45,3.6 and 7.2 mg/ml.Point out the application of described expression product at preparation antibacterials, immunostimulant, fodder additives.
Claims (2)
1. Scylla paramamosain antibacterial peptide hyastatin gene clone and proteins encoded restructuring, it is characterized in that: described Scylla paramamosain antibacterial peptide Hyastatin gene, there is base sequence in sequence table SEQ ID NO.1, this gene is the expressed sequence tag screening from Scylla paramamosain hemolymph cDNA library, this sequence total length 837 bp, comprise the open reading frame (ORF) of 396 bp, 131 amino acid of encoding, the 5' non-coding head of district 105 bp, the 3' non-coding head of district 336 bp, have polyadenylic acid tailing signal and polyadenylic acid tail;
Described Scylla paramamosain antibacterial peptide Hyastatin gene coded protein, there is the aminoacid sequence shown in SEQ ID NO.2, this molecular weight of albumen is 70547.7KDa, iso-electric point is 5.04, and wherein the 106-153 of encoding sequence is signal peptide sequence, and mature peptide molecular weight is 29358.0, iso-electric point is 5.05, amino acid has the peculiar region of Penaeidins family: N end for Pro/Arg district, and C end contains 6 Cys, forms 3 intramolecular disulfide bonds.
2. Scylla paramamosain antibacterial peptide hyastatin gene clone according to claim 1 and proteins encoded are recombinant expressed, in the application of preparation antibacterials, immunostimulant, fodder additives.
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| CN104151414A (en) * | 2014-08-20 | 2014-11-19 | 厦门大学 | Preparation method of green mud crab antibacterial peptide SpHyastatin and application thereof |
| CN104151414B (en) * | 2014-08-20 | 2017-06-20 | 厦门大学 | The preparation method of Scylla paramamosain antibacterial peptide SpHyastatin and application |
| CN105238809A (en) * | 2015-11-04 | 2016-01-13 | 黑龙江八一农垦大学 | Expressing method of antimicrobial peptide CC31 in bacillus subtilis |
| US11254717B2 (en) | 2018-06-20 | 2022-02-22 | Xiamen University | Antimicrobial peptide Sparamosin from Scylla paramamosain and application thereof |
| WO2019241938A1 (en) * | 2018-06-20 | 2019-12-26 | 厦门大学 | Scylla paramamosain antibacterial peptide sparamosin and application thereof |
| WO2020103511A1 (en) * | 2018-11-21 | 2020-05-28 | 厦门大学 | Scylla paramamosain antibacterial peptide scyreprocin and application thereof |
| US11512120B2 (en) | 2018-11-21 | 2022-11-29 | Xiamen University | Antimicrobial peptide Scyreprocin of Scylla paramamosain and method thereof |
| CN112724221A (en) * | 2021-01-26 | 2021-04-30 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamplin58-82And uses thereof |
| CN112724221B (en) * | 2021-01-26 | 2022-03-15 | 厦门大学 | Scylla paramamosain antibacterial peptide Spamplin58-82And uses thereof |
| WO2022268115A1 (en) * | 2021-06-25 | 2022-12-29 | 厦门大学 | Scylla paramamosain antibacterial polypeptide spampcin 56-86 and application thereof |
| US12102086B2 (en) | 2021-06-25 | 2024-10-01 | Xiamen University | Antimicrobial peptide Spampcin56-86 from Scylla paramamosain and applications thereof |
| CN115058430A (en) * | 2022-06-12 | 2022-09-16 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
| CN115058430B (en) * | 2022-06-12 | 2023-06-09 | 宁波大学 | Scylla paramamosain 5-HT2 receptor gene and application thereof |
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