CN105859862A - Crassostrea hongkongensis antimicrobial peptide sa-lectin as well as recombinant expression method and application thereof - Google Patents
Crassostrea hongkongensis antimicrobial peptide sa-lectin as well as recombinant expression method and application thereof Download PDFInfo
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- CN105859862A CN105859862A CN201610335666.1A CN201610335666A CN105859862A CN 105859862 A CN105859862 A CN 105859862A CN 201610335666 A CN201610335666 A CN 201610335666A CN 105859862 A CN105859862 A CN 105859862A
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- lectin
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- concha ostreae
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- antibacterial peptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The invention discloses crassostrea hongkongensis antimicrobial peptide sa-lectin as well as a recombinant expression method and application thereof. The gene coding sequence of antimicrobial peptide sa-lectin is shown in SEQ ID NO: 1, in which 156 amino acids are coded, and is shown in SEQ ID NO: 2, in which 16 amino acids at the end N are signal peptide sequences. According to the crassostrea hongkongensis antimicrobial peptide sa-lectin, a prokaryotic expression vector for removing the signal peptide sequence is constructed, so that sa-lectin recombined proteins are expressed and purified and can suppress growth of various types of gram-positive bacteria and gram-negative bacteria; furthermore, by the combination of sialic acid, an important effect is achieved in immune defense of shellfish.
Description
Technical field
The present invention relates to shellfish technical field of pharmaceuticals, be specifically related to a kind of Hong Kong Concha Ostreae antibacterial peptide sa-lectin and recombinant expression method thereof and application.
Technical background
Shellfish culture occupies very important status in China coast socio-economic development, is one of the most most important grass economic category, has huge development potentiality.Hong Kong Concha Ostreae (Crassostrea hongkongensis), belong to Mollusca, Bivalvia, Margarita mesh, Ostreidae animal, be China Coastal Areas of South China most important shellfishery animal.But, breeding scarcity causes Concha Ostreae immunocompetence low, and massive mortality even occurs in illness outbreak, is the Main Bottleneck of restriction shellfish industry development.
Therefore, the antibacterial ability how improving shellfish is the effective means solving present problems.
Summary of the invention
It is an object of the invention to solve the existing issue of current shellfish immunocompetence deficiency etc., from the cDNA of Hong Kong Concha Ostreae, identify a kind of sialic acid binding lectin salectin gene, obtained total length ORF changing gene by clone.And then by building its prokaryotic expression carrier, it is thus achieved that the recombiant protein of its solubility.Antibacterial assay shows that this recombiant protein has the ability of the growth of effective suppression gram positive bacteria and negative bacterium, and also has the ability of bound sialic acid.So, this patent not only can effectively improve the resistance against diseases of shellfish, but also can be that exploitation novel antimicrobial peptide provides material.
A kind of Hong Kong Concha Ostreae antibacterial peptide sa-lectin, its gene coded sequence is as shown in SEQ ID NO:1；Its aminoacid sequence is as shown in SEQ ID NO:2.
The recombinant expression method of Hong Kong Concha Ostreae antibacterial peptide sa-lectin of the present invention comprises the steps:
(1) extraction of RNA；
(2) structure of cDNA library；
(3) structure of prokaryotic expression carrier: design the primer containing sa-lectin gene coded sequence for a pair, primer two ends are separately added into BamH-I and Xho-I site and protection base；Use this that primer is carried out PCR amplification, amplified production and pMALTMCarrier uses BamH-I and Xho-I to carry out double digestion respectively, and reclaims purification digestion products；Connect the PCR fragment after enzyme action and pMALTMCarrier；Conversion, picking positive colony, sequence verification carrier is correct；
(4) prokaryotic expression of recombiant protein and purification.
Hong Kong Concha Ostreae antibacterial peptide sa-lectin of the present invention application in improving shellfish immunocompetence.
Compared with prior art, there is advantages that
The present invention uses prokaryotic expression method to prepare salectin gene and removes the recombiant protein of signal peptide sequence, is experimentally verified that, this albumen can suppress the growth of multiple gram positive bacteria and gram negative bacteria, and can be with bound sialic acid.
Accompanying drawing explanation
Fig. 1 is to express and the salectin recombiant protein of purification, wherein, and M: albumen Marker；Lane1: recombinant expression protein MBP-salectin；Lane2: MBP after purification；The destination protein MBP-salectin that arrow instruction is recombinant expressed；
Fig. 2 is the antimicrobial spectrum of salectin；By detecting the antibacterial bacteriostasis at the light absorption value analysis salectin of 600 nm: gram negative bacteria (A) V.alginolyticus
(B) E. coli and gram positive bacteria (C) S. aureus and (D) B. thuringiensis；Fig. 2 shows that recombiant protein salectin can effectively suppress the growth of gram negative bacteria and gram positive bacteria；
Fig. 3 is that Native PAGE electrophoresis showed selectin is with sialic binding ability；Wherein, Lane 1:MBP；Lane 2:MBP+containing sialic albumen fetuin；Lane 3/6: fetuin；Lane 4: recombiant protein MBP-salectin；Lane 5: recombiant protein MBP-salectin
+ fetuin；What arrow referred to is MBP dimer；Fig. 3 shows that selectin can be with bound sialic acid.
Detailed description of the invention
For further illustrating the technological means of the present invention, novelty and purpose effect, in conjunction with actual illustrated embodiments, but following example are exemplary, are only used for explaining that this invents, and are not considered as limiting the invention.
Embodiment
1
1. RNA extracts
The extraction of total serum IgE uses Trizol (Invitorgen) to extract, and specifically comprises the following steps that (1) takes 50mg and is organized in the mortar of Liquid nitrogen precooler, after being clayed into power by tissue, transfers to, in the centrifuge tube equipped with 1ml Trizol, blow and beat, mix；(2) adding 200 μ L chloroforms, acutely shake 40s, room temperature places 5min；(3) 4 DEG C, 12,000 × g is centrifuged 10 min, draws supernatant and is transferred to newly manage；(4) isopropanol of 0.5ml, mixing ,-80 DEG C of precipitates overnight are added；(5) 4 DEG C, 12,000 × g is centrifuged 10 min, abandons supernatant；(6) 75% twice precipitation of ethanol wash；(7) abandon supernatant, add 100 μ L DEPC water dissolution RNA.
2. cDNA library builds
CDNA library builds and uses SMART cDNA
Library construction Kit(Clontech), specifically comprise the following steps that (1) uses MMLV to synthesize cDNA Article 1 chain；(2) LD PCR is used to expand cDNA；(3) E.C. 3.4.21.64 digestion purification cDNA product；(4) Sfil digestion, uses Chroma Spin400 to carry out product size separation；(5) connect cDNA to λ TripEx2 carrier, after bacteriophage lambda is packed, convert to host cell；(6) detection cDNA library titre.
3. Prokaryotic expression vector construction
(1) design is contained the primer of salectin ORF for a pair (forward primer sequence is GACTGGATCCTATGGATATGTCTATCACAGGTC; reverse primer sequences is GACTCTCGAGTTAaataattctaattaaggtaccg), the two ends of primer are separately added into BamH-I and Xho-I site and protection base；(2) use this that primer is carried out PCR amplification；(3) PCR primer and pMALTMCarrier uses BamH-I and Xho-I to carry out double digestion respectively, and reclaims purification digestion products；(4) PCR fragment after enzyme action and pMAL are connectedTMCarrier；(5) converting, picking positive colony, sequence verification carrier is correct, carries out follow-up test.
4. the prokaryotic expression of recombiant protein and purification
(1) the LRR prokaryotic expression carrier built is converted to BL21 (DE3)；(2) select monoclonal and cultivate to OD=0.6, adding IPTG to final concentration of 0.1 mM,
22 DEG C, abduction delivering under conditions of 180rmp；(3) collecting bacterium solution after 4h, 4 DEG C, 12,000 × g is centrifuged 10 min；(4) Lysozyme is added to final concentration 2 mg/ml, then ultrasonication；(5) use Amylose Resin purification of recombinant proteins, analyze purified product at 12% SDS-PAGE glue.
5. antimicrobial spectrum analysis
(1) all of bacterium to be measured grows to logarithmic (log) phase in LB culture medium, is then diluted to 104CFU/mL；(2) taking the bacterium to be measured after 50 μ L dilutions and add 50 μ L recombiant proteins, protein concentration is 60 ng/ μ L；(3) 37 ° of C hatch 3 h；(4) add 900 μ L LB culture medium, continue to cultivate；(5) respectively cultivate 2h to 6h detection cell at the light absorption value of 600 nm, draw its growth curve.
6. sialic acid binding analysis
(1) at 50 mM
In pH 8.0 Tris-HCl buffer, it is separately added into 20 ng recombiant protein MBP-salectin and the fetuin of excess 100 times；(2) room temperature combines 3 h；(3) 2% Native PAGE gel combine coomassie brilliant blue staining analysis and combine product.
Claims (5)
1. Hong Kong Concha Ostreae antibacterial peptide sa-lectin, its gene coded sequence is as shown in SEQ ID NO:1.
2. Hong Kong Concha Ostreae antibacterial peptide sa-lectin described in claim 1, its aminoacid sequence is as shown in SEQ ID NO:2.
3. include the prokaryotic expression carrier of Hong Kong Concha Ostreae antibacterial peptide sa-lectin described in claim 1.
4. the recombinant expression method of Hong Kong Concha Ostreae antibacterial peptide sa-lectin described in claim 1, it is characterised in that comprise the steps:
(1) extraction of RNA；
(2) structure of cDNA library；
(3) structure of prokaryotic expression carrier: design the primer containing sa-lectin gene coded sequence for a pair, primer two ends are separately added into BamH-I and Xho-I site and protection base；Use this that primer is carried out PCR amplification, amplified production and pMALTM
Carrier uses BamH-I and Xho-I to carry out double digestion respectively, and reclaims purification digestion products；Connect the PCR fragment after enzyme action and pMALTMCarrier；Conversion, picking positive colony, sequence verification carrier is correct；
(4) prokaryotic expression of recombiant protein and purification.
5. the application in improving shellfish immunocompetence of Hong Kong Concha Ostreae antibacterial peptide sa-lectin described in claim 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586596A (en) * | 2018-04-04 | 2018-09-28 | 中国科学院南海海洋研究所 | A kind of oyster cell apoptogene SMAC genes and its application in preparing pathological examination diagnostic reagent |
| CN112707961A (en) * | 2021-02-04 | 2021-04-27 | 中国科学院南海海洋研究所 | Shellfish antibacterial peptide P-AMP153 and application thereof |
| CN113234132A (en) * | 2021-04-12 | 2021-08-10 | 浙江理工大学 | Scylla paramamosain C-type lectin and preparation method and application thereof |
| CN114106103A (en) * | 2022-01-24 | 2022-03-01 | 中国科学院南海海洋研究所 | Antibacterial peptide P-AMP108 from shellfish and application thereof in preparation of medicine for treating acne |
Citations (2)
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| CN104474557A (en) * | 2014-11-25 | 2015-04-01 | 中国科学院海洋研究所 | Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity |
| CN105585626A (en) * | 2016-02-03 | 2016-05-18 | 广东海洋大学 | Pinctada martensi lectin PmCLEC-1 and application thereof |
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Patent Citations (2)
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| CN104474557A (en) * | 2014-11-25 | 2015-04-01 | 中国科学院海洋研究所 | Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity |
| CN105585626A (en) * | 2016-02-03 | 2016-05-18 | 广东海洋大学 | Pinctada martensi lectin PmCLEC-1 and application thereof |
Non-Patent Citations (2)
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586596A (en) * | 2018-04-04 | 2018-09-28 | 中国科学院南海海洋研究所 | A kind of oyster cell apoptogene SMAC genes and its application in preparing pathological examination diagnostic reagent |
| CN108586596B (en) * | 2018-04-04 | 2020-02-14 | 中国科学院南海海洋研究所 | Oyster apoptosis gene SMAC gene and application thereof in preparation of pathological detection diagnostic reagent |
| CN112707961A (en) * | 2021-02-04 | 2021-04-27 | 中国科学院南海海洋研究所 | Shellfish antibacterial peptide P-AMP153 and application thereof |
| CN112707961B (en) * | 2021-02-04 | 2022-06-10 | 中国科学院南海海洋研究所 | Shellfish antibacterial peptide P-AMP153 and application thereof |
| CN113234132A (en) * | 2021-04-12 | 2021-08-10 | 浙江理工大学 | Scylla paramamosain C-type lectin and preparation method and application thereof |
| CN114106103A (en) * | 2022-01-24 | 2022-03-01 | 中国科学院南海海洋研究所 | Antibacterial peptide P-AMP108 from shellfish and application thereof in preparation of medicine for treating acne |
| CN114106103B (en) * | 2022-01-24 | 2022-04-15 | 中国科学院南海海洋研究所 | Antibacterial peptide P-AMP108 from shellfish and application thereof in preparation of medicine for treating acne |
| WO2022262385A1 (en) * | 2022-01-24 | 2022-12-22 | 中国科学院南海海洋研究所 | Shellfish-derived antimicrobial peptide p-amp108 and use thereof in preparation of drug for treating acne |
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Application publication date: 20160817 |