CN105950636B - A kind of trachidermus fasciatus creatine kinase TfM-CK gene, trachidermus fasciatus TfM-CK recombinant protein and its application - Google Patents

Abstract

The present invention provides a kind of trachidermus fasciatus creatine kinase TfM-CK gene, trachidermus fasciatus TfM-CK recombinant protein and its applications, belong to gene engineering technology field, the cDNA full length sequence of trachidermus fasciatus creatine kinase TfM-CK gene is not only obtained, trachidermus fasciatus TfM-CK recombinant protein is also obtained by prokaryotic expression.The present invention provides a kind of trachidermus fasciatus creatine kinase TfM-CK gene, sequence 1474bp includes 1146bp gene open reading frame.The present invention is used as fish meal immunity additive to enhance fish disease premunition.

Description

A kind of trachidermus fasciatus creatine kinase TfM-CK gene, trachidermus fasciatus TfM-CK recombinant protein and It is applied

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of cDNA of trachidermus fasciatus creatine kinase TfM-CK gene Full length sequence, the trachidermus fasciatus TfM-CK recombinant protein obtained by it by prokaryotic expression and its application.

Background technique

Trachidermus fasciatus (Trachidermus fasciatus) also known as four-gill perch, are under the jurisdiction of Rockfish shape mesh (Scorpaeniforme), Cottidae (Cottidae), trachidermus fasciatus category (Trachidermus) are a kind of typical drop Hai Hui The small fishes of trip property, entire growth period face two kinds of more complicated living environments of seawater and freshwater, are once distributed widely in me State is coastal.In recent years, trachidermus fasciatus is destroyed water pollution and due to building river water conservancy etc. to inhabit, breed and migration road Line causes the species population quantity to fall sharply, and distributed area also sharply reduces, therefore, research, the cultivation and application for developing trachidermus fasciatus, Have great importance.

Creatine kinase (Creatine Kinase, CK) belongs to highly conserved phosphagen kinase families, being capable of catalytic phosphatase Phosphoric acid and ADP in creatine combine the reversible reaction for generating ATP.It is widely present in invertebrate and vertebrate.In ridge In Vertebrate body, CK is unique phosphoric acid Clotogen, is typically found in energetic supersession vigorous tissue and cell, be one with it is thin Energy operation intracellular, contraction of muscle, ATP regeneration have the important kinases of direct relation.Clinically, human serum creatine kinase is diagnosis The important indicator of lesion tissue.Although nobody proposes that CK is the acute phase protein of mammal, some reports show that it can It can be related to acute phase response (the acute phage reponse APR) of mammal.Nearly all animal by wound and APR can all be started when the external injuries such as infection, synthesis APP (acute phage protein, APP) carries out quick self-protection, The invasion for resisting microorganism, promotes the reparation etc. of wound, and APR is attributed to the innate immune system of animal.

There are four types of isoenzymes forms, i.e. brain type, flesh type, hydridization type and Mitochondrial form by CK.Currently, the research of CK is mostly concentrated In terms of mammal, the especially flesh type CK most study of people, and it clinically has very high application value.Existing research Show that brain type CK and muscularity CK two types are distributed in fish, such as exists in carp (Cyprinus carpio) Muscularity CK, and there are brain type CK in rainbow trout (Oncorhynchus mykiss).For flesh type CK, in electric eel It is only sent out in (Electrophorus electricus), zebra fish (Danio rerio) and mandarin fish (Siniperca chuatsi) etc. Show a kind of flesh type CK, however in channel catfish (Ictalurus punctatus), the black fin ice fish in the arctic Then discovery has 2 kinds of different flesh type CK in (Chaenocephalus aceratus) etc., or even 3 kinds of differences are obtained in carp Flesh type CK.Although being found different type CK gene in fish, functional study is substantially at the stage not yet carried out. Gorgeous, Fan Ningning research of nearest Gao Yuanyuanan etc. is sent out by the research to lancelet (Branchiostoma belcheri Gray) Existing, lancelet CK is a kind of stress response protein, has activity of lectin.In the work of early period, we are from trachidermus fasciatus skin The information of creatine kinase CK is obtained in the protein spectrum analysis of obvious up-regulated expression after mucus stimulation, therefore is chosen as immune protein It conducts further research.As a kind of migratory small fishes, complicated living environment leads to its extensive adaptability and supports Drag, therefore using trachidermus fasciatus as research object, clone obtains CK full length gene sequence to carry out fish creatine kinase in immune side The functional study in face, and thus to obtain a kind of source of fish recombinant protein that can be used as fish meal immunity additive or immunoactivator, This will have far reaching significance for this field.

Summary of the invention

The purpose of the present invention is to provide a kind of trachidermus fasciatus creatine kinase TfM-CK genes, pass through prokaryotic expression system by it Obtained trachidermus fasciatus TfM-CK recombinant protein and its application are expressed, trachidermus fasciatus creatine kinase TfM-CK gene is not only obtained CDNA full length sequence also obtains trachidermus fasciatus TfM-CK recombinant protein by prokaryotic expression, to be applied to the immune addition of fish meal Enhance fish disease premunition in terms of agent.

An aspect of of the present present invention provides a kind of trachidermus fasciatus creatine kinase TfM-CK gene, sequence 1474bp, Comprising 1146bp gene open reading frame, nucleotide sequence is as follows:

Wherein, the ATG that underscore indicates in sequence is initiation codon, and the TAA of underscore mark is terminator codon, under AATAAA shown in scribing line is tailing signal.

Another aspect provides it is a kind of using the trachidermus fasciatus creatine kinase TfM-CK gene encode albumen, Including 381 amino acid, amino acid sequence is as follows:

Wherein, N-terminal structural domain is from the 24th amino acids residue to the 99th amino acids residue, and C-terminal structural domain is from 120 be amino acid residue to the 367th amino acids residue.

Another aspect of the invention provides a kind of trachidermus fasciatus TfM-CK recombinant protein, the pine as described in above-mentioned technical proposal River perch creatine kinase TfM-CK gene is obtained by prokaryotic expression.In the technical scheme, used protokaryon table It is e. coli bl21 (DE3), expression vector pet30a up to bacterial strain.

It is yet another aspect of the present invention to provide a kind of trachidermus fasciatus TfM-CK genes as described in above-mentioned technical proposal to prepare Application in fish feed additive.

It is yet another aspect of the present invention to provide a kind of trachidermus fasciatus TfM-CK genes as described in above-mentioned technical proposal to prepare Treat or prevent the application in aquiculture disease drug.

It is yet another aspect of the present invention to provide a kind of, and the trachidermus fasciatus TfM-CK recombinant protein as described in above-mentioned technical proposal exists Prepare the application in fish feed additive.

It is yet another aspect of the present invention to provide a kind of, and the trachidermus fasciatus TfM-CK recombinant protein as described in above-mentioned technical proposal exists Preparation treats or prevents the application in aquiculture disease drug.

The present invention has filled up trachidermus fasciatus by the acquisition of the cDNA full length sequence of trachidermus fasciatus creatine kinase TfM-CK gene The vacancy of TfM-CK gene studies.Meanwhile it can the conjunction of artificial synthesized or genetically modified organism according to trachidermus fasciatus TfM-CK gene order At biologically active recombinant protein, facilitates us and understand the genome structure of trachidermus fasciatus TfM-CK gene, analyze the base The expression of cause and the function of albumen.In addition, inventor is also constructed by trachidermus fasciatus TfM-CK gene order has obtained trachidermus fasciatus TfM-CK gene recombinant protein, the immunity additive that can be used as in fish meal effectively apply in aquaculture.

Detailed description of the invention

Fig. 1 is expression pattern of the TfM-CK gene in each tissue after the stimulation of Vibrio anguillarum provided by the embodiment of the present invention 2 Variation diagram, wherein error line is by the resulting standard deviation for statistical analysis of independent experiment three times；

Fig. 2 is that trachidermus fasciatus creatine kinase TfM-CK recombinant protein sugar provided by the embodiment of the present invention 5 combines quantitative table, In, test institute's value is that average value is tested in 3 repetitions；

Fig. 3 is TfM-CK recombinant protein provided by the embodiment of the present invention 6 to the staphylococcus aureus for having agglutination (gram-positive bacteria) and Vibrio anguillarum (Gram-negative bacteria) and not the agglutination schematic diagram of the Pseudomonas aeruginosa of agglutination；

Fig. 4 is that TfM-CK recombinant protein provided by the embodiment of the present invention 7 is aggregated comparison diagram to the inhibition of sugar；

Fig. 5 is to inject Vibrio anguillarum, Vibrio anguillarum+trachidermus fasciatus creatine kinase TfM-CK respectively provided by the embodiment of the present invention 8 Carp death rate schematic diagram after recombinant protein.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.

Embodiment 1: the acquisition of the cDNA full length sequence of trachidermus fasciatus creatine kinase TfM-CK gene:

By the pine under the post-stimulatory trachidermus fasciatus Skin mucus (experimental group) of Vibrio anguillarum (V.anguillarum) and normal condition River perch Skin mucus (control group) carries out polyacrylamide gel electrophoresis (SDS-PAGE), by comparing the difference of two histones, Protein band by experimental group relative to control group up-regulated expression is analyzed by mass spectrometry, as the result is shown trachidermus fasciatus M type creatine kinase In contain FEEILTR, QKRGTGGVDT, ASVGGVFDIS peptide fragment, according to nucleotide sequence corresponding to known protein sequence And other species downloaded on the website NCBI: spot head fish (Hexagrammos agrammus, AY583132.1), ten lines, six line Fish (Hexagrammos decagrammus, AY583138.1), long line Greenling (Hexagrammos lagocephalus, AY583136.1), greenling (Hexagrammos otakii, AY583133.1), cross wires Greenling (Hexagrammos Octogrammus, AY583134.1), hickie Greenling (Hexagrammos stelleri, AY583137.1), Qiao Shi shut out father Fish (Jordania zonope, AY583146.1), leptocottus armatus (Leptocottus armatus, AY583147.1), ink Green flat Rockfish (Sebastes atrovirens, AY583131.1), stellerina xyosterna (Stellerina xyosterna, AY583148.1), zebra fish (Danio rerio, BC056706.1), Ontario salmon (Salmo salar, BT044639.1), Sperling (Osmerus mordax, BT074432.1), red porgy (Pagrus major, GU135652.1), bottom Medaka (Fundulus heteroclitus, AB290197.1), great flounder (Platichthys stellatus, GU062902.2), channel catfish (Ictalurus punctatus, AF227794.1), pike (Esox lucius, BT079098.1), rainbow trout (Oncorhynchus mykiss, EZ907109.1), headband ice fish (Chaenocephalus Aceratus, AY161313.1), Tilapia mossambica (Oreochromis mossambicus, AY034098.1) and elegant jessamine sculpins Creatine kinase homologous sequence design primer CKF1, the CKR1 of (Rhamphocottus richardsonii, AY583145.1) and CKF2, CKR2, using the cDNA of trachidermus fasciatus musculature as template carry out PCR (PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min, 94 DEG C denaturation 45s, 56 DEG C of renaturation 45s, 72 DEG C of extensions 30s, 35 recycle, 72 DEG C of overall elongation 10min.).Tract is obtained through sequencing Section designs CKF3, CKR3 according to segment and above-mentioned homologous sequence, after PCR after sequencing obtains sequence fragment, according to segment and upper Homologous sequence design CKF4, CKR4, CKF5 are stated, and anchor R of CKF5 and 3 ', 5 ' Primer and CKR4 are matched and carry out PCR, PCR product is connect with pMD-18-T carrier (TaKaRa biotech company) after purification, and is transformed into Escherichia coli sense By in state (DH5 α), after plate is coated with, chooses bacterium colony and carry out PCR detection, will test result is that positive bacterial strain serves Hai Sheng Work company is sequenced, and sequencing primer is M13-47 and RV-M.Be sequenced, splice after obtain trachidermus fasciatus creatine kinase TfM-CK base The cDNA full length sequence (referring to sequence table<400>1) of cause, and it is encoded, obtain corresponding amino acid sequence (ginseng See sequence table<400>2).Prediction molecular weight of albumen is 42985.8Da, and theoretical isoelectric point (pI) is 6.44.SMART structural domain is pre- Survey show TfM-CK include two structural domains, one be N-terminal ATP-gua_PtransN structural domain, one is C-terminal ATP- Gua_Ptrans structural domain.The primer sequence is referring to table 1.

Primer sequence used in the process of 1 trachidermus fasciatus creatine kinase TfM-CK gene cDNA full length sequence of table obtains

Embodiment 2: real-time fluorescence quantitative PCR (qRT-PCR) detects the Tissue distribution and Vibrio anguillarum stimulation of TfM-CK gene Expression pattern variation afterwards

According to primer CKRTF, the CKRTR for cloning resulting TfM-CK gene cDNA sequence design qRT-PCR, with control group CDNA with each time point of stimulation group is template, carries out qRT-PCR.Specific reaction system and step referring to Premix Ex Taq^TMSpecification.

It is obtained by above-mentioned test, trachidermus fasciatus TfM-CK gene is in muscle, skin after Vibrio anguillarum (V.anguillarum) stimulation Equal up-regulated expression in skin, spleen and head-kidney, wherein in muscle until for 24 hours when expression quantity start on be adjusted to 12 times of left sides of control group The right side, this may be to need to adjust immunity function in body after being invaded and harassed due to body by extraneous pathogen to cope with the sense of bacterium Dye can transfer internal various immune factors rapidly after fish body adapts to this stimulation to resist the invasion of pathogen；And trachidermus fasciatus Skin, in spleen and head-kidney then respectively in 12h, for 24 hours, 2h when TfM-CK gene expression amount on be adjusted to highest, skin, spleen and Being immunized for head-kidney and fish is closely bound up, and TfM-CK gene is as Energy Metabolism Enzyme in immune organ and group after being stimulated by pathogen Knit middle expression quantity up-regulation.It is specific as shown in Figure 1, wherein histogram shows the experiment analysis results of qRT-PCR, and " * " indicates real Test significance difference anisotropic (* P < 0.05 of the group with 0h；P < 0.01 *), error line is by the institute for statistical analysis of independent experiment three times The standard deviation obtained.TfM-CK gene illustrates TfM-CK base in the increasing for expression quantity by the post-stimulatory each immuning tissue of Vibrio anguillarum Because being a kind of intracorporal acute phase protein of trachidermus fasciatus, play an important role in the inherent immunity of trachidermus fasciatus, therefore can It is used to prepare treatment, prevention aquiculture disease drug or fish feed additive.

Embodiment 3: expression, purifying and the concentration mensuration of trachidermus fasciatus creatine kinase TfM-CK recombinant protein

1) screening of bacterial strain is expressed

It extracts expression vector pet30a and constructs the plasmid in successful DH5 α bacterium, and be transferred to expression bacterial strain BL21 (DE3) in, screening positive clone is carried out.TfM-CK prokaryotic expression bacterial strain is filtered out, through 37 DEG C, 200rpm shaken cultivation 12- 16h。

2) taking temperature for recombinant protein reaches

The TfM-CK prokaryotic expression bacterial strain strain for cultivating 12-16h is turned into culture about 3-4h according to 1: 100 ratio, is added eventually Concentration is to carry out SDS-PAGE after the thiogalactoside (IPTG) of 0.5mM is induced, and will express preferable bacterial strain and adds 1/10 Volume glycerol (80%), -70 DEG C of conservations.

3) the extensive expression and purification of recombinant protein

The bacterial strain of conservation is inoculated into the LB culture medium containing 100 μ g/mL kanamycins according to 1: 100 ratio, 37 DEG C, 200rpm is incubated overnight, and turns to continue to cultivate in the LB liquid medium that culture contains kanamycins (100 μ g/mL) to 300mL to go forward side by side Row induction, and purified using the His-tag affinity column of GenScript according to specification, the purifying protein that will be obtained By Urea Gradient dialysis renaturation, concentrated rear recombinant protein concentration is 1.7mg/mL.It is dense to recombinant protein with Bradford method Degree is measured, and specific steps are referring to the raw work Bradford determination of protein concentration kit specification in Shanghai.

Embodiment 4: the measurement of the enzymatic activity of trachidermus fasciatus creatine kinase TfM-CK recombinant protein

Creatine kinase CK can be catalyzed atriphos (ATP) and creatine reaction generates phosphocreatine, and phosphocreatine is quickly Molybdenum blue can be generated under hydrolysis, ammonium molybdate and reduction, and atriphos (ATP) and adenosine diphosphate (ADP) (ADP) are very stable, Will not and ammonium molybdate reaction the vigor of enzyme therefore can be calculated according to the amount for generating Phos.

The enzyme activity that creatine kinase (CK) testing cassete specification carries out recombinant protein TfM-CK after renaturation is built up referring to Nanjing Measurement.The enzyme activity (U/ml) of TfM-CK is calculated according to standard curve Y=7.4491 × absolute OD value -0.0716.By TfM-CK Enzyme activity brings formula into: CK vigor (U/mgprot)=according to standard curve calculates resulting CK enzyme activity (U/mL)/to test sample This protein concentration (mgprot/mL).Enzyme activity after the TfM-CK renaturation finally asked is 3.1U/mg.According to enzyme activity determination knot Fruit shows trachidermus fasciatus creatine kinase TfM-CK recombinant protein bioactivity with higher provided by the embodiment of the present application.

Embodiment 5: trachidermus fasciatus creatine kinase TfM-CK recombinant protein sugar combines test

Bind directly activity of the TfM-CK recombinant protein to sugar is measured using enzyme linked immunosorbent assay (ELISA), Selected polysaccharide are as follows: peptide glycan (Peptidoglycans, PGN), lipopolysaccharides (Lipopolysaccharides, LPS), rouge Teichoic acid (Lipoteichoic acid, LTA).Polysaccharide is dissolved with sterile water, concentration is 80 μ g/mL, ultrasonication (20% Power, 2s, 3 times).Specific step is as follows:

(1) wrapper sheet: 50 μ L polysaccharide are added in 96 orifice plates, is placed in 25 DEG C of constant incubators and dries overnight.

(2) fixed: next morning goes in 60 DEG C of hybrid heaters and toasts 30min.

(3) close: the PBS, 37 DEG C of closing 2h that 200 μ L contain 5% skimmed milk power is added in every hole.

(4) board-washing: outwelling confining liquid, and the PBST (20%Tween-80: PBS=1: 100) that 200 μ L are added in every hole is washed 4 times, Each 5min.

(5) protein binding: the albumen (albumen uses the PBS containing 1% skimmed milk power to dilute) of 50 μ L is added in every hole, and control group adds The BSA for entering 50 μ L various concentration gradients, is incubated for 3h at room temperature.

(6) board-washing: outwelling albumen, and the PBST (20%Tween-80: PBS=1: 100) that 200 μ L are added in every hole washes 4 times, often Secondary 5min.

(7) add primary antibody: by the antibody of TfM-CK according to 1: 300 dilution proportion in the PBS containing 1% skimmed milk power, often 100 μ L are added in hole, react 1-2h under room temperature.

(8) board-washing: outwelling primary antibody, and the PBST (20%Tween-80: PBS=1: 100) that 200 μ L are added in every hole washes 4 times, often Secondary 5min.

(9) add secondary antibody: by horseradish peroxidase-labeled goat-anti rabbit (secondary antibody) according to 1: 3000 dilution proportion in containing 1% In the PBS of skimmed milk power, 100 μ L are added in every hole, react 1h under room temperature.

(10) board-washing: outwelling secondary antibody, and the PBST (20%Tween-80: PBS=1: 100) that 200 μ L are added in every hole is washed 4 times, Each 5min.

(11) it develops the color: developing the color according to the raw work IL-TMB colour reagent box specification in Shanghai, develop the color 5- under the conditions of 37 DEG C Light absorption value is read at 10min, microplate reader 450nm.

As a result as it can be seen that the present embodiment ELISA method is to 3 kinds of sugar: peptide glycan (peptidoglycans, PGN), lipopolysaccharides (lipopolysaccharides, LPS), lipoteichoicacid (lipoteichoic acid, LTA) progress sugar bind directly reality It tests, as a result as shown in Fig. 2, sugared Binding experiment shows: trachidermus fasciatus creatine kinase TfM-CK recombinant protein can be with three of the above sugar Specific binding, and as the increase binding ability of protein concentration shows saturability.This illustrates trachidermus fasciatus creatine kinase TfM-CK recombinant protein can be used as pattern recognition receptors and play a role, in this case, recombinant protein can be combined by sugar etc. Identify pathogenic bacteria, so that the immunological effect of mediate downstream act on, for example (,) it is agglutination, solidification, induced phagocytosis, activating complement, sharp Promoting blood circulation platelet reinforces natural killer cell activity.

Embodiment 6: the bacteria agglutination test of trachidermus fasciatus creatine kinase TfM-CK recombinant protein

8 kinds of bacteriums in bacteriostatic experiment are selected to carry out bacterial agglutination experiment, by microbionation 37 in 3mL fluid nutrient medium DEG C (28 DEG C of Vibrio anguillarum) is incubated overnight, and turns culture 3-4h or so to logarithmic growth phase, 6000rpm, 5min according to 1: 100 ratio Centrifugation, is resuspended, and bacteria concentration is adjusted to 3 × 106 cell/mL with TBS after being washed twice with TBS.Recombinant protein is added 96 In orifice plate, then 25 μ L bacteriums are added in every 25 μ L of hole, be incubated at room temperature 1h, and negative control group replaces recombinant protein with BSA.It is real The above each group is observed under inverted microscope after testing and is taken pictures.

The result shows that: TfM-CK recombinant protein can be aggregated Vibrio anguillarum (V.anguillarum), staphylococcus aureus (S.aureus), bacillus thuringiensis (B.thuringiensis) and Friedlander's bacillus (K.pneumoniae), and it is right In bacillus megaterium (B.megaterium), bacillus subtilis (B.subtilis), Escherichia coli (E.coli) and green pus Bacillus (P.aeruginosa) is without agglutination.As shown in figure 3, it lists TfM-CK recombinant protein to the gold for having agglutination Staphylococcus aureus (gram-positive bacteria) and Vibrio anguillarum (Gram-negative bacteria) and the not Pseudomonas aeruginosa of agglutination It is aggregated schematic diagram.

Embodiment 7: the inhibition of the sugar of trachidermus fasciatus creatine kinase TfM-CK recombinant protein is aggregated experiment

96 orifice plates are added in 25 μ L PGN of TfM-CK recombinant protein (200 μ g/mL, 25 μ L) and various concentration, LPS, LTA The Vibrio anguillarum of 25 μ L is added after middle incubation at room temperature 1h, is incubated at room temperature 1h, negative control group replaces recombinant protein with BSA, positive Control group only aggravates histone and bacterium, observes 96 orifice plates under inverted microscope after experiment and takes pictures.

The result shows that: after three kinds of sugar are added, the agglutination phenomenon of bacterium obviously weakens, but there is no complete inhibitions to be aggregated, As shown in Figure 4.

In conjunction with the embodiments the bacteria agglutination test of trachidermus fasciatus creatine kinase TfM-CK recombinant protein provided by 6,7 and sugar Inhibit test it is found that the TfM-CK recombinant protein that the embodiment of the present invention obtains is a kind of antibacterial factor with activity of lectin, Not only it can be used as one mode identification receptor (PRR) (referring to embodiment 5), but also can be used as effector and play a role (referring to reality Apply example 6,7).

Embodiment 8: the bacterium infection test of trachidermus fasciatus creatine kinase TfM-CK recombinant protein

Vibrio anguillarum shaken cultivation and is turned into culture to after logarithmic growth phase at a temperature of 28 DEG C, is resuspended after washing 2 times with TBS To 1% volume.The similar carp of Individual Size (Cyprinus carpio) raising in the fresh water of inflation, is adapted at room temperature It is tested after a week.Experiment carp is divided into 3 groups in parallel, injects Vibrio anguillarum, 0.65%NaCl solution (negative control), pine respectively River perch creatine kinase TfM-CK recombinant protein (1.5mg/ml)+Vibrio anguillarum (1:1 mixing), the every 12 hours statistics death rates, until 96 hours.

As a result as shown in figure 5, trachidermus fasciatus creatine kinase TfM-CK recombinant protein is intracorporal in trachidermus fasciatus after Vibrio anguillarum stimulation Immunization experiment shows to inject Vibrio anguillarum+trachidermus fasciatus creatine kinase TfM-CK recombinant protein compared to Vibrio anguillarum group is only injected Lethality of the group in 96 hours is compared and is reduced to 35%.Since 0.65%NaCl solution is negative control, without death, therefore This curve is not shown in Fig. 5.

It is understood that Vibrio anguillarum is conditioned pathogen, when aquiculture animal is under undesirable environmental condition, meet with When unfavorable stimulation or injury, Vibrio anguillarum can induce the generation of aquaculture creature disease.But it is in conjunction with the embodiments thin given by 8 Bacterium infection experiment can obviously reduce carp it is found that after trachidermus fasciatus creatine kinase TfM-CK recombinant protein is added into Vibrio anguillarum Therefore trachidermus fasciatus creatine kinase TfM-CK recombinant protein can be added to fish as additive and raised by the lethality in 96 hours In material, when feeding aquatic livestock as fish meal, its lethality can be not only reduced, but also combine above-described embodiment it is found that Songjiang Perch creatine kinase TfM-CK recombinant protein also has Immune discrimination effect, therefore the immunity energy of aquiculture animal also can be improved Power.

Claims (5)

1. a kind of trachidermus fasciatus creatine kinase TfM-CK gene treats or prevents the application in aquiculture disease drug in preparation, It is characterized in that, the sequence of the trachidermus fasciatus creatine kinase TfM-CK gene is 1474bp, is read comprising 1146bp gene open Frame, nucleotide sequence are as follows:

Wherein, the ATG that underscore indicates in sequence is initiation codon, and the TAA of underscore mark is terminator codon, underscore The AATAAA shown is tailing signal；

The trachidermus fasciatus creatine kinase TfM-CK gene is for treating or preventing by Vibrio anguillarum V.anguillarum, golden yellow Portugal Grape coccus S.aureus, bacillus thuringiensis B.thuringiensis or Friedlander's bacillus K.pneumoniae cause Aquiculture disease.

2. a kind of trachidermus fasciatus TfM-CK gene is preparing the application in fish feed additive, which is characterized in that the trachidermus fasciatus flesh The sequence of acid kinase TfM-CK gene is 1474bp, includes 1146bp gene open reading frame, and nucleotide sequence is as follows:

Wherein, the ATG that underscore indicates in sequence is initiation codon, and the TAA of underscore mark is terminator codon, underscore The AATAAA shown is tailing signal.

3. a kind of trachidermus fasciatus TfM-CK recombinant protein is preparing the application in fish feed additive, which is characterized in that the Songjiang Perch TfM-CK recombinant protein is obtained by trachidermus fasciatus creatine kinase TfM-CK gene by prokaryotic expression, the trachidermus fasciatus The sequence of creatine kinase TfM-CK gene is 1474bp, includes 1146bp gene open reading frame, nucleotide sequence is such as Under:

Wherein, the ATG that underscore indicates in sequence is initiation codon, and the TAA of underscore mark is terminator codon, underscore The AATAAA shown is tailing signal.

4. a kind of trachidermus fasciatus TfM-CK recombinant protein treats or prevents the application in aquiculture disease drug, feature in preparation It is, the trachidermus fasciatus TfM-CK recombinant protein is obtained by trachidermus fasciatus creatine kinase TfM-CK gene by prokaryotic expression It arriving, it includes 1146bp gene open reading frame that the sequence of the trachidermus fasciatus creatine kinase TfM-CK gene, which is 1474bp, Nucleotide sequence is as follows:

Wherein, the ATG that underscore indicates in sequence is initiation codon, and the TAA of underscore mark is terminator codon, underscore The AATAAA shown is tailing signal；

The trachidermus fasciatus TfM-CK recombinant protein is for treating or preventing by Vibrio anguillarum V.anguillarum, Staphylococcus aureus Water caused by bacterium S.aureus, bacillus thuringiensis B.thuringiensis or Friedlander's bacillus K.pneumoniae Produce the farming disease harms.

5. application according to claim 3 or 4, which is characterized in that protokaryon table employed in the prokaryotic expression system It is e. coli bl21 (DE3), expression vector pet30a up to bacterial strain.