CN105861475B - A kind of recombinant protein of fish MBL associated serine protease 2 and its application - Google Patents
A kind of recombinant protein of fish MBL associated serine protease 2 and its application Download PDFInfo
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- CN105861475B CN105861475B CN201610305025.1A CN201610305025A CN105861475B CN 105861475 B CN105861475 B CN 105861475B CN 201610305025 A CN201610305025 A CN 201610305025A CN 105861475 B CN105861475 B CN 105861475B
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- serine protease
- recombinant protein
- associated serine
- mbl
- csmasp1
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/6408—Serine endopeptidases (3.4.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21106—Hepsin (3.4.21.106)
Abstract
The present invention relates to field of immunology, the recombinant protein of specifically a kind of fish (Cynoglossus semilaevis) MBL associated serine protease 2 and its application.Fish MBL associated serine protease 2 CsMASP1 recombinant protein is the MBL associated serine protease 2 CsMASP1 recombinant protein of Cynoglossus semilaevis, it is using Cynoglossus semilaevis cDNA as template, PCR amplification MBL associated serine protease 2 CsMASP1 gene is carried out with primer MASPF1 and MASPR1, amplified production is recombinant protein through inducing expression, purifying.The MBL associated serine protease 2 being capable of significant immune cell activated.
Description
Technical field
The present invention relates to field of immunology, specifically a kind of fish (Cynoglossus semilaevis) MBL associated serine protease 2
Recombinant protein and its application.
Background technique
Lectin complement approach is an important channel of complement activation, and the activation of lectin pathway is in host's innate immunity
Important defense reaction is played in the process.MBL associated serine protease 2 (mannose-binding lectin-
Associated serine protease, MASP) be lectin pathway central element.In lectin pathway, mannose
Binding lectin (mannose-binding lectin, MBL) and the identification of pathogenic microorganism surface receptor molecule combine, into one
Step sequential activation MASP in such a way that Ca2+ is relied on, then c4 cleavage and C2, form C3 convertase, and C3 convertase is cracked into C3
C3a and C3b, C3b can form C5 convertase in conjunction with C4b2a.C5 is cracked into C5a and C5b by C5 convertase, C5b and C6, C7,
C8, C9 form membrane attack complex, to play natural anti-infectious immunity effect.Meanwhile MASP also has thrombin activity, it can
Crack Hageman factor and fibrinogen, discharge chemotactic factor (CF) fibrinopeptide B, the latter be leucocyte, fibroblast and
The chemotactic factor (CF) of monocyte.But the research of fish MASP is now also in the stage identified with derived components, function
Deng all unclear.
Summary of the invention
It is an object of that present invention to provide a kind of recombinant protein of fish MBL associated serine protease 2 and its applications.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of recombinant protein of fish MBL associated serine protease 2, it is characterised in that: fish MBL Associated Serine egg
White enzyme CsMASP1 recombinant protein is the MBL associated serine protease 2 CsMASP1 recombinant protein of Cynoglossus semilaevis, is sliding with half
Tongue sole cDNA is template, carries out PCR amplification M BL associated serine protease CsMASP1 base with primer MASPF1 and MASPR1
Cause, amplified production are recombinant protein through inducing expression, purifying, and the MASPF1 is 5 '-
GATATCATGGTGGAACTCAGGGCGTTATA-3′；MASPR1 is 5 '-GATATCTGTAAAGAGAGTGAGGGACTCAG-3 '.
A kind of application of the recombinant protein of fish MBL associated serine protease 2, the MBL associated serine protease 2 of the Cynoglossus semilaevis
Application of the CsMASP1 recombinant protein in preparation activation peripheral blood lymphocytes preparation.
The MBL associated serine protease 2 CsMASP1 recombinant protein of the Cynoglossus semilaevis, is to be with Cynoglossus semilaevis cDNA
Template carries out PCR amplification MBL associated serine protease 2 CsMASP1 gene, amplified production warp with primer MASPF1 and MASPR1
Inducing expression, purifying are recombinant protein, and the MASPF1 is 5 '-GATATCATGGTGGAACTCAGGGCGTTATA-3 '；
MASPR1 is 5 '-GATATCTGTAAAGAGAGTGAGGGACTCAG-3 '.
The present invention has the advantage that MBL associated serine protease 2 of the invention, it can be from molecular level and cellular water
Head up display writes immune cell activated.
Detailed description of the invention
Fig. 1 is MBL associated serine protease 2 recombinant protein (being named as rCsMASP1) electricity provided in an embodiment of the present invention
Swimming figure.Swimming lane 1, molecular weight standard；Swimming lane 2, MBL associated serine protease 2 rCsMASP1.
Fig. 2 is that recombination MBL associated serine protease 2 rCsMASP1 provided in an embodiment of the present invention expresses immunogene
Effect.Cynoglossus semilaevis peripheral blood lymphocytes (peripheral blood leukocytes, PBL) and rCsMASP1 are incubated for
Pass through the expression quantity of absolute fluorescence quantitative PCR detection immunogene afterwards.
Specific embodiment
Below 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.
Involved routinely experimental method is all made of following method in embodiments of the present invention:
1. plasmid extraction, DNA (PCR) product purification, DNA fragmentation are recycled all from gel using " Tiangeng biochemical technology (north
Capital) Co., Ltd " corresponding reagent box.
2. Escherichia coli Hanahan method (Sambrook and Russell:Molecular Cloning:A
Laboratory Mannual.Cold Spring Harbor Laboratory Press 2001)。
3. all restriction enzymes and ligase are purchased from " knob Great Britain Bioisystech Co., Ltd ", Beijing.
Embodiment 1
The preparation of MBL associated serine protease 2 recombinant protein rCsMASP1
1) building of the plasmid pCsMASP1 of MBL associated serine protease 2 recombinant protein rCsMASP1 is expressed:
MBL associated serine protease 2 CsMASP1 of the invention is Cynoglossus semilaevis MBL associated serine protease 2, sequence
Column have announced (GenBank accession number 008316896.1):
MTRCCVLVLYFLLLRGSVCVELRALYGSFTSPDFPQPYPDHQHMQWNIIVPDGHRVKLYFTHFSVEPSD
QCEYDYIQVLAEGNETLRFCGEENKNHESTPGNTIILSAGNLMSVVFRSDYSNEGRFTGFRAFYTSEDIDECLSKVD
GEKVCDHFCHNFIGGYYCSCRQGFHLHANNRSCTGPCHNQVLTSPSGVLTSPGYPNPYPPMSQCDHTIRLPEGHRIF
LDFLSPFDIEGHPNVPCPYVMLKISTVGQEYGPFCGSTPPARIDTGSYQVHIQFRSDTSGKNRGWKMNYTSAKAESL
TLFT
CsMASP1 is made of 304 amino acid, and there are two CUB structural domain (amino acid 8-117 and 165-276) for tool.
Using Cynoglossus semilaevis cDNA as template, PCR amplification is carried out with primer MASPF1 and MASPR1 and mends CsMASP1 gene.PCR
Condition are as follows: 94 DEG C of 60s initial denaturation template DNAs, then 94 DEG C of 40s, 62 DEG C of 60s, 72 DEG C of 65s, again 72 after 30 circulations
DEG C extension 7-10min.The corresponding reagent box of PCR product Tiangeng purifies.By expression vector pET259 (building process referring to
Hu YH, Zheng WW, Sun L.Identification and molecular analysis of a ferritin
subunit from red drum (Sciaenops ocellatus).Fish Shellfish Immunol 2010；28:
678-86) with 5.3kb segment is recycled after restriction enzyme EcoRV digestion, by the PCR product T4DNA of itself and above-mentioned purifying
Ligase connection, connection liquid are transformed into bacillus coli DH 5 alpha, cultivate 18- on the LB culture medium containing kanamycins (100ug/ml)
24 hours, screening transformant extracted plasmid, was named as pCsMASP1.By DNA sequencing analytical proof pCsMASP1 be containing
The expression plasmid of the MBL associated serine protease 2 CsMASP1 sequence of above-mentioned C5 structural domain.
The LB constituent is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride,
97.5% distilled water.The MASPF1 is 5 '-GATATCATGGTGGAACTCAGGGCGTTATA-3 '；MASPR1 is 5 '-
GATATCTGTAAAGAGAGTGAGGGACTCAG-3′。
2) expression and purity of MBL associated serine protease 2 CsMASP1 recombinant protein
Above-mentioned plasmid pCsMASP1 conventional method is converted into e. coli bl21 (DE3) and (is purchased from " Tiangeng biochemistry section
Skill Co., Ltd ", Beijing), cultivated 18-24 hours on the LB solid medium of (50ug/ml) containing kanamycin, picking turns
Beggar is named as BL21/pCsMASP1.BL21/pCsMASP1 is trained in the LB liquid of (50ug/ml) containing kanamycin
It supports and is incubated overnight in base；The LB liquid of 100ml fresh (50ug/ml) containing kanamycin is added in culture solution after taking 1ml to stay overnight
In body culture medium, revolving speed 200rpm shakes culture to OD at 37 DEG C600Be 0.6, be added the IPTG of final concentration of 1mM, 20 DEG C after
It is continuous that culture 14h is shaken with revolving speed 160rpm, then with 5000g, 4 DEG C of centrifugation 10min, bacterium solution is collected, 5ml lysate is added,
Room temperature on shaking table in slowly shaking 1-2 hours, until bacteria suspension becomes clarification.By bacterium solution with 10000g, 4 DEG C of centrifugation 30min,
Recycle supernatant.Albumen affinity column His Trap HP Columns in supernatant (is purchased from U.S. GE Healthcare
Company) recovery purifying obtains rCsMASP1 albumen.By the albumen of purifying through SDS-PAGE electrophoresis detection (electrophoresis 25- under 8v/cm voltage
30min, electrophoresis 2-2.5h under subsequent 15v/cm voltage), measure its molecular size range (referring to Fig. 1).It was found that its protein matter
It measures identical as MBL associated serine protease 2 CsMASP1.
The lysate is the 10mM NaH of final concentration2PO4, 10mM Tris and 8M urea, pH 8.0.
Embodiment 2
Effect of the rCsMASP1 to respiratory burst
Cynoglossus semilaevis peripheral blood lymphocytes (peripheral blood is prepared using Percoll method
Leukocytes, PBL), referring specifically to document Zhou ZX, Zhang J, Sun L.Poly (I: C) induces antiviral
immune responses in Japanese flounder(Paralichthys olivaceus)that require
TLR3 and MDA5 and is negatively regulated by Myd88.PLoS One 2014；9:e112918.It will
Cell is placed in 96- hole plate (every hole 10 containing L-15 culture medium (being purchased from Thermo Scientific HyClone, Beijing)5
A cell), the rCsMASP1 to final concentration of 80ug/ml of the purifying of above-described embodiment 1 is added in every hole.Same body is added in control cell
Long-pending PBS.By cell in 22 DEG C of heat preservation 1h.Then with the respiratory burst activity of conventional method detection cell.Referring specifically to document
Chung S, Secombes CJ.Analysis of events occurring within teleost macrophages
during the respiratory burst.Comp Biochem Physiol 1988；89:539-544.The result shows that with
Control cell is compared, and the respiratory burst activity with the rCsMASP1 cell handled is 2.1 times of control cell, significant (P <
0.01) it is higher than control cell.
The PBS constituent is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%
Na2HPO4.12H2O, 0.024%NaH2PO4, surplus is water.
Embodiment 3
Effect of the rCsMASP1 to cell migration
The rCsMASP1 albumen that above-described embodiment 1 purifies is diluted to 80ug/ml, as rCsMASP1 dilution in PBS
Liquid.24 hole Costar Transwell are added in 600ul rCsMASP1 dilution or PBS (control) and (are purchased from Coming
Costar Co., the U.S.) plate lower slot in, by 100ul (105Cell) above-described embodiment 2 obtain PBL be added 24 holes
In the upper slot of Costar Transwell, 22 DEG C of heat preservation 40min.Then migrated by micro- sem observation to the PBL cell of lower slot
Number.Referring specifically to document Li YX, Sun JS, Sun L.An inflammatory CC chemokine of CynoglosSus
semilaevis is involved in immune defense against bacterial infection.Fish
Shellfish Immunol 2011；31 (3): 446-52.The result shows that compared with the control, the cell of rCsMASP1 induction moves
It moves index and increases 4.5 times, significant difference (P < 0.01), these results illustrate, rCsMASP1 of the invention has chemotaxis,
PBL migration can be remarkably promoted.
Embodiment 4
The effect that rCsMASP1 expresses immunogene
By the rCsMASP1 dilution or PBS (control) of 100ul above-described embodiment 3 and 100ul (105Cell) above-mentioned implementation
The PBL mixing that example 2 obtains, 22 DEG C of heat preservation 1h.Using EZNA Total RNA kit (be purchased from Omega Bio-tek,
Doraville, the U.S.) total serum IgE is extracted, immunogene table is detected for cDNA preparation and absolute fluorescence quantitative PCR (qRT-PCR)
It reaches.Referring specifically to document Zheng WJ, Sun L.Evaluation of housekeeping genes as references
for quantitative real time RT-PCR analysis of gene expression in Japanese
flounder(Paralichthys olivaceus).Fish Shellfish Immunol.2011；30:638-45.It is detected
Immunogene are as follows: interleukin-11 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), hole R sample receptor 9 (TLR9), tumour
Necrosis factor-alpha (TNF α), C reactive protein (CRP) and Myd88.The result shows that rCsMASP1 can significantly increase all inspections
The expression of cls gene (see Fig. 2).These results indicate that rCsMASP1 has significant immunoregulation effect.
In conclusion rCsMASP1 can significantly increase respiratory burst, cell migration and the immunogene expression of PBL,
Illustrate that rCsMASP1 has the application of activation PBL.
Claims (2)
1. a kind of application of the recombinant protein of fish MBL associated serine protease 2, it is characterised in that: the MBL phase of Cynoglossus semilaevis
Close serine protease CsMASP1 recombinant protein preparation enhancing lymphocyte from peripheral blood of fish respiratory burst, cell migration with
And the application in immunogene expression preparation；
The amino acid sequence of the MBL associated serine protease 2 CsMASP1 of Cynoglossus semilaevis is as shown in sequence 1.
2. the application of the recombinant protein of fish MBL associated serine protease 2 according to claim 1, it is characterised in that: institute
The MBL associated serine protease 2 CsMASP1 recombinant protein for stating Cynoglossus semilaevis is using Cynoglossus semilaevis cDNA as template, with drawing
Object MASPF1 and MASPR1 carry out PCR amplification MBL associated serine protease 2 CsMASP1 gene, amplified production through inducing expression,
Purifying is recombinant protein, and the MASPF1 is 5 '-GATATCATGGTGGAACTCAGGGCGTTATA -3 '；MASPR1 is
5’- GATATCTGTAAAGAGAGTGAGGGACTCAG-3’。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509809A (en) * | 2013-09-25 | 2014-01-15 | 中山大学 | Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof |
| CN105541987A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院海洋研究所 | Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509809A (en) * | 2013-09-25 | 2014-01-15 | 中山大学 | Branchiostoma belcheri chitin-binding associated serine protease CASP gene for identifying chitin and application thereof |
| CN105541987A (en) * | 2016-02-24 | 2016-05-04 | 中国科学院海洋研究所 | Recombinant protein of cynoglossus semilaevis complement component C5a and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CsMAP34, a teleost MAP with dual role: A promoter of MASP-assisted complement activation and a regulator of immune cell activity;Mo-fei Li，et al;《Scientific Reports》;20161223;1-14 * |
| Transcriptome analysis revealed changes of multiple genes involved in immunity in Cynoglossus semilaevis during Vibrio anguillarum infection;Xiang Zhang,et al;《Fish & Shellfish Immunology》;20141225;第43卷;209-218 * |
| XP_008316896.1;unknown;《GENBANK》;20140619;1 * |
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