CN109576275A - A kind of Cynoglossus semilaevis antibacterium disease related gene and its application method - Google Patents
A kind of Cynoglossus semilaevis antibacterium disease related gene and its application method Download PDFInfo
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Abstract
The present invention provides a kind of antibacterium disease related gene screened from Cynoglossus semilaevis, and the nucleotides sequence of the gene is classified as SEQ ID NO:1, and the sequence of the albumen of coding is SEQ ID NO:2.Gene provided by the invention is related to Cynoglossus semilaevis antibacterium disease, and the expression quantity in intestines is related to fish body disease resistance, and expression is higher, and fish body disease resistance is stronger；The albumen of its in-vitro recombination expression can obviously inhibit the proliferation of several Gram-negative bacterias, therefore, have application value in terms of the disease-resistant family selective breeding of Cynoglossus semilaevis, disease control, feed addictive and fungicide.
Description
Technical field
The invention belongs to aquatic products Biotechnology in Genetic Breeding fields, and in particular to a kind of Cynoglossus semilaevis antibacterium disease related gene and
Its application method.
Background technique
Cynoglossus semilaevis (Cynoglossus semilaevis) is mainly distributed on China's Coastal Areas, is the important sea in China
The economic cultured fishes of water, it is delicious meat, full of nutrition, it is deep to be liked by the majority of consumers, there is the good market demand and support
Grow potentiality to be exploited.However, the reasons such as intensive aquaculture model and environmental pollution of high density, cause Cynoglossus semilaevis ascites, rotten
The bacteriosis such as fin, rotten tail are frequently broken out, and especially skin caused by Vibrio harveyi etc. festers the most serious, are caused
Huge economic loss seriously hinders the development of cynoglossus semilaevis cultivation industry.Currently, for the anti-of fish Vibrio harveyi disease
It controls, depends on antibiotic, but the problems such as the abuse of drug can not only generate medicament residue, drug resistance and environmental pollution, it is right
There is also potential threats for human health.In recent years, people urgently find always the drug that can replace conventional antibiotic, and
With the appearance of more and more antibiotics resistance bacterial strains, so that antibacterial protein is in culture fishery, pharmaceuticals industry and food additive
The fields such as agent are added widely to be paid attention to.
Antibacterial protein is the large biological molecule with antibacterial activity encoded by gene, is the important set of life system of defense
At part.Its main function is the invasion for resisting pathogenic microorganism, improves the disease resistance of organism itself, safeguards vital movement
Continuity and normal operation.Antibacterial protein not only resists effect to gram-positive bacteria and Gram-negative bacteria, moreover it is possible to inhibit
Or kill fungi, mould and virus etc..People have isolated and purified from microorganism, plant, animal and human body to a variety of antibacterial eggs
White and synthesized some antibacterial peptides using genetic engineering recombinant technique, the antibacterial protein of separate sources becomes in aquaculture field
It obtains more and more important.Resist studies have shown that adding antibacterial peptide Mytilin in the breeding feed of Penaeus monodon and can significantly improve it
Virus infection ability；Antibacterial peptide is added in feed can significantly improve the disease resistance of litopenaeus vannamei and Penaeus Vannmei；
Recombinant antibacterial peptide Chelonianin can efficiently control the inflammation generation of infection vibrio harveyi Tilapia mossambica and can improve it and survive
Rate.The research of antibacterial protein makes fast progress in recent years, and increasingly mature with protein research technology, antiseptic protein is
An effective way as the exploitation of disease-resistant new gene.
Have some researchs about Cynoglossus semilaevis gene involved in immunity at present to report, mainly passes through homologous clone method
Obtain the homologous gene for the gene involved in immunity reported in mammal.Such as interferon, agglutinin, histocompatibility complex
Family, cytokine family, complement factor, tumor necrosis factor, chemotactic factor (CF), lysozyme, transcription factor, actin activation
Albumen (dctn5), signal transduction and transcriptional activation activate sub (stat5), R-spondins secretion whiting etc..However, due to kind
The presence of group specificity, the albumen of these homologous genes encodings not necessarily have the function of anti pathologic immunity in fish such as Cynoglossus semilaevis
Can, especially not necessarily have the effect for resisting Cynoglossus semilaevis specific pathogen bacterium.In addition, above-mentioned gene involved in immunity is in half sliding-tongue
There is not been reported for the expression and difference of the disease-resistant family of sole and susceptible family, is difficult to be developed as Cynoglossus semilaevis antibacterium disease
The significant gene of ability.So the related gene of the anti-specific pathogen bacterium of Cynoglossus semilaevis itself is screened, it is preferably sliding from half
Tongue sole itself is screened.In consideration of it, in conjunction with the full-length genome of the disease-resistant family of Cynoglossus semilaevis and not disease-resistant family weight sequencing data
New tissue is screened using bioinformatics techniques such as whole-genome associations (GWAS) with immuning tissue's transcript profile data
It is very important for gene specific expressed, with antibacterium disease correlation function and disease-resistant family predominant expression.Further
The function and its mechanism of action in anti pathologic immunity of such Cynoglossus semilaevis antibacterium disease related gene are studied, it is explored
The technological approaches applied in Cynoglossus semilaevis breeding for disease resistance and disease prevention and control etc. is extremely urgent, can be Cynoglossus semilaevis breeding for disease resistance
It provides fundamental basis and genetic marker or gene product with disease prevention and control.This is for the fish such as Cynoglossus semilaevis breeding for disease resistance and disease
Prevention and control are of great significance and application value.
Summary of the invention
The present invention provides a kind of Cynoglossus semilaevis antibacterium disease related gene and its for the first time as premunition evaluation mark
Using preparation and reorganization albumen and the bacteriostatic activity for identifying recombination expression product.Gene is provided for the disease-resistant fine-variety breeding of Cynoglossus semilaevis
Sequence and genetic marker, and the research and development for green feed additive and antibacterial agent provide technical method.
The present invention provides a kind of antibacterium ospc gene screened from Cynoglossus semilaevis, and the sequence of the gene coded protein is
SEQ ID NO:2；
The gene, a kind of specific nucleotides sequence are classified as SEQ ID NO:1；
Gene provided by the present invention is applicable in the marker gene for doing Cynoglossus semilaevis antibacterium disease Genealogy screening.
The present invention also provides application of the above-mentioned antibacterium disease related gene in the detection of Cynoglossus semilaevis premunition；
It is to detect the gene in Cynoglossus semilaevis the present invention provides a kind of method for detecting above-mentioned antibacterium disease related gene
The amount expressed in intestines；Its expression quantity is higher, and antibacterium disease ability is stronger.
The detection primer that the method uses, a kind of sequence information are as follows:
Upstream primer: 5 '-GACCAACTGCCACCATACTCC-3 ' (SEQ ID NO:3)
Downstream primer: 5 '-TCGTCCTCCAAGCCAAACC-3 ' (SEQ ID NO:4)；
The present invention provides the recombinant strains for recombinantly expressing above-mentioned albumen；
Albumen of the invention is used to prepare the product of Cynoglossus semilaevis Vibrio harveyi disease prevention and treatment；
Albumen of the invention is also used to prepare feed addictive and antibacterial agent.
Gene provided by the invention is related to immune disease-resistance, and the albumen of in-vitro recombination expression can obviously inhibit gram
The proliferation of negative bacterium, therefore, Cynoglossus semilaevis disease control, feed addictive and in terms of have application value.
Detailed description of the invention
Fig. 1: the amino acid sequence of cDNA full length sequence and the open reading frame coding of Cynoglossus semilaevis antibacterium disease related gene
Column:
SEQ ID NO:1: Cynoglossus semilaevis antibacterium disease Related cDNAs full length sequence.
SEQ ID NO:2: the amino acid sequence of Cynoglossus semilaevis antibacterium disease related open reading frame codes.
Fig. 2: expression of the antibacterium disease related gene in each tissue of Cynoglossus semilaevis health adult fish:
Wherein, 2-1:RT-PCR half-quantitative detection antibacterium disease related gene is in each tissue of Cynoglossus semilaevis health adult fish
Expression.The english abbreviation wherein organized: L: liver；Sp: spleen；K: kidney；I: intestines；Gi: the gill；H: heart；Br: brain；
St: stomach；Bl: blood；Hk: head-kidney.
Expression of the 2-2:qRT-PCR quantitative detection gene in each tissue of Cynoglossus semilaevis health adult fish.It is wherein horizontal
The different tissue of coordinate representation Cynoglossus semilaevis, ordinate indicates expression quantity of the gene in different tissues, wherein the English respectively organized
The same Fig. 2-1 of text abbreviation.
Fig. 3: expression of the Cynoglossus semilaevis antibacterium disease related gene in Vibrio harveyi premunition linked groups point
Analysis.Abscissa indicates different time points after infection, and ordinate indicates expression quantity of the antibacterium disease related gene in different tissues.
6 tissues are had detected altogether, comprising: intestines (Intestine), the gill (Gill), skin (Skin), kidney (Kidney), liver
(Liver), spleen (Spleen).
Fig. 4: the inducing expression of the recombinant protein of Cynoglossus semilaevis antibacterium disease related gene is isolated and purified and is identified:
(A) recombinant protein inducing expression, M: albumen marker, 1: the non-inducing expression of control group, 2: control group inducing expression,
3: the non-inducing expression of recombinant protein group, 4: recombinant protein group inducing expression；
(B) recombinant protein soluble analysis and purifying, M: large molecular weight protein marker, 1: supernatant after ultrasonic disruption,
2: being precipitated after ultrasonic disruption；
(C) after recombinant protein purification and dialysis, M: macromolecule marker, 1: recombinant protein after purifying and dialysis.
Fig. 5: the external bacteriostatic activity analysis chart of recombinant protein:
(1) recombinant protein of antibacterium disease related gene inhibits the signal of Vibrio harveyi (Vibrio harveyi) effect
Figure, 0 is PBS as negative control group；1 is the ampicillin of 10mg/L as positive controls；2,3,4 is dense for 3 differences
The recombinant protein of degree is as experimental group: wherein 2 be 0.6mg/ml, and 3 be 0.12mg/ml, and 4 be 0.054mg/ml.
(2) recombinant protein of antibacterium disease related gene inhibits vibrio parahemolyticus (Vibrio Parahemolyticus)
Effect diagram, 0 is PBS as negative control group；1 is the ampicillin of 10mg/L as positive controls；2,3,4 be 3
The recombinant protein of a various concentration is as experimental group: wherein 2 be 0.6mg/ml, and 3 be 0.12mg/ml, and 4 be 0.054mg/ml.
(3) recombinant protein of antibacterium disease related gene inhibits tarda (Edwardsiella tarda) effect to show
It is intended to, 0 is PBS as negative control group；1 is the ampicillin of 10mg/L as positive controls；2,3,4 be 3 differences
The recombinant protein of concentration is as experimental group: wherein 2 be 0.6mg/ml, and 3 be 0.12mg/ml, and 4 be 0.054mg/ml.
Fig. 6: expression of the Cynoglossus semilaevis antibacterium disease related gene in 2014 are disease-resistant and susceptible family is respectively organized:
Wherein 6-1:RT-PCR half-quantitative detection Cynoglossus semilaevis antibacterium disease related gene is disease-resistant in Cynoglossus semilaevis in 2014
Expression quantity in respectively being organized with susceptible family.Before the English alphabet abbreviation respectively organized plus r indicates disease-resistant family, and preceding plus s indicates easy
Feel family, L: liver, Sp: spleen；K: kidney；Gi: the gill；I: intestines；Bl: blood.
6-2:qRT-PCR quantitative detection Cynoglossus semilaevis antibacterium disease related gene Cynoglossus semilaevis in 2014 it is disease-resistant with it is susceptible
Family respectively organize in expression quantity.Wherein abscissa indicates that Cynoglossus semilaevis different tissues, ordinate indicate gene in different tissues
Expression quantity.SF (Susceptible family) indicates susceptible family, is indicated with black cylindricality, RF (Resistant
Family it) indicates disease-resistant family, is indicated with grey cylindricality；Gi: the gill；I: intestines；Bl: blood；K: kidney；Sp: spleen；L: liver.
Fig. 7: Cynoglossus semilaevis antibacterium disease related gene is in Cynoglossus semilaevis in 2017 is disease-resistant and susceptible family is respectively organized
Expression:
7-1:RT-PCR half-quantitative detection Cynoglossus semilaevis antibacterium disease related gene is disease-resistant each with susceptible family in 2017
Expression quantity in tissue.Before the English alphabet abbreviation wherein organized plus r indicates disease-resistant family, and preceding plus s indicates susceptible family, L: liver
It is dirty, Sp: spleen；K: kidney；Gi: the gill；I: intestines.
Expression of the 7-2:qRT-PCR quantitative detection Cynoglossus semilaevis gene in 2017 disease-resistant and susceptible family is respectively organized
Amount.Wherein abscissa indicates that the different tissue of Cynoglossus semilaevis, ordinate indicate gene in the expression quantity of different tissues.SF
(Susceptible family) indicates susceptible family, is indicated with black cylindricality, and RF (Resistant family) indicates disease-resistant
Family is indicated with grey cylindricality；Gi: the gill；I: intestines；K: kidney；Sp: spleen；L: liver.
Specific embodiment
It is clear in order to be more clear the purpose of the present invention, technology path and advantage, the present invention is lifted with reference to the accompanying drawing
Example is described in detail.
Embodiment 1: the clone of Cynoglossus semilaevis antibacterium disease related gene full-length cDNA and its sequence analysis
Applicant Chen Songlin etc. is first by the Cynoglossus semilaevis screened in the past anti-Vibrio harveyi patient and his family system and easily
Sense family resurveys sequence using conventional method progress full-length genome and is associated with (GWAS) analysis with full-length genome, it was found that with Cynoglossus semilaevis
The SNP marker of the anti-Vibrio harveyi disease linkage of characters, and further navigate to the SNP site in one segment, then should
Segment is verified by RT-PCR and RACE-PCR amplification, obtains the full length cDNA sequence of gene.
(1) experimental method: Trizol method extracts the synthesis of RNA, cDNA, PCR amplification
Trizol method extracts RNA: taking the tissue block of about 100mg or so, is put into the nothing for having added 500 μ L Trizol reagents
In enzyme centrifuge tube, ground on ice with grinding rod；After organizing to grind, then 500 μ L Trizol reagents are added, stood 4 after 10min
DEG C/12000rpm centrifugation 10min, abandon precipitating；200 μ L chloroforms are added, standing 5min is allowed to mix well after being aggressively shaken 30s, and 4
DEG C, 12000rpm is centrifuged 15min；Isometric isopropanol is added in Aspirate supernatant, is stored at room temperature 10min after mixing well, and 4
DEG C, 12000rpm is centrifuged 10min, abandons clear liquid；Again with 75% ethanol washing twice, 12000rpm is centrifuged 3min and small at 4 DEG C
The heart abandons supernatant；It is placed at room temperature for dry 5min and eliminates ethyl alcohol as far as possible；Appropriate RNase-Free H is added2O, RNA precipitate is abundant
Dissolution；Total serum IgE quality, RNA/DNA spectrophotometric determination RNA purity and concentration are detected with agarose gel electrophoresis；It will extract
RNA be placed on -80 DEG C of preservations.
CDNA synthesis: the RNA of extraction being operated according to TaKaRa reverse transcription reagent box specification and carries out reverse transcription, synthesis
CDNA-20 DEG C of preservation.
PCR amplification: it based on the fragment sequence that SNP marker defines, is disposably amplified in longer by RT-PCR
Between segment, then 5 ' ends and 3 ' end sequences are amplified with RACE round pcr.
RACE PCR reaction is divided into two-wheeled.Specific step is as follows:
One wheel: Touchdown PCR reaction system and reaction condition are as follows:
Two wheels: nested PCR reaction system and reaction condition are as follows:
* the PCR product of the first round dilutes 50 or 100 times of amplification templates as the second wheel.
PCR product gel extraction is taken turns by two, PCR product is connect with cloning vector pEASY-T1 after purification, by positive colony
It is sequenced.Gene cloning step is operated according to pEASY-T1 carrier specification.(3) acquisition of full-length cDNA and homology analysis
The splicing and comparison analysis of sequence: the sequence that sequencing obtains is spliced with DNA Star software, is finally obtained
Full length cDNA sequence；It is found with ORF finder and translates open reading frame；The cDNA overall length for finally obtaining the gene is
1835bp (SEQ ID NO:1), 5 ' the decomplier areas including 21bp, the open reading frame of 1545bp and the 3 ' decompliers of 269bp
Area.The open reading frame of the gene encodes the albumen (SEQ ID NO:2) (Fig. 1) of 514 amino acid.
Embodiment 2: Cynoglossus semilaevis antibacterium disease related gene is analyzed in the expression of normal fish different tissues
(1) it designs quantitative primer and verifies the specificity of primer
According to antibacterium disease related gene sequence purpose of design gene quantification amplimer, regular-PCR amplification, electrophoresis are carried out
Detection, cloning and sequencing, if electrophoretic band is single bright, and negative control, without band, cloning and sequencing analysis is purpose gene order,
Tentatively conclude this primer specific；In addition machine on quantitative fluorescent PCR, observation peak figure, CT value and amplification efficiency, if fluorescence are carried out again
Quantitative PCR finds that peak figure is single, and the amplification efficiency of reference gene and target gene is almost consistent, it was demonstrated that primer specific,
It can be used to carry out real-time quantitative PCR experiment.The quantitative primer of Actin gene is originated from the article that the laboratory Chen Songlin has been delivered.
Primer sequence is as follows:
Upstream primer: 5 '-GACCAACTGCCACCATACTCC-3 '
Downstream primer: 5 '-TCGTCCTCCAAGCCAAACC-3 '
(2) antibacterium disease related gene is detected in the expression water of different tissues with quantitative fluorescent PCR and semiquantitive PCR method
It is flat
Take 9 of healthy Cynoglossus semilaevis fish to organize first, respectively liver, spleen, kidney, intestines, the gill, head-kidney, stomach, brain,
Blood extracts RNA and reverse transcription into cDNA with Trizol method.
Quantitative fluorescent PCR: quantitative fluorescent PCR reaction detection antibacterium disease related gene is carried out at each group with above-mentioned primer
Relative expression quantity in knitting.Reaction system are as follows: 10 μ L SYBR, 0.4 μ L primer-F, 0.4 μ LPrimer-R, 0.4 μ L Rox
RD II, 1 μ L cDNA template, 7.8 μ L ddH20.Reaction condition are as follows: 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.
Semiquantitive PCR: the cDNA obtained using reverse transcription is carried out first with the quantitative primer of reference gene actin as template
Common PCR reaction, electrophoresis detection.Template concentration and dosage are adjusted, until the PCR product electrophoretic band brightness one of each tissue
It causes.Then target gene common PCR reaction, electrophoresis detection are carried out with the concentration and dosage adjusted.
Two kinds of PCR reaction results are shown: other than having very high expression in the tissue such as intestines and the gill, gene is in Cynoglossus semilaevis
Expression quantity in remaining tissue is extremely low (Fig. 3).
Embodiment 3: phase of the Cynoglossus semilaevis antibacterium disease related gene after infecting Vibrio harveyi in immunological relevant tissue
To expression analysis
The Vibrio harveyi saved using the laboratory Chen Songlin carries out Vibrio harveyi infection experiment to Cynoglossus semilaevis.If
Control group and experimental group are set, control group injects the sterile PBS of 0.01mol/L, and experimental group injection concentration is 1.0 × 104The Kazakhstan of cfu
Vickers vibrios, injection dosage are about 0.1mL/10g.0h, 12h after injection, for 24 hours, 48h, 72h, six time points of 96h, point
Liver, spleen, kidney, intestines, the gill and skin 6 tissues are not taken.Antibacterium disease related gene is detected with the method for quantitative fluorescent PCR
Expression quantity in above-mentioned 6 tissues.The primer, reagent and experimental method are same as above.
The results show that the expression for the antibacterium disease related gene respectively organized after injecting Vibrio harveyi is different
The up-regulation of degree: wherein in intestines, increasing always in the expression quantity of 12h to 48h, antibacterium disease related gene, with when 12h most
It is high；In the gill, in addition to 12h and for 24 hours other than, the expression quantity of 48h to 96h gene is constantly in increasing state；In spleen, kidney and skin
In, the expression quantity of gene is nearly always at propradation from 12h to 96h, and when 72h reaches peak；In liver, in addition to 12h
Outside, the expression quantity of antibacterium disease related gene is higher than control group to 96h from for 24 hours.In addition to parenteral, the expression of remaining 5 tissues
Amount reaches highest in 72h.These results indicate that after Vibrio harveyi stimulation, other than intestines and the gill, this hair under normal circumstances
The expression quantity of antibacterium disease related gene is also obvious in the extremely low immunological relevant tissue of bright antibacterium disease related gene expression amount
It increases, shows Cynoglossus semilaevis antibacterium disease related gene to Vibrio harveyi infected with strong immunologic defence function (Fig. 3).
Embodiment 4, the in-vitro recombination expression of screening-gene of the present invention and its product bacteriostatic activity analysis (1) Cynoglossus semilaevis are anti-
The construction method of bacterial disease related gene recombinant expression carrier:
Using the flat end Pfu enzyme of hi-fi, compiled by the intact proteins that PCR reacts amplification antibacterium disease related gene
Code region sequence.PCR product after purification passes through T4 connection enzymatic treatment, is inserted into Peasy E1 prokaryotic expression carrier.It is correct through being sequenced
Afterwards, the recombinant expression plasmid in positive colony bacterial strain is extracted.Recombinant expression plasmid is converted to competence BL21 (DE3) plyS work
In journey bacterium.It is induced, is purified, after dialysis and renaturation, obtained recombination expression product antibacterium disease relevant recom-binant protein, as a result see
Fig. 4.Realize that the concrete scheme of above-mentioned target is as follows:
1. the construction method of antibacterium disease related gene recombinant expression carrier:
Mixing cDNA is knitted as template using Cynoglossus semilaevis multiple groups, and the area antibacterium disease related gene ORF is carried out using high fidelity enzyme
Sequence clone, obtains protein-coding region.PCR product purifying, is inserted into pMD18-T carrier, is sequenced.Sequence is correctly cloned
It is connect with Quan Shijin peasy E1 expression vector, obtains the related recombinant vector of E1- antibacterium disease, sequencing confirmation positive colony mentions
Take purpose plasmid.E1- antibacterium disease related plasmids are converted into competence BL21 (DE3) plyS bacterium, correct standby is sequenced
With.
2. the inducing expression of recombinant protein and the analysis of expression product
The monoclonal of BL21 (DE3) plyS-E1- antibacterium disease related gene bacterial strain is seeded to 1ml and contains 50 μ of final concentration
In the LB liquid medium of g/mL AMP+, after 37 DEG C of 200rpm culture 8h, 200ML ammonia benzyl is seeded to by the inoculative proportion of 1:500
In the LB culture medium of resistance, expand culture (condition is same as above) to OD600For 0.4-0.6.The IPTG for adding final concentration 1mM, using 28
DEG C, 200rpm condition induce 6-12h.Three kinds of bacterium solution, control group empty carrier after taking 1ml inducing expression and the bacterium solution not induced etc.
Bacterium solution carries out the SDS-PAGE electrophoresis detection of protein expression simultaneously, the results showed that, the egg of variant expression between 50KD-70KD
There is (Fig. 4 A) in informal voucher band.By the bacterium solution after induction at 4 DEG C, 8000r/min is centrifuged 5-10min, collects whole thallus, then plus
Entering lysate, (lysate: being added combination buffer 10ml, DNAase I 20U, PMSF 400 μ l (50mM) in every g thallus, molten
400 μ l (10mg/ml) of bacterium enzyme.Wherein, combination buffer preparation method is as follows: sodium phosphate 3.8g, chlorine being added in every 1L sterile water
Change sodium 14.61g, imidazoles 1.02g), lysis at room temperature half an hour, then ultrasonication 20min in ice bath, condition are ultrasound
0.5s suspends 2s.2 × SDS loading is added according to the ratio of 1:1 in supernatant of bacteria solution liquid and broken bacterial sediment
Buffer boils 5min；SDS-PAGE electrophoresis detection expression of results, the results showed that protein content is bright in broken bacterial sediment
Aobvious to be higher than supernatant, antibacterium disease relevant recom-binant protein mainly exists in the form of inclusion body, as a result sees Fig. 4 B.
3. the purifying and verifying of Cynoglossus semilaevis antibacterium disease relevant recom-binant protein
Above-mentioned bacterial sediment is urea-denatured using 7M, after precipitating basic dissolution, supernatant is collected by centrifugation, uses
HisTrap FF crude purification column purity analysis is used after 0.45 μm of filtering with microporous membrane, concrete operations are as follows:
Firstly, cleaning purification column with 3-5mL aseptic deionized water, 5ml combination buffer (20mM/L is then used
Na3PO4,0.5M NaCl, 20mM/L imidazoles) it is incubated for purification column, the supernatant comprising destination protein of collection is slowly flowed across pure
Change column, flow velocity 1-2mL/min is then incubated for purification column using the cleaning of 5mL combination liquid, then uses the elution buffer of 5mL
(20mM/L Na3PO4,0.5M NaCl, 500mM/L imidazoles) elution destination protein.Destination protein after purification is collected, is used
The SDS-PAGE electrophoresis of 12% concentration carries out albumen size detection.The urea that destination protein after confirmation uses concentration gradient to reduce
It dialyses, albumen is assisted to be folded, obtain recombinant protein, electrophoresis result is shown as single band (see Fig. 4 C).Using BCA
Protein quantification kit carries out quantitative analysis to the albumen after dialysis.
4. the Cynoglossus semilaevis antibacterium disease relevant recom-binant protein bacteriostatic activity analysis of purifying
The detection of recombinant protein bacteriostatic activity is carried out using the method for Oxford cup inhibition zone.Select Gram-negative bacteria: love
Moral Fahrenheit bacterium (Edwardsiella tarda), vibrio parahemolyticus (Vibrio Parahemolyticus) and Vibrio harveyi
Three kinds of pathogenics such as (Vibrio harveyi) carry out fungistatic effect detections, by above-mentioned Bacteria culturing to logarithmic phase,
Diluting bacterium solution to concentration with PBS solution is 107~108A/ml is spare.Common culture medium is made, with a thickness of 0.5cm or so, is put
Oxford cup after entering 5 sterilizings is spare.Pathogen bacterium solution is dropped to 40 degree or so not solidified solid mediums with temperature to mix,
It pours on the solid plate of included 0.5cm depth, production becomes sandwich culfure base.10mg/L is separately added into Oxford cup
Ampicillin, 0.6mg/ml, 0.12mg/ml, the recombinant protein of 0.054mg/ml.28 DEG C or 37 DEG C (according to bacterium kind
Depending on class) size of inhibition zone is observed and measured after incubator culture 12h.The result shows that recombinant protein is to tarda
(Edwardsiella tarda), vibrio parahemolyticus (Vibrio Parahemolyticus) and Vibrio harveyi (Vibrio
Harveyi obvious bacteriostatic activity) is shown.Recombinant protein is to tarda (Edwardsiella tarda), parahemolyticas
The bacteriostatic activity of vibrios (Vibrio Parahemolyticus) and Vibrio harveyi (Vibrio harveyi) is with protein concentration
Increase and enhances.When low concentration, under same protein concentration, suppression of the recombinant protein to Vibrio harveyi (Vibrio harveyi)
Bacterium activity is minimum, but when protein concentration is higher (0.6mg/mL), and recombinant protein is to tarda (Edwardsiella
Tarda), vibrio parahemolyticus (Vibrio Parahemolyticus) and Vibrio harveyi (Vibrio harveyi) is antibacterial
Activity is higher.When recombinant protein concentration is 0.054mg/mL to the fungistatic effect of Vibrio harveyi (Vibrio harveyi)
Very weak (Fig. 5).
Embodiment 5: antibacterium disease related gene expression is horizontal in the disease-resistant family of Cynoglossus semilaevis and susceptible family different tissues
Analysis
1, the production of the disease-resistant and susceptible family of Cynoglossus semilaevis: it is sliding that half is established using the method invented before applicant Chen Songlin
The disease-resistant family of tongue sole and susceptible family (Chen Songlin etc., 2010).
2, the measurement of disease-resistant family and susceptible family different tissues antibacterium disease related gene expression level
(1) liver, spleen, kidney, intestines, the gill of the disease-resistant family of Cynoglossus semilaevis in 2014 and each 3 fishes of susceptible family are taken
It is organized with blood 6.RNA extraction, quantitative PCR the primer, reagent and experimental method are the same as previously described.Quantitative fluorescent PCR
Shown with semiquantitive PCR result: liver, kidney, in spleen antibacterium disease related gene expression in disease-resistant family and
There is no significant difference in susceptible family, and the expression of the antibacterium disease related gene in the intestines of disease-resistant family fish, the gill is high
In susceptible family (Fig. 6).
The detection of antibacterium disease related gene expression level in (2) 2017 years disease-resistant familys and susceptible family different tissues
Take the disease-resistant family of Cynoglossus semilaevis in 2017 and the liver of each 5 fishes of susceptible family, spleen kidney, intestines and 5, gill tissues.
RNA extraction method, quantitative PCR the primer, reagent and the same previous step of experimental method.Quantitative fluorescent PCR and semiquantitive PCR knot
Fruit shows: liver, kidney, the expression of gene does not have significant difference in disease-resistant family and susceptible family in spleen,
The expression quantity of gene is higher than susceptible family fish in the intestines and the gill of disease-resistant family fish, especially in intestines the most significant (Fig. 7).Thus it weighs
The result for the disease-resistant family and susceptible family in 2014 of appearing again.
By related to each tissue antibacterium disease of susceptible family to the disease-resistant family of Cynoglossus semilaevis in 2017 to 2014
The detection of gene expression dose shows exist just between the expression and premunition of the antibacterium disease related gene in intestines and the gill
Correlativity.It can be used using the height of antibacterium disease related gene expression level in intestines or the gill as an index of premunition
In disease-resistant family selective breeding.
(3) application method of the expression of antibacterium disease related gene as premunition evaluation index in Cynoglossus semilaevis intestines
The intestines of tongue sole families fish are acquired, extract RNA, reverse transcription is at cDNA.
Gene expression dose analysis is carried out according to general measure PCR method, using actin as internal reference, draws expression water
Flat to scheme, the expression of the antibacterium disease related gene in more different family Cynoglossus semilaevis adult fish intestines, expression is higher, resists
Sick power is stronger.The adult fish chosen in the horizontal high family of antibacterium disease related gene expression is cultivated into parent population for raising up seed,
The Cynoglossus semilaevis fry of antibacterium disease ability raising can be produced.Method of the invention is for the first time by antibacterium disease dependency basis in intestines
Index of the expression of cause as fish body antibacterium disease merit rating thereby establishes one kind of Cynoglossus semilaevis premunition detection
Molecular method, this method Cynoglossus semilaevis premunition test and disease-resistant family selective breeding in there is major application value, can also be with
It is promoted and applied on other fish breeding for disease resistance.
Sequence table
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>a kind of Cynoglossus semilaevis antibacterium disease related gene and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1835
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agtgtcttct tgcccgcagc catgctgggc ctttctaaac aagcctcgct ctacctgagt 60
cttcttctat ttctgtccct ccaatcctca gtcctcaccc ttactgggac cgcgagccag 120
tgccagtctg ctccctttgt accaggttac aatctggcag gagagggctt taacatagtc 180
actctgcgcc gacaaggagc ctacgtgatt gatatgagga cccacctcaa cccgaatggc 240
acctgtacac tttaccacaa ccctcttcaa ggcaacgcgt tgcaaaaagt gccactctct 300
gtgcatgact ggagatcctt ccgtcagtgc actgcatctc tccacggaag ctaccacagc 360
tctaacagca cactaattga aacatacacc agtctagaca gccatgactg gaagtccgga 420
ctgaatataa gtaaacttgg tggcttaaat gttggtggat caagttccag tgcctacagc 480
tttgcttcaa ccaggagcag agaagaccag cacattttca gccttcacta catgtactgc 540
aaccactact cataccgagt gtcaaacaga ccaaccctga gctctgagtt caggggggat 600
ttggaccaac tgccaccata ctccgcttca acaaaggctg attacaggag aattatagag 660
acatacggca cacattacat caacaaggtt tggcttggag gacgatacag aaggctgtca 720
gctatccgta catgtttgtc cagattgaat ggcttgtcaa cttatcaggc acatgactgc 780
ttgtctctgg gaatcaaatt aagcctgaag ataattcaag gttcatcaga aacacatacc 840
tgctccaaga tcttggagaa catggacact gctgcatcat acagcgctgg gctccaccag 900
cacgtcacag aggtaacagg aggaaatggt tggctcggcg aattttcact ctctggtatt 960
gacgcctctg gctatgaaac atggctgcgc agcctcaaag atcaccctga tgttattcac 1020
tattctctga gaccactgta cgagctggca ctatcgtggt caactatgat tggactgtat 1080
ttggccacac atgattacct caaagaacat ggcgtcagtt ctgggaccag taatccaaga 1140
tgtagcggtc gtcctaacct tgattacaac tgctgtccca tcgagacccg cagaggaaat 1200
ctaagggtga ccatcgtccg tgcttggggt ctgaaaggag atcctgtcgg gaagacagag 1260
gcgtatgtga agatgtggta cggtcaacat taccgtagga cccgtatgat ccgatcaaac 1320
tccccccgtt ggaattcaga ttatgacctt ggaaatgttt atgcccattt gagtcttaag 1380
attgaagtct gggatgaaga tgtgttcaga gacgaccgtc tggggtcatg tgtgaggaac 1440
ctgagacagg ggtcacacac ttttatctgt tcagtccaaa gagggggaat agagatcaga 1500
tactcactca cctgtgaccg ccatctgact ggaaaccagt gccaggacta cagaccagtt 1560
ccatagaaac tcagtctgaa tgaaagaaac aatgaaacat tatattagca tttccactgt 1620
atgagcatta gctttcccga tttttgatca atatcttttc taataaatga catgataggt 1680
gttggtgaaa gttaaaaagg tctgatctat gtcactgtga ttttagatga tttagtaact 1740
gtctacttca tcactttact ttgctaaaaa cagctaataa atacacagaa atgattttaa 1800
gcatcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1835
<210> 2
<211> 514
<212> PRT
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Met Leu Gly Leu Ser Lys Gln Ala Ser Leu Tyr Leu Ser Leu Leu Leu
1 5 10 15
Phe Leu Ser Leu Gln Ser Ser Val Leu Thr Leu Thr Gly Thr Ala Ser
20 25 30
Gln Cys Gln Ser Ala Pro Phe Val Pro Gly Tyr Asn Leu Ala Gly Glu
35 40 45
Gly Phe Asn Ile Val Thr Leu Arg Arg Gln Gly Ala Tyr Val Ile Asp
50 55 60
Met Arg Thr His Leu Asn Pro Asn Gly Thr Cys Thr Leu Tyr His Asn
65 70 75 80
Pro Leu Gln Gly Asn Ala Leu Gln Lys Val Pro Leu Ser Val His Asp
85 90 95
Trp Arg Ser Phe Arg Gln Cys Thr Ala Ser Leu His Gly Ser Tyr His
100 105 110
Ser Ser Asn Ser Thr Leu Ile Glu Thr Tyr Thr Ser Leu Asp Ser His
115 120 125
Asp Trp Lys Ser Gly Leu Asn Ile Ser Lys Leu Gly Gly Leu Asn Val
130 135 140
Gly Gly Ser Ser Ser Ser Ala Tyr Ser Phe Ala Ser Thr Arg Ser Arg
145 150 155 160
Glu Asp Gln His Ile Phe Ser Leu His Tyr Met Tyr Cys Asn His Tyr
165 170 175
Ser Tyr Arg Val Ser Asn Arg Pro Thr Leu Ser Ser Glu Phe Arg Gly
180 185 190
Asp Leu Asp Gln Leu Pro Pro Tyr Ser Ala Ser Thr Lys Ala Asp Tyr
195 200 205
Arg Arg Ile Ile Glu Thr Tyr Gly Thr His Tyr Ile Asn Lys Val Trp
210 215 220
Leu Gly Gly Arg Tyr Arg Arg Leu Ser Ala Ile Arg Thr Cys Leu Ser
225 230 235 240
Arg Leu Asn Gly Leu Ser Thr Tyr Gln Ala His Asp Cys Leu Ser Leu
245 250 255
Gly Ile Lys Leu Ser Leu Lys Ile Ile Gln Gly Ser Ser Glu Thr His
260 265 270
Thr Cys Ser Lys Ile Leu Glu Asn Met Asp Thr Ala Ala Ser Tyr Ser
275 280 285
Ala Gly Leu His Gln His Val Thr Glu Val Thr Gly Gly Asn Gly Trp
290 295 300
Leu Gly Glu Phe Ser Leu Ser Gly Ile Asp Ala Ser Gly Tyr Glu Thr
305 310 315 320
Trp Leu Arg Ser Leu Lys Asp His Pro Asp Val Ile His Tyr Ser Leu
325 330 335
Arg Pro Leu Tyr Glu Leu Ala Leu Ser Trp Ser Thr Met Ile Gly Leu
340 345 350
Tyr Leu Ala Thr His Asp Tyr Leu Lys Glu His Gly Val Ser Ser Gly
355 360 365
Thr Ser Asn Pro Arg Cys Ser Gly Arg Pro Asn Leu Asp Tyr Asn Cys
370 375 380
Cys Pro Ile Glu Thr Arg Arg Gly Asn Leu Arg Val Thr Ile Val Arg
385 390 395 400
Ala Trp Gly Leu Lys Gly Asp Pro Val Gly Lys Thr Glu Ala Tyr Val
405 410 415
Lys Met Trp Tyr Gly Gln His Tyr Arg Arg Thr Arg Met Ile Arg Ser
420 425 430
Asn Ser Pro Arg Trp Asn Ser Asp Tyr Asp Leu Gly Asn Val Tyr Ala
435 440 445
His Leu Ser Leu Lys Ile Glu Val Trp Asp Glu Asp Val Phe Arg Asp
450 455 460
Asp Arg Leu Gly Ser Cys Val Arg Asn Leu Arg Gln Gly Ser His Thr
465 470 475 480
Phe Ile Cys Ser Val Gln Arg Gly Gly Ile Glu Ile Arg Tyr Ser Leu
485 490 495
Thr Cys Asp Arg His Leu Thr Gly Asn Gln Cys Gln Asp Tyr Arg Pro
500 505 510
Val Pro
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gaccaactgc caccatactc c 21
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tcgtcctcca agccaaacc 19
Claims (10)
1. a kind of antibacterium disease related gene screened from Cynoglossus semilaevis, which is characterized in that the gene coded protein
Amino acid sequence is SEQ ID NO:2.
2. gene as described in claim 1, which is characterized in that the nucleotides sequence of the gene is classified as SEQ ID NO:1.
3. application of the gene described in claim 1 as the marker gene of Cynoglossus semilaevis antibacterium disease Genealogy screening.
4. application of the gene described in claim 1 in the detection of Cynoglossus semilaevis premunition.
5. a kind of method for detecting gene expression amount described in claim 1, which is characterized in that the method is detection gene
The amount expressed in Cynoglossus semilaevis intestines, expression is higher, and disease resistance is stronger.
6. method as claimed in claim 5, which is characterized in that the detection primer that the method uses, upstream primer
Sequence is SEQ ID NO:3, and the sequence of downstream primer is SEQ ID NO:4.
7. a kind of recombinant strains, which is characterized in that the recombinant strains are for recombinantly expressing described in claim 1
Gene coding albumen.
8. a kind of albumen, which is characterized in that the amino acid sequence of the albumen is SEQ ID NO:2.
9. application of the albumen according to any one of claims 8 in the product for preparing the prevention and treatment of Cynoglossus semilaevis Vibrio harveyi disease.
10. application as claimed in claim 9, which is characterized in that the product is feed addictive or antibacterial agent.
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| CN110684099A (en) * | 2019-11-16 | 2020-01-14 | 浙江万里学院 | Expression method and application of chemotactic factor protein of Chinese softshell turtle |
| CN110734486A (en) * | 2019-10-30 | 2020-01-31 | 青岛大学 | Cynoglossus semilaevis agglutinin family collectins disease-resistant gene |
| CN110964092A (en) * | 2019-12-26 | 2020-04-07 | 大连海洋大学 | Novel antibacterial peptide with antibacterial effect from shrimp, magnolia and stramonium linnaeus |
| CN111944913A (en) * | 2020-09-04 | 2020-11-17 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis disease-resistant breeding gene chip and application thereof |
| CN114990243A (en) * | 2022-06-21 | 2022-09-02 | 中国水产科学研究院黄海水产研究所 | Method for screening marker combination of anti-vibriosis harveyi cynoglossus semilaevis and screening disease-resistant individual of cynoglossus semilaevis |
| CN115011712A (en) * | 2022-06-10 | 2022-09-06 | 南方海洋科学与工程广东省实验室(湛江) | Biomarker for vibrio harveyi infection |
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| CN110734486A (en) * | 2019-10-30 | 2020-01-31 | 青岛大学 | Cynoglossus semilaevis agglutinin family collectins disease-resistant gene |
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| CN111944913A (en) * | 2020-09-04 | 2020-11-17 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis disease-resistant breeding gene chip and application thereof |
| CN115011712A (en) * | 2022-06-10 | 2022-09-06 | 南方海洋科学与工程广东省实验室(湛江) | Biomarker for vibrio harveyi infection |
| CN115011712B (en) * | 2022-06-10 | 2023-03-10 | 南方海洋科学与工程广东省实验室(湛江) | Biomarker for vibrio harveyi infection |
| CN114990243A (en) * | 2022-06-21 | 2022-09-02 | 中国水产科学研究院黄海水产研究所 | Method for screening marker combination of anti-vibriosis harveyi cynoglossus semilaevis and screening disease-resistant individual of cynoglossus semilaevis |
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