CN105510598A - Application of crassostrea gigas CgNatterin-3 recombinant protein - Google Patents
Application of crassostrea gigas CgNatterin-3 recombinant protein Download PDFInfo
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- CN105510598A CN105510598A CN201511025559.0A CN201511025559A CN105510598A CN 105510598 A CN105510598 A CN 105510598A CN 201511025559 A CN201511025559 A CN 201511025559A CN 105510598 A CN105510598 A CN 105510598A
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- cgnatterin
- recombinant protein
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- long oyster
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
Abstract
The invention belongs to the technical field of molecular biology and relates to application of a crassostrea gigas CgNatterin-3 recombinant protein. The crassostrea gigas CgNatterin-3 recombinant protein is obtained by using an in-vitro recombination expression technology, and pathogen associated molecular model combination activity, and pathogenic bacteria identification and agglutination activity of the crassostrea gigas CgNatterin-3 recombinant protein are claimed. The recombinant protein provided by the invention can be used for developing a pathogenic bacteria identification preparation.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to a kind of application of long oyster CgNatterin-3 recombinant protein.
Background technology:
Invertabrate lacks adaptive immune system (adaptiveimmunity), and inherent immunity (innateimmunity) therefore only can be relied on to resist cause of disease invasion.Inherent immunity kills and wounds mainly through antibacterial peptide/albumen and phagocytosis and removes pathogen, is wherein bring out immunoreactive committed step to the identification of pathogen.Cause of disease is in invasion host processes, infect with it and copy, a series of conservative element can be produced, be called pathogen-associated molecular pattern (pathogenassociatedmolecularpatterns, PAMPs), these PAMPs effectively can be identified by the pattern recognition receptors (patternrecognitionreceptors, PRRs) on host immune cell surface, and then induce a series of immune cascades to react.At present, more than ten class identification receptors have been found in invertabrate, as peptidoglycan recognition protein (PGRP) and Toll-like receptor etc., they are by completing the identification to various pathogenic microorganism in conjunction with different pathogen-associated molecular patterns, the generation of inducing cytokine and inflammation, antibacterial peptide expression, activate in a series of innate immune response such as phenol oxidase cascade reaction and phagocytosis and play a significant role.
Pattern recognition receptors (PRRs) is the identification molecule that a class is mainly expressed in inherent immunity cell surface, one or more PAMPs of identifiable design.PRRs is perception cause of disease invasion " outpost " in inherent immunity, is encoded by a limited number of germline gene, very conservative in evolution, also shows that this receptoroid is very important to the existence of biosome.Mutual identification and the effect of the pathogen-associated molecular pattern (PAMPs) on itself and causal organism surface are the keys starting innate immune response.Compare with adaptive immunity medium size lymphocyte acceptor, PRRs has four features: (1) is all encoded by germline gene; (2) composition ground is expressed; (3) tachysynthesis is caused to reply; (4) various pathogen can be identified.The research of pattern recognition receptors is the forward position hot fields of immunological investigation in recent years always, therefore the excavation of PRRs molecule and utilization are contributed to the mechanism of our clear and definite oyster immune system recognition pathogen, disclose oyster immune system bring out and remove the rule of pathogen, final is that oyster disease control, medicament research and development and fine-variety breeding are provided fundamental basis.
Summary of the invention
The object of the present invention is to provide a kind of application of long oyster CgNatterin-3 recombinant protein.
For achieving the above object, the present invention adopts technical scheme to be:
An application for long oyster CgNatterin-3 gene recombinant protein, described recombinant protein c gNatterin-3 is as the application in pathogen identification preparation.
The application of described long oyster CgNatterin-3 gene recombinant protein, described long oyster CgNatterin-3 recombinant protein has pathogen-associated molecular pattern binding activities and pathogen identification and agglutination activity, can be used for exploitation pathogen identification preparation.
The preparation of described long oyster CgNatterin-3 gene recombinant protein:
(1) with long oyster CgNatterin-3 code area for template, adopt P1 and P2 primer to carry out pcr amplification, stand-by;
Described primer P1 and P2 is respectively P1:5 '-ATGGCAGAGTGGGTATC-3 ';
P2：5’-CTAAATGACTTTATACAG-3’；
(2) pcr amplification product is connected by T4 ligase with by pEASY-E1 carrier, transforms, order-checking qualification recon;
(3) above-mentioned carrier construction is proceeded in Transetta (DE3) expression strain carry out Fiber differentiation, then purifying, namely obtain the recombinant protein of CgNatterin-3.
The advantage that the present invention has:
Long oyster CgNatterin-3 in the present invention is new ant algorithms identification receptor, and its recombinant protein has pathogen-associated molecular pattern binding activities and pathogen identification and agglutination activity.To the research of long oyster CgNatterin-3 albumen, be conducive to deep announcement long oyster inherent immunity mechanism of action, fully excavate and utilize marine source genetic resources, the disease control for marine economy cultivated animals provides important substance basis and theories integration.
Accompanying drawing explanation
The long oyster CgNatterin-3 gene recombinant protein that Fig. 1 provides for the embodiment of the present invention has stronger binding activities figure to pathogen-associated molecular pattern.
Fig. 2 has the activity figure of combination and aggegation bacterium and fungi for long oyster CgNatterin-3 gene recombinant protein that the embodiment of the present invention provides.
Embodiment:
In experimental example below, the invention will be further elaborated, but the present invention is not limited thereto.
The present invention utilizes in-vitro recombination expression technology to obtain long oyster CgNatterin-3 recombinant protein, and (No. Genbank is: EKC36293.1), discloses the pathogen-associated molecular pattern binding activities of described long oyster CgNatterin-3 gene recombinant protein, pathogen identification and agglutination activity.The recombinant protein utilizing the present invention to obtain can be used for exploitation pathogen identification preparation.
Embodiment 1: the external RT-PCR of long oyster CgNatterin-3 gene coding region is expressed, and comprises the following steps:
1, the structure of recombinant vector
The recombinant expression carrier adopted in the present invention is the pEASY-E1 prokaryotic expression carrier of Beijing Quan Shi King Company.By round pcr, use gene-specific primer P1 (5 '-ATGGCAGAGTGGGTATC-3 ') and P2 (5 '-CTAAATGACTTTATACAG-3 ').Increase the coding domain segment of long oyster CgNatterin-3 gene.Reaction conditions is: first 94 DEG C of denaturations 5 minutes, then enter following circulation: 94 DEG C of sex change 30 seconds, and 50 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, carry out 32 circulations altogether, last 72 DEG C of extensions 10 minutes.PCR primer purifying is reclaimed, is connected with pEASY-E1 carrier.After transforming, bacterium colony PCR screens and checks order, and extracts positive colony plasmid, completes the structure of carrier.
2, the expression of recombinant protein
With Transetta (DE3) for restructuring expressive host bacterial strain, be transformed into by the recombinant vector of above-mentioned structure in expressive host bacterial strain, picking monoclonal, extract plasmid, sequence verification reading frame is correct.Picking monoclonal, is inoculated in 5mLLB fluid nutrient medium, cultivates 12 ~ 16 hours in 37 DEG C of concussion shaking tables, and then with in the ratio of 1:100 (v/v) inoculation 200mLLB fluid nutrient medium, 37 DEG C are cultured to OD600:0.5 ~ 0.7.Add IPTG, make final concentration reach 0.5mMmL
-1, 16 DEG C are continued cultivation 20 hours.4 DEG C, 6000rpm, centrifugal 10min, collect thalline, frozen for subsequent use in-20 DEG C.Get 1mL bacterium liquid centrifugal, after supernatant discarded, add 80 μ L water and 20 μ L5 × albumen sample-loading buffer, 100 DEG C of boiling water boil 10 minutes, and SDS-PAGE detects expression product.
3, the purifying of recombinant protein
Mannose affinity column purifying is adopted by above-mentioned gained e. coli total protein to obtain recombinant protein, and desalination of dialysing.
Concrete operation step is as follows:
Mannose affinity column purifying obtains recombinant protein
(1) mannose affinity column, 1 × 10cm, bed volume is 7mL;
(2) with TBS damping fluid (20mMTris-base, 150mMNaCl, pH7.2), balance 3 ~ 5 bed volumes, flow velocity is 1mLmin
-1;
(3) thalline is resuspended in TBS damping fluid (20mMTris-base, 150mMNaCl, pH7.2), ultrasonication, and with 0.45 μm of membrane filtration cell pyrolysis liquid, loading, flow velocity is 1mLmin
-1;
(4) 10 bed volumes washed by TBS damping fluid 1 (20mMTris-base, 150mMNaCl, pH7.2), and flow velocity is 1mLmin
-1;
(5) TBS damping fluid 2 (20mMTris-base, 150mMNaCl, 200mM mannose, pH7.2) wash-out, flow velocity is 1mLmin
-1, collect eluent, SDS-PAGE protein isolate also carries out coomassie brilliant blue staining, the purification effect of detection fusion albumen.
(6) purifying protein is added in bag filter (3kDa aperture), be placed in distilled water and dialyse, dialyse 4 hours at every turn or spend the night, dialyse 4 times altogether, namely obtain long oyster CgNatterin-3 gene recombinant protein.
Embodiment 2: the pathogen-associated molecular pattern binding activities of long oyster CgNatterin-3 prokaryotic recombinant protein is analyzed
Long oyster CgNatterin-3 is in conjunction with the enzyme-linked immunosorbent assay (ELISA) of pathogen-associated molecular pattern: by pathogen-associated molecular pattern lipopolysaccharides, peptide glycan, mannan and β-1,3-glucosan wraps by 96 orifice plates (20 μ g/ hole) respectively, 4 DEG C are spent the night, after add 3%BSA in room temperature close 1 hour.CgNatterin-3 albumen is dissolved in PBS damping fluid, adds variable concentrations recombinant C gNatterin-3 albumen (10.00,5.00,2.50,1.25,0.63,0.32 and 0.16 μMs) to every hole, incubated at room 1 hour.PBST (PBS, 0.1%Tween-20) wash 3 times, what add rat source can identify that the antibody of CgNatterin-3 albumen is hatched, room temperature 1 hour, PBST (PBS, 0.1%Tween-20) washes 3 times, and rear horseradish peroxidase-labeled two is anti-hatches, room temperature 1 hour, PBST washes 3 times.Add horseradish peroxidase substrate to react, after 15 minutes, add 2M concentrated sulphuric acid cessation reaction, microplate reader detects every hole reactant liquor at 450nm place light absorption value (see Fig. 1).
As shown in Figure 1, enzyme-linked immunosorbent assay shows result, CgNatterin-3 to lipopolysaccharides, peptide glycan, mannan and β-1,3-glucosan there is higher binding activities.Wherein A is CgNatterin-3 and combined with lipopolysaccharide dissociation constant 1.2 × 10
-6m; B be CgNatterin-3 with peptide glycan in conjunction with dissociation constant 2.1 × 10
-7m; C be CgNatterin-3 with mannan in conjunction with dissociation constant 4.5 × 10
-8m; D is CgNatterin-3 and β-1,3-glucan binding domian dissociation constant 1.6 × 10
-7m.
Embodiment 3: the activity analysis of long oyster CgNatterin-3 combination and aggegation bacterium and fungi
By the Vibrio splindidus of cultivation, Escherichia coli, staphylococcus aureus, bacillus subtilis, Ye Luoweiya yeast and Pichia pastoris carry out collected by centrifugation, and 4% paraformaldehyde fixes 30 minutes, PBS washs thalline 3 times, and is resuspended in by thalline containing FITC (final concentration 0.1mgmL
-1) 0.01MNaHCO
3in solution, lucifuge hatches 1 hour.PBS washs 3 times, is then resuspended in PBS by bacterium.CgNatterin-3 and different thalline distinguish incubated at room 1 hour.PBS washs 3 times, adds the antibody that can identify CgNatterin-3 albumen in rat source, incubated at room 1 hour.PBS washs 3 times, adds green fluorescent label two and resists, incubated at room 1 hour.PBS washs 3 times, respectively with fluorescent microscope and flow cytomery CgNatterin-3 to the aggegation of different bacterium with in conjunction with effect (see Fig. 2).
Result such as Fig. 2 shows, and CgNatterin-3 conspicuousness can combine also aggegation Vibrio splindidus, Escherichia coli, staphylococcus aureus, bacillus subtilis, Ye Luoweiya yeast and Pichia pastoris.Wherein, A represent CgNatterin-3 can aggegation Gram-negative bacteria Vibrio splindidus, Escherichia coli, gram-positive bacteria staphylococcus aureus, bacillus subtilis, fungi Ye Luoweiya yeast and Pichia pastoris.B represents that CgNatterin-3 can in conjunction with Gram-negative bacteria Vibrio splindidus, Escherichia coli, gram-positive bacteria staphylococcus aureus, bacillus subtilis, fungi Ye Luoweiya yeast and Pichia pastoris.
Claims (2)
1. an application for long oyster CgNatterin-3 gene recombinant protein, is characterized in that: described recombinant protein c gNatterin-3 is as the application in pathogen identification preparation.
2., by the application of long oyster CgNatterin-3 gene recombinant protein according to claim 1, it is characterized in that: the preparation of described long oyster CgNatterin-3 gene recombinant protein:
(1) with long oyster CgNatterin-3 code area for template, adopt P1 and P2 primer to carry out pcr amplification, stand-by;
Described primer P1 and P2 is respectively P1:5 '-ATGGCAGAGTGGGTATC-3 ';
P2：5’-CTAAATGACTTTATACAG-3’；
(2) pcr amplification product is connected by T4 ligase with by pEASY-E1 carrier, transforms, order-checking qualification recon;
(3) above-mentioned carrier construction is proceeded in Transetta (DE3) expression strain carry out Fiber differentiation, then purifying, namely obtain the recombinant protein of CgNatterin-3.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201511025559.0A CN105510598A (en) | 2015-12-31 | 2015-12-31 | Application of crassostrea gigas CgNatterin-3 recombinant protein |
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| CN201511025559.0A CN105510598A (en) | 2015-12-31 | 2015-12-31 | Application of crassostrea gigas CgNatterin-3 recombinant protein |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586186A (en) * | 2007-02-23 | 2012-07-18 | 贝勒研究院 | Activation of human antigen-presenting cells through CLEC-6 |
| CN104861056A (en) * | 2015-06-16 | 2015-08-26 | 中国科学院海洋研究所 | Recombinant protein CgC1qDC-1 of pacific oyster complement molecule, as well as preparation and application of recombinant protein CgC1qDC-1 |
-
2015
- 2015-12-31 CN CN201511025559.0A patent/CN105510598A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586186A (en) * | 2007-02-23 | 2012-07-18 | 贝勒研究院 | Activation of human antigen-presenting cells through CLEC-6 |
| CN104861056A (en) * | 2015-06-16 | 2015-08-26 | 中国科学院海洋研究所 | Recombinant protein CgC1qDC-1 of pacific oyster complement molecule, as well as preparation and application of recombinant protein CgC1qDC-1 |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "Crassostrea gigas unplaced genomic scaffold482，whole genome shotgun sequence GenBank：JH816607.1", 《NCBI》 * |
| GENBANK: "Natterin-3[Crassostrea gigas] GenBank:EKC36293.1", 《NCBI》 * |
| ZHAOQUN LIU ET AL.: "The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas", 《FISH & SHELLfiSH IMMUNOLOGY》 * |
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Application publication date: 20160420 |