CN104987378B - A kind of c-type agglutinin encoding gene and its albumen prepare and application - Google Patents
A kind of c-type agglutinin encoding gene and its albumen prepare and application Download PDFInfo
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- CN104987378B CN104987378B CN201510420056.7A CN201510420056A CN104987378B CN 104987378 B CN104987378 B CN 104987378B CN 201510420056 A CN201510420056 A CN 201510420056A CN 104987378 B CN104987378 B CN 104987378B
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Abstract
The present invention relates to field of molecular microbiology, specifically a kind of c-type agglutinin (C type lectin) and its preparation and application.C-type agglutinin is shown in the amino acid sequence in sequence table SEQ ID No.1.Preparation method：Using the flat rockfish cDNA of Xu Shi as template PCR amplifications c-type agglutinin gene, plasmid pLecC is built；By pLecC conversion e. coli bl21 Transetta (DE3), after carrying out induced expression to transformant, supernatant is collected, up to recombinant C-type agglutinin after concentration.The c-type agglutinin has efficient agglutination to Vibrio, and through c-type agglutinin, treated that bacterium significantly reduces the infection ability of the flat rockfish of Xu Shi.Therefore, which is expected to the prevention applied to fish bacterial disease.
Description
Technical field
The present invention relates to molecular biology and field of molecular microbiology, specifically a kind of c-type agglutinin (C-type
Lectin) encoding gene and its albumen are prepared and applied.
Background technology
Agglutinin is a kind of carbohydrate-binding protein, is widely present in microorganism and animals and plants.C-type agglutinin is agglutinin man
The important member of race, all collectins and selectin belong to c-type agglutinin, are present in most animals body.Such is solidifying
Collection element is characterized mainly in that them containing there are one common sugar identification structural domain (carbohydrate-recognition
domain,CRD).As a kind of important pattern recognition receptors, c-type agglutinin can be with the specific glycan molecule knot of antimicrobial surface
It closes and plays agglutination, played an important role in the innate immunity of host versus microbial infection.In addition, c-type agglutinin
After being reacted with cause of disease, body can be stimulated to generate a series of immune response, including enhancing phagocytosis, chemotaxis is stimulated and lived
Property the generation of the oxygen and release of cell factor, part c-type agglutinin may also participate in antigen submission and the absorption of dendritic cells mediation.
Therefore, c-type agglutinin can be used as immunomodulator or disease control drug in culture fishery.
The content of the invention
Present invention aims at provide a kind of c-type agglutinin encoding gene and its albumen preparation and application.
Specific embodiment
With reference to embodiment, the invention will be further described.Embodiment is intended to carry out citing description to the present invention, and
It is non-to limit the invention in any form.
Embodiment 1
The c-type agglutinant protein of the present invention is the amino acid sequence in sequence table SEQ ID No.1, and encoding gene is sequence
Base sequence in list SEQ ID No.2
Sequence table SEQ ID No.1 are：
ADCPEGEASSLKLQLNLLRNRFRHLCDQYSNLATNCSAPVIPCAQCPEGWLVVGDQCFLLTTDRDDYSN
STNKCAEIGAHLAILTTKEQHDAVEKEGKNIGGIYTYYWIGLTDIETEGDWRWVDNSKLRTPFWEAPEPNNHLSGGP
EGEDCAVVQSYTQLWHDVPCSFTYPRICQMDAILPQ
(a) sequence signature：
● length：182
● type：Amino acid sequence
● chain：It is single-stranded
● topological structure：Linearly
(b) molecule type：Protein
(c) assume：It is no
(d) antisense：It is no
(e) initial source：The flat rockfish of Xu Shi
(f) structure feature：The albumen is expected containing there are one CRD structural domains (being made of amino acid 46-175).
Sequence table SEQ ID No.2 are：
CTCATCGGCTTGTTGGCCTCCTCTCAGGCTGCAGACTGTCCAGAGGGAGAGGCGTCCTCTCTGAAGCTG
CAACTAAACTTGCTGAGGAATCGCTTCAGACACCTGTGTGACCAATACTCCAACCTGGCAACCAACTGCTCAGCTCC
AGTGATCCCCTGCGCCCAGTGTCCTGAAGGGTGGCTTGTCGTTGGGGACCAATGCTTCCTCCTCACCACTGACAGGG
ACGACTATTCTAATAGTACAAATAAGTGTGCAGAGATAGGAGCCCATCTGGCCATCTTGACCACCAAAGAACAGCAT
GATGCCGTGGAAAAAGAAGGCAAAAACATTGGAGGGATATACACGTACTACTGGATCGGACTGACTGACATTGAGAC
TGAAGGAGACTGGAGATGGGTGGACAACTCAAAACTTCGAACCCCATTTTGGGAGGCGCCGGAGCCAAACAACCACC
TGTCCGGTGGGCCGGAAGGAGAGGACTGTGCGGTGGTGCAGAGCTACACTCAGTTATGGCACGATGTTCCCTGTTCC
TTCACGTACCCACGAATCTGTCAGATGGACGCCATCCTGCCCCAG
(a) sequence signature：
● length：576
● type：Base sequence
(b) molecule type：Gene
(c) assume：It is no
(d) antisense：It is no
(e) initial source：The flat rockfish of Xu Shi
Embodiment 2
The preparation method of c-type agglutinin：
(1) structure of plasmid pLecC：
The corresponding amino acid sequence of c-type agglutinin encoding gene is analyzed, to remove encoded signal peptide and terminate close
The gene order of code is stencil design forward primer LecCF1 and reverse primer LecCR1.Using the flat rockfish cDNA of Xu Shi as template, use
Primer LecCF1 and LecCR1 carry out PCR amplification.PCR conditions are：94 DEG C of 60s pre-degeneration template DNAs, then 94 DEG C of 40s, 54 DEG C
60s, 72 DEG C of 40s are changed to 94 DEG C of 40s, 61 DEG C of 60s, 72 DEG C of 40s after 5 cycles, again in 72 DEG C of extensions after 25 cycles
7min.PCR product connects after purification with carrier pEasy-T1 (being purchased from " Quan Shijin (Beijing) Bioisystech Co., Ltd ") in room temperature
It connects 10 minutes, containing ampicillin (100 μ g/ml), Xgal (40 μ g/ml), isopropyl after connection mixed liquor conversion Escherichia coli
When culture 18-24 is small on the LB culture mediums of base-β-D- thiogalactosides (24 μ g/ml), screening transformant extraction plasmid, i.e.,
For plasmid pT1LecC.
By above-mentioned plasmid pT1LecC and plasmid pET259 (plasmid pET259 building process referring to Zheng WJ, Hu YH,
Sun L.Cloning and analysis of a ferritin subunit from turbot(Scophthalmus
maximus).Fish Shellfi sh Immunol.2010a；28 (5-6), 829-836.) restriction enzyme is used respectively
0.58kb and 5.4kb segments are recycled after EcoRV and SwaI digestions, this two segments T4DNA ligases are connected, connection liquid turns
Escherichia coli are dissolved into, when culture 18-24 is small on the LB culture mediums containing kanamycins (50 μ g/ml), screening transformant extraction matter
Grain is pLecC.
The LecCF1 is 5 '-gatatcCTCATCGGCTTGTTGGC-3 '；LecCR1 is 5 '-
gatatcCTGGGGCAGGATGGC-3’。
(2) expression and preparation of c-type agglutinin：
The plasmid pLecC conversion e. coli bl21 Transetta (DE3) of step (1) (are purchased from " Quan Shijin (Beijing)
Bioisystech Co., Ltd "), it cultivates, sieves on the LB culture mediums of the kanamycins containing 50 μ g/ml and the chloramphenicol of 50 μ g/ml
It is BL21/pLecC to select transformant.By BL21/pLecC in 37 DEG C of trainings in the LB culture mediums containing kanamycins and chloramphenicol
It supports to OD600For 0.6, add in the isopropyl-β-D-thiogalactoside of 1mM, 37 DEG C continue shaken cultivation 4-5 it is small when, then use
The nickel-nitrilotriacetic acid affinity column purification of recombinant proteins of GE Healthcare (U.S.) company.
The recombinant protein of purifying is subjected to n terminal amino acid sequencing, it was demonstrated that it is respectively amino acid sequence in sequence table SEQ ID No.1
Shown c-type agglutinin.
(3) c-type agglutinin is to the agglutination activity of bacterium
It is prepared by Vibrio vulnificus and fish enteron aisle vibrios.Vibrio vulnificus and fish enteron aisle vibrios are cultivated in LB culture mediums respectively extremely
OD600 is 0.5, then room temperature centrifuge 10 minutes, collect thalline, be suspended in TBS-Ca2+ buffer solutions (50mM Tris-Cl,
100mM NaCl, 10mM CaCl2, pH 7.5) in final concentration of 2x109cfu/ml.
The agglutination activity of agglutinin LecC.By the 10 above-mentioned bacterium solutions of μ l and the LecC (500 μ g/ml) or PBS (control) of equivalent
Mixing, when 25 DEG C of heat preservations 1 are small.Then will mixing drop on glass slide, in micro- Microscopic observation.Observe the results show recombinant C
Type agglutinin has stronger aggegation effect for Vibrio vulnificus and fish enteron aisle vibrios.
(4) application of the c-type agglutinin in disease control
10 turbot (every weighs about 10g) are randomly divided into 2 groups, every group 5.A and B groups are respectively designated as by this 2 groups.
3) the middle Vibrio vulnificus prepared is resuspended in TBS-Ca2+ buffer solutions to 2x106cfu/ml, 50 μ l bacteria suspensions is taken, adds in 50 μ l
For recombinant C-type agglutinin to final concentration of 500 μ g/ml or PBS (control), 25 DEG C keep the temperature 1h.Then every fish belly chamber of A groups is noted
Penetrate the mixed liquor of 100 μ l LecC and Vibrio vulnificus.100 μ l PBS and wound arc are injected intraperitoneally in every fish of B groups (control group)
The mixed liquor of bacterium.12 it is small when after will A and B groups fish anesthesia after take liver organization, be homogenized after weighing, homogenate be coated on LB solids
Tablet, when 28 DEG C of culture 32-48 are small.Bacterium colony, which counts, to be found, compared with PBS groups, the processing of recombinant C-type agglutinin causes wound arc
Bacterium reduces by 93.5% to the infection ability of the flat rockfish of Xu Shi.Therefore, which is expected applied to the anti-of fish bacterial disease
It controls.
The influence (× 104CFU/g) of table one, LecC to Vibrio vulnificus infection ability
| PBS groups | LecC groups |
| 6.82±0.31 | 0.45±0.05 |
Claims (3)
1. a kind of c-type agglutinant protein, which is characterized in that c-type agglutinant protein is the amino acid in sequence table SEQ ID No.1
Shown in sequence.
2. a kind of preparation method of c-type agglutinant protein described in claim 1, it is characterised in that
(1) structure of plasmid pLecC
Using the flat sturgeon cDNA of Xu Shi as template, PCR amplification is carried out with primer LecCFl and LecCRl, PCR product after purification with carrier
PEasy-T1 is built into plasmid pT1LecC, and pT1LecC and plasmid pET259 is used restriction enzyme EcoRV and SwaI respectively
0.58kb and 5.4kb segments are recycled after digestion, the T4DNA ligases connection of this two segments is built into plasmid pLecC；
(2) expression and preparation of c-type agglutinin
By the plasmid pLecC of step (1) conversion e. coli bl21 Transetta (DE3), containing kanamycins and chloramphenicol
Transformant BL21/pLecC is screened on LB culture mediums, by BL21/pLecC in the LB culture mediums containing kanamycins and chloramphenicol
In 37 DEG C of cultures, the protein induced expression of progress of isopropyl-β-D-thiogalactose former times is added in, then uses nickel-
Nitrilotriacetic acid affinity column purification of recombinant proteins is amino acid sequence in sequence table SEQ ID No.1
Shown c-type agglutinin；
Wherein, the sequence of the primer described in step (1) is：
LecCFl：5'-gatatcCTCATCGGCTTGTTGGC-3'；
LecCRl：5'-gatatcCTGGGGCAGGATGGC-3'.
3. a kind of application of c-type agglutinant protein described in claim 1 in the drug for preparing anti-Vibrio vulnificus.
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| CN106243200A (en) * | 2016-10-19 | 2016-12-21 | 上海市农业科学院 | A kind of Cordyceps militaris (L.) Link. agglutinant protein Lectin ccm4 and its preparation method and application |
| CN115925861B (en) * | 2022-10-13 | 2024-02-20 | 海南大学 | C-lectin CaCTL4E gene, protein, vector, recombinant cell of humpback bass and application thereof |
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