CN102586261A - Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof - Google Patents
Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof Download PDFInfo
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- CN102586261A CN102586261A CN201210064577XA CN201210064577A CN102586261A CN 102586261 A CN102586261 A CN 102586261A CN 201210064577X A CN201210064577X A CN 201210064577XA CN 201210064577 A CN201210064577 A CN 201210064577A CN 102586261 A CN102586261 A CN 102586261A
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Abstract
The invention belongs to the technical field of molecular biology and particularly relates to a portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof. The PtCrustin-2 gene DNA is amplified from portunus trituberculatus by utilizing a cDNA library and an RACE technology and the gene exerts important effect in the aspect of immunologic defence of the portunus trituberculatus. The recombinant PtCrustin-2 protein has obvious bacteriostatic activity on Gram negative bacteria and Gram positive bacteria. The minimum inhibitory concentrations of the gene on vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus and micrococcus luteus are 24.75 MuM, 12.37 MuM, 49.51 MuM and 49.51 MuM respectively. The gene does not have obvious bacteriostatic effect on fungi pichia pastoris. Basis is laid for diseases prevention and treatment on the portunus trituberculatus, gene auxiliary breeding and development of feed additives.
Description
Technical field
The invention belongs to technical field of molecular biology, a kind of specifically Portunus trituberculatus Miers PtCrustin-2 gene and proteins encoded and application.
Background technology
Crustin is a kind of cationic antibacterial peptide that is rich in cysteine residues of in the crustacean body, finding.Its molecular weight size is 7-14kDa, and iso-electric point is between 7.0-8.7, and the N end comprises signal peptide, and the C end comprises WAP (whey acidic protein) structural domain, and this structural domain is made up of four disulfide linkage cores (4-DSC) that 8 Cys form.According to the intermediate sequence plot structure between signal peptide and WAP structural domain, Crustin can be divided into three types of I-III.I type Crustin mainly is present in crab, lobster and the crayfish; II type and III type Crustin mainly are found in the prawn.
Crustin is named as carcinin at first, is from the granular hemocyte of Carcinus maenas, to find, and gram-positive microorganism is had the obvious suppression effect.At present existing more than 70 Crustin sequence reported in different crustaceans, comprises crab, lobster, prawn and crayfish etc.Research shows that the Crustin albumen of these natural purifying or vitro recombination all has the effect of obvious suppression Gram-positive bacteria growing; And all be high-caliber constitutive expression, prompting Crustin plays a significant role in crustacean inherent immunity system.
Advantages such as Portunus trituberculatus Miers (Portunus trituberculatus) is a kind of important marine products economic crab, and is abundant owing to its nutritive value, that growth is rapid have become the important sea farming kind of China.But along with continuous expansion of breed scale and intensification degree improve constantly, the various diseases that is caused by bacterium, fungi etc. is on the rise, and causes enormous economic loss for the swimming crab aquaculture.The continuous outburst of disease and the variety of the cause of disease press for from the start with antibacterials of research its disease-resistant mechanism and development of new of the immune defense factor of Portunus trituberculatus Miers self.
Up to the present, the research about Portunus trituberculatus Miers Crustin is still few.Therefore, identification, qualitative antimicrobial effect molecule-Crustin are to the immune defence mechanism of research Portunus trituberculatus Miers and carry out disease control and have important theory and practice significance.
Summary of the invention
The purpose of this invention is to provide a kind of Portunus trituberculatus Miers PtCrustin-2 gene and proteins encoded and application.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of Portunus trituberculatus Miers PtCrustin-2 gene is shown in the base sequence among the sequence table SEQ ID No.1.
Portunus trituberculatus Miers PtCrustin-2 gene coded protein is among the sequence table SEQ ID No.2 shown in the aminoacid sequence.
The application of Portunus trituberculatus Miers PtCrustin-2 gene: the recombination expression product of said Portunus trituberculatus Miers PtCrustin-2 gene coded protein is used to prepare antibacterials, immunostimulant, fodder additives, sanitas or preservation agent.
The recombination expression product of said Portunus trituberculatus Miers PtCrustin-2 gene coded protein is used to prepare the antibacterial medicines of Gram-negative bacteria, gram-positive microorganism.Said Gram-negative bacteria is vibrio alginolyticus, Pseudomonas aeruginosa, and gram-positive microorganism is streptococcus aureus, micrococcus luteus.
The advantage that the present invention had:
The present invention utilizes expressed sequence tagging method (EST), RACE technology from Portunus trituberculatus Miers, to be cloned into PtCrustin-2 gene cDNA full length sequence; Be cloned in pET32a (+) expression vector through the gene fragment of round pcr amplification coding PtCrustin-2 mature peptide and with it, realize in-vitro recombination expression at e. coli bl21 (DE3)-plysS.Recombinant protein is after TALON column purification and dialysis; Gram-negative bacteria vibrio alginolyticus, Pseudomonas aeruginosa, gram-positive microorganism streptococcus aureus and micrococcus luteus are had significant bacteriostatic activity, and minimal inhibitory concentration is respectively 24.75 μ M, 12.37 μ M, 49.51 μ M, 49.51 μ M; And the fungi pichia spp is not had the obvious suppression effect.
Gene of the present invention and recombinant protein thereof are mainly used in drug manufacture, immunostimulant, fodder additives and sanitas and preservation agent production; Can also the basis be provided for further research Portunus trituberculatus Miers immune defence mechanism in addition, and reference be provided for the disease control of Portunus trituberculatus Miers and gene assist-breeding.
Description of drawings
Gene amplification product electrophorogram (wherein, M:DNA marker, 1: the gene amplification product of mature peptide) of the Portunus trituberculatus Miers PtCrustin-2 encoding mature peptide of the purifying that Fig. 1 provides for instance of the present invention.
Fig. 2 is the Portunus trituberculatus Miers PtCrustin-2 recombinant protein electrophorogram of inducing of providing of instance of the present invention and purifying (M wherein: albumen marker; 1: do not induce expressed proteins in the thalline; 2:IPTG induces the back expressed proteins; 3: purified recombinant albumen).
Embodiment
Among the following embodiment the present invention is done further elaboration, but the invention is not restricted to this.
The cDNA sequence clone of Portunus trituberculatus Miers PtCrustin-2 among the present invention comprises the following steps:
A) purifying of the extraction of the total RNA of Portunus trituberculatus Miers and mRNA;
B) Portunus trituberculatus Miers cDNA library construction;
C) the extensive mensuration of Portunus trituberculatus Miers cDNA library est sequence;
D) screening of the homology analysis of Portunus trituberculatus Miers est sequence and PtCrustin-2 gene fragment;
E) the RACE amplification obtains the complete sequence of PtCrustin-2 and the checking of complete sequence.
Embodiment 1.
Portunus trituberculatus Miers PtCrustin-2 gene is the base sequence shown in the SEQ ID No.1.
Sequence table SEQ ID No.1 is:
(a) sequence signature
● length: 629bp (useful length 58-405)
● type: base sequence
● chain: strand
● topological framework: linearity
(b) molecule type: double-stranded DNA
(c) suppose: not
(d) antisense: not
(e) initial source: Portunus trituberculatus Miers (Portunus trituberculatus)
(f) specificity title: CDS
The structure concrete operations are following:
1. the purifying of the extraction of the total RNA of Portunus trituberculatus Miers and mRNA: utilize the Trizol reagent of Invitrogen company to extract the total RNA of Portunus trituberculatus Miers, utilize the Oligitex mRNA purification kit purified mRNA of QIAGENE company.
2. Portunus trituberculatus Miers cDNA library construction: the Creator Smart cDNA Library Construction Kit test kit operation instruction construction cDNA library that utilizes Clontech company.With the mRNA behind the purifying is template, SMART IV Oligonucleotide (5 ' AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG 3 ') and CDS III/3 ' PCR Primer (5 ' ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) 30N_
1N 3 ') is primer, under ThermoScript II (MMLV reverse transcriptase) effect, transcribes synthetic cDNA first chain.With 5 ' PCR Primer (5 ' AAGCAGTGGTATCAACGCAGAGT 3 ') and CDS III/3 ' PCR Primer (5 ' ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) 30N_
1N 3 ') be that primer is through synthetic cDNA second chain of long distance (LD-PCR).Double-stranded cDNA (ds cDNA) digests 20min through Proteinase K (0.8mg/ml) under 45 ℃ of conditions; Carrying out enzyme with the SfiI enzyme then cuts; Enzyme is cut product through 1.5% agarose gel electrophoresis, utilizes the QIAEX II Agarose Gel Extraction Kit of QIAGEN company to reclaim the fragment of 1-3kb.The fragment that reclaims is connected the back purifying with carrier pDNR-LIB, transforms through electricity and import competent escherichia coli cell, 220rpm/min cultivates 1h under 37 ℃ of conditions, adds the glycerine of final volume 20%, is the original library of full-length cDNA.
3. the extensive mensuration of Portunus trituberculatus Miers cDNA library est sequence: screening positive clone in the library; Use carrier universal primer M13F (5 ' TGTAAAACGACGGCCAGT 3 ') on the ABI3730xl sequenator, to carry out sequencing; The parent mass peak map file that obtains (* .ab1, * .abd file) data are converted into sequential file (* .seq) and quality document (* .seq.qual) through the Phred routine processes, and the numerical value that provides according to quality document confirms to obtain the word error probability of sequence; Remove low-quality base; With the carrier sequence in the cross-match program mask data, from the data that obtain, choose continuous base quality greater than Q13 (accuracy rate is greater than 95%) and length greater than the sequence of 100bp as the EST data, concrete " expressed sequence (EST) data analysis handbook (Hu Songnian work; Press of Zhejiang University, 2005).
4. the screening of the homology analysis of Portunus trituberculatus Miers est sequence and PtCrustin-2 gene fragment: whole effectively EST data that will obtain are carried out the cluster splicing; Generate Contigs and Singletons; Respectively Contigs that is obtained and Singletons are carried out BLASTn and BLASTx analysis in DB; The result is presented at the sequence of having found in the est sequence with off-lying sea swimming crab Portunus pelagicus Crustin gene similarity higher (76%), has confirmed the est sequence of Portunus trituberculatus Miers PtCrustin-2 gene according to the similarity analysis result.
5. the clone of Portunus trituberculatus Miers PtCrustin-2 gene cDNA full length sequence: according to the est sequence design special primer F1 (5 ' GGGACGCCTCCTATATCTGACA 3 ') and R1 (5 ' GGTCCGATTCTGAGATACTCA 3 ') of PtCrustin-2 dna homolog, utilize carrier universal primer M13F (5 ' TGTAAAACGACGGCCAGT 3 ') and M13R (5 ' CAGGAAACAGCTATGACC 3 ') to carry out 3 ' and the amplification of 5 ' end respectively.The PCR product detects with 1.5% agarose gel electrophoresis; Reclaim test kit with Axygen glue and carry out recovery of PCR product and purifying; Be connected with pMD-18T carrier (the precious biotechnology in Dalian ltd) again; Transformed into escherichia coli competence Trans1-T1 (Beijing Quanshijin Biotechnology Co., Ltd) then; Select positive colony and check order with carrier primer M13-47 and RV-M, the gained result is spliced through Phred/Phrap software, obtains Portunus trituberculatus Miers PtCrustin-2 full length gene cDNA sequence and sees SEQ ID No.1.
6. the checking of Portunus trituberculatus Miers PtCrustin-2 gene cDNA total length: design a pair of primers F 2 (5 ' GCCTCGCTCTCATCTTTGCTCCAG 3 ') and R2 (5 ' CATAGTGACATCAAAACGC 3 ') on the PtCrustin-2 full length sequence of order-checking splicing are the checking that template is carried out total length with cDNA.Order-checking and analysis are with 5.
3 ' RACE amplification reaction system and reaction conditions:
25 μ l reaction systems:
Be reflected among the TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.) and carry out, reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 56 ℃ of annealing 50s, 72 ℃ are extended 1min, 34 circulations; 72 ℃ are extended 10min.
5 ' RACE amplification reaction system and reaction conditions:
Be reflected among the TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.) and carry out, reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 56 ℃ of annealing 50s, 72 ℃ are extended 1min, 34 circulations; 72 ℃ are extended 10min.
The PCR reaction system and the reaction conditions of total length checking are:
25 μ l reaction systems:
Be reflected among the TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.) and carry out, reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 58 ℃ of annealing 50s, 72 ℃ are extended 1min, 34 circulations; 72 ℃ are extended 10min.
Sequence table SEQ ID No.1 is cloned into PtCrustin-2 gene cDNA total length 629p from Portunus trituberculatus Miers, ORFs 348bp wherein, and 5 ' non-translational region 57bp, 3 ' non-translational region 224bp has the polyadenylic acid tail.
Embodiment 2.
The said base sequence of Portunus trituberculatus Miers PtCrustin-2 sequence table SEQ ID No.1, described aminoacid sequence such as sequence table SEQ ID No.2 are said.
Sequence table SEQ ID No.2 is:
It has complete proteins encoded and contains 98 amino acid, and wherein the 1-20 of an encoding sequence amino acid is a signal peptide, and mature peptide comprises 95 amino acid, and the molecular weight of prediction is 10.58kDa, and iso-electric point is 6.74.Mature peptide has typical type I Crustin mode configuration, comprises a zone (Cys-rich region) and a WAP structural domain (WAP domain) that is rich in halfcystine.Wherein, be rich in the zone of halfcystine 4 cysteine residues and arrange comparatively conservatively, be C-(X3)-C-(X8-12)-C-C pattern; The WAP structural domain comprises 8 cysteine residues, and they can form four disulfide linkage cores (4-DSC).
The acquisition of Portunus trituberculatus Miers PtCrustin-2 recombinant protein, concrete operations are following:
The cDNA sequence corresponding according to SEQ ID No.2; Design contains the Auele Specific Primer F3 (5 ' GGATCCGGGACGCCTCCTATATCTGACA 3 ') and the R3 (5 ' CTCGAGTTAATGTGGGTCAGCAATCAGGC 3 ') of restriction enzyme BamHI and XhoI restriction enzyme site; Gene fragment (referring to Fig. 1) through round pcr amplification coding PtCrustin-2 mature peptide; Be reflected among the TaKaRa PCR Thermal Cycler Dice Model TP600 (Takara Bio Inc.) and carry out, reaction conditions is: 94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 58 ℃ of annealing 50s, 72 ℃ are extended 30s, 34 circulations; Last 72 ℃ are extended 10min.Cut through enzyme then it is cloned in pET32a (+) expression vector, be transformed into e. coli bl21 (DE3)-plysS, after order-checking confirmed that expression cassette is correct, the inoculation positive colony was in the LB substratum, and 37 ℃ of shaking culture are to O.D.
600=0.4-0.6, adding IPTG is that 1mM induces centrifugal collection thalline after 4 hours to final concentration.Thalline is handled 30-60min (each 2s, 2s at interval), the centrifugal supernatant that removes, collecting precipitation (containing the recombinant protein inclusion body) with UW 180W under condition of ice bath.After the urea dissolving of deposition with 8M, utilize the TALON column purification recombinant products of Clontech company.Purified recombinant albumen is transferred in the dialysis tubing; Under 4 ℃ of conditions; Dialysis in containing dialysis renaturation solution (pH=8.0) 2mM reduction Triptide, 0.2mM oxidation Triptide, 1mM EDTA, 50mM Tris-HCl, 50mM NaCl, 10% glycerine and 1% glycocoll and the urea (6M, 5M, 4M, 3M, 2M, 1M, 0M) that gradient reduces; Make the recombinant protein renaturation, dialyse 2 times at the damping fluid of 50mM Tris-HCl (pH=8.0) at last, to remove other composition in the solution.The recombinant protein of dialysis after the renaturation concentrates through the centrifugal evaporating pipe of the Microsep of PALL company Advance ultrafiltration, and the concentration of using the BCA determination of protein concentration test kit of green skies company to record Portunus trituberculatus Miers PtCrustin-2 recombinant protein is 2.80mg/ml (referring to Fig. 2).
Embodiment 3.
The extracorporeal bacteria inhibitor test of Portunus trituberculatus Miers PtCrustin-2 recombinant protein:
1. cultivation of mikrobe and preparation: vibrio alginolyticus is with 28 ℃ of TSB substratum; Pseudomonas aeruginosa is with 37 ℃ of TSB substratum, and streptococcus aureus is with 37 ℃ of LB substratum, and micrococcus luteus is with 37 ℃ of LB substratum; Pichia spp is with 28 ℃ of YPD substratum; When above-mentioned each bacterial strain makes bacteria concentration reach logarithmic phase with shaking table 220rpm/min cultivation, use 50mM Tris-HCl (pH=8.0) damping fluid dilution thalline respectively, make its every milliliter colony count in the bacterium liquid be about 1 * 10 respectively
3
2. recombinant protein PtCrustin-2 bacteriostatic activity is measured: utilize the foregoing description gained recombinant protein PtCrustin-2 with Tris-HCl (50mM; PH=8.0) behind the gradient dilution (1,1/2,1/4,1/8,1/16,1/32,1/64,1/128), get 50 μ l be added to aseptic flat 96 orifice plates (Costar, Fisher) in; 50 μ l Tris-HCl (50mM; PH=8.0) be made as contrast, add the good bacteria suspension of 50 μ l dilution then, and mixing.The hole that only adds 50 μ l bacterium liquid is a blank well.96 orifice plates add corresponding substratum 150 μ l after hatching 3 hours under the culture temperature of bacterium liquid, blank well adds substratum 200 μ l, incubated overnight.96 orifice plates of solubilization algae vibrios, Pseudomonas aeruginosa, pichia spp are reading under the visible light of 560nm at wavelength, and 96 orifice plates that add streptococcus aureus and micrococcus luteus are reading under the visible light of 600nm at wavelength.Find that the foregoing description recombinant protein PtCrustin-2 has the obvious suppression effect to Gram-negative bacteria vibrio alginolyticus and Pseudomonas aeruginosa, gram-positive microorganism streptococcus aureus and micrococcus luteus, minimal inhibitory concentration is respectively 24.75 μ M, 12.37 μ M, 49.51 μ M, 49.51 μ M; And the fungi pichia spp is not had the obvious suppression effect.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. Portunus trituberculatus Miers PtCrustin-2 gene, it is characterized in that: Portunus trituberculatus Miers PtCrustin-2 gene is shown in the base sequence among the sequence table SEQ ID No.1.
2. the described Portunus trituberculatus Miers PtCrustin-2 of claim 1 gene coded protein is characterized in that: said PtCrustin-2 gene coded protein is among the sequence table SEQ ID No.2 shown in the aminoacid sequence.
3. application by the described Portunus trituberculatus Miers PtCrustin-2 of claim 1 gene, it is characterized in that: the recombination expression product of said Portunus trituberculatus Miers PtCrustin-2 gene coded protein is used to prepare antibacterials, immunostimulant, fodder additives, sanitas or preservation agent.
4. by the application of the described Portunus trituberculatus Miers PtCrustin-2 of claim 3 gene, it is characterized in that: the recombination expression product of said Portunus trituberculatus Miers PtCrustin-2 gene coded protein is used to prepare the antibacterial medicines of Gram-negative bacteria, gram-positive microorganism.
5. by the application of the described Portunus trituberculatus Miers PtCrustin-2 of claim 4 gene, it is characterized in that: said Gram-negative bacteria is vibrio alginolyticus, Pseudomonas aeruginosa, and gram-positive microorganism is streptococcus aureus, micrococcus luteus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937406A (en) * | 2017-11-30 | 2018-04-20 | 宁波大学 | A kind of application of Portunus trituberculatus Miers novel C rustin genes and its recombinant protein |
| CN109134635A (en) * | 2018-08-07 | 2019-01-04 | 内蒙古科技大学 | A kind of antibacterial peptide and the preparation method and application thereof with composite bio-active |
| CN110317813A (en) * | 2019-07-17 | 2019-10-11 | 中国科学院海洋研究所 | Portunus trituberculatus Miers c-type agglutinin PtCLec2 gene and its coding albumen and application |
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2012
- 2012-03-13 CN CN201210064577XA patent/CN102586261A/en active Pending
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| 刘媛: "三疣梭子蟹（Portunus trituberculatus）cDNA文库构建、EST分析及抗脂多糖因子研究", 《中国博士学位论文全文数据库农业科技辑》 * |
| 岳峰: "三疣梭子蟹在氨氮胁迫下免疫应答与解毒代谢机制的研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937406A (en) * | 2017-11-30 | 2018-04-20 | 宁波大学 | A kind of application of Portunus trituberculatus Miers novel C rustin genes and its recombinant protein |
| CN109134635A (en) * | 2018-08-07 | 2019-01-04 | 内蒙古科技大学 | A kind of antibacterial peptide and the preparation method and application thereof with composite bio-active |
| CN109134635B (en) * | 2018-08-07 | 2021-07-23 | 内蒙古科技大学 | Antibacterial peptide with composite bioactivity and preparation method and application thereof |
| CN110317813A (en) * | 2019-07-17 | 2019-10-11 | 中国科学院海洋研究所 | Portunus trituberculatus Miers c-type agglutinin PtCLec2 gene and its coding albumen and application |
| CN110317813B (en) * | 2019-07-17 | 2022-12-27 | 中国科学院海洋研究所 | Portunus trituberculatus C-type lectin PtCLec2 gene, and coding protein and application thereof |
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Application publication date: 20120718 |