CN104587463B - Activating effect and its application of the fish NKEF A recombinant proteins to adaptability humoral immunity - Google Patents
Activating effect and its application of the fish NKEF A recombinant proteins to adaptability humoral immunity Download PDFInfo
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Abstract
The activating effect to adaptability humoral immunity and its application the invention discloses a kind of fish NKEF A recombinant proteins, described application is that can promote the immunopotentiator of fish body fluid immunocompetence for preparing, and the amino acid sequence of the fish NKEF A recombinant proteins is SEQID NO:Shown in 1；The NKEF A recombinant proteins that the present invention is provided the advantage is that as adaptability humoral immunity reinforcing agent：(1) dosage is small, efficiency high；With 1:100 (NKEF A albumen:Antigen) NKEF A recombinant proteins dosage can play significant immunologic enhancement；(2) easily prepared immune mixed liquor：NKEF A recombinant proteins are soluble proteins, are easily mixed with antigen, overcome conventional mineral oil emu class preparation and are difficult to emulsify the shortcoming of antigen；(3) it is safe；Different from bacteriotoxin or compound formulation, NKEF A are the albumen of host itself, and the potential toxic side effect produced is imported in the absence of allogenic material.
Description
(1) technical field
The present invention relates to aquatic wholesale market and aquiculture disease prevention and control field, more particularly to a kind of natural killer cell
Enhancer A (Natural Killer Enhancer Factor A, NKEF-A) promotes the new of fish adaptability humoral immunity
Function and its application.
(2) background technology
With fish production fast development, Aquatic farming animals disease becomes increasingly conspicuous, and seriously constraining aquatic products industry health can
Sustainable development.Come for a long time due to lacking effective pest control method, various antibiotic, insecticide are widely used, cause cultivation
Environment and aquatic products pollution, aquatic food is on the hazard safely, while the drug resistance that antibiotic is produced exacerbates aquatic livestock again
Disease occurs.Therefore, it is one to develop natural, safety and efficient disease control of aquatic animal preparation and its application technology
Very urgent task.The exploitation of fish immunity disease-resistant gene are fundamentally to solve disease control of aquatic animal, are ensured
The effective way of aquatic food safety, represents the Main way of the green and environmentally friendly aquaculture pattern of current development.
But the long-term next research both at home and abroad to aquatic livestock immune disease-resistance gene is relatively weak, therefore the immune disease-resistance gene number developed
Amount is still extremely limited.
Natural killer cell enhancer (NKEF) is initially found in human red blood cells, and because of it, to strengthen natural killer thin
The cellulotoxic effect of born of the same parents (Natural Killer, NK) and named.Recent study shows that NKEF is except with enhancing NK
Outside cytoactive, also with the function such as anti-oxidant.But whether there are other biological functions to it, such as whether exempting to adaptability
Epidemic disease has regulatory function unclear.
There is the New function for promoting fish adaptability humoral immunity the invention firstly discloses NKEF-A, it is found that it can be notable
Fish adaptability humoral immunity is improved, effective preventing and treating to fish diseases is realized.Functional mechanism research shows that NKEF-A can pass through
The TLR4 receptor activation NF- κ B inflammatory signals paths on antigen presenting cell (APC) surface, up-regulation costimulatory signal molecule CD40,
CD80/86 and CD83 etc. expression, promotes CD4+T cell is Proliferative Activated and generation of IgM antibody, is adapted to so as to play enhancing
Property humoral immune function.The present invention not only enriches fish immunity and gained knowledge, and is opened for the immune protection of fish diseases
New way, has broad application prospects.
(3) content of the invention
It is an object of the present invention to provide a kind of new opplication of fish NKEF-A recombinant proteins, specially fish NKEF-A restructuring eggs
The application of white promotion fish body fluid immunocompetence, NKEF-A recombinant proteins are coordinated with Aeromonas hydrophila whole-cell vaccines to be made
With so as to improve the effect of prevention fish bacterial hueppe's disease.
The technical solution adopted by the present invention is：
The present invention provides a kind of fish NKEF-A recombinant proteins and is preparing the Immune-enhancing effect of enhancing fish body fluid immunocompetence
Application in agent, the amino acid sequence of the fish NKEF-A recombinant proteins is SEQ ID NO:(nucleotides sequence is classified as shown in 1
SEQ ID NO:Shown in 2), its purposes is the adaptability humoral immune response for promoting bacterin preparation, including but not limited to albumen
The adaptability humoral immune response of the inductions such as the full bacterium inactivated vaccine of antigen vaccine, bacterium.Described enhancing fish humoral immunity
Immunopotentiator is the preparation for the treatment of fish septicemia, and the preparation is by Aeromonas hydrophila whole-cell vaccines and with SEQ ID
NO:The recombinant protein of amino acid sequence shown in 1 is constituted；The method of administration of the preparation uses hypodermic mode, the tool
There are SEQ ID NO:The recombinant protein consumption of amino acid sequence shown in 1 is 0.1~0.25 μ g/ tails, and the Aeromonas hydrophila is complete
Cell vaccine consumption is 5 × 105~5 × 106CFU/ tails, effect was produced in 35 days.
NKEF-A recombinant proteins promote the functional mechanism of fish adaptability humoral immunity to be that the factor can promote antigen submission
The costimulatory signal molecules such as cell (APC) expression CD40, CD80/86 and CD83, strengthen CD4+T cell is Proliferative Activated and IgM
The generation of antibody.
The present invention obtains filefish using escherichia coli prokaryotic expression system (pET28a and Rosetta BL21) vivoexpression
NKEF-A recombinant proteins, this recombinant protein are tested with exogenous antigen albumen and the full bacterium inactivated vaccine co-immunization of bacterium respectively dynamic
Thing zebra fish.Using technologies such as quantitative fluorescent PCR (qPCR) and flow cytometries, detection antigen presenting cell (APC) and T, B
Lymphocyte activity, detects that B cell produces the change of antibody (IgM) using EUSA (ELISA).It was found that
NKEF-A recombinant proteins can remarkably promote the transcriptional level of the active marker molecule of APC and T, bone-marrow-derived lymphocyte, promote T, B lymph
Cell is bred and B cell produces the titre of antibody (IgM).Prove that NKEF-A recombinant proteins can be by promoting immunocyte
The approach such as activation and antibody generation, activates the adaptability humoral immunity of body, strengthens immunity of organisms, improve to bacterium infection
Function is resisted, increases body survival rate.
The adaptive immunity reinforcing agent used at present in the mankind and mammal has the cells such as interleukins, interferon
The factor, the compound such as aluminium salt, saponin(e, MF59, liposome, mineral oil, Bordetella pertussis, muramyl dipeptide, LA,
Bacteriotoxin (such as comma bacillus enterotoxin and escherichia coli enterotoxin) bacterial structure component or metabolic secretion product etc..It is preferable
Immunopotentiator should have no toxic side effect, there is significant activation to immune system, be easy to produce and use.It is true
On, above-mentioned preparation can also fully meet these requirements without any type, because most of preparations derive from bacterial product
Or there is different degrees of safety issue in compound, therefore use.
Compared with prior art, the NKEF-A recombinant proteins that provide of the present invention are as adaptability humoral immunity reinforcing agent, its
Advantage is：(1) dosage is small, efficiency high；With mass ratio 1:100 (NKEF-A albumen：Antigen) NKEF-A recombinant proteins
Dosage can play significant immunologic enhancement, and the mass ratio of conventional mineral oil emu class preparation and antigen is 1:1；
(2) easily prepared immune mixed liquor；NKEF-A recombinant proteins are soluble proteins, are easily mixed with antigen, overcome traditional ore deposit
Thing oil emu class preparation is difficult to emulsify the shortcoming of antigen；(3) it is safe；It is different from bacteriotoxin or compound formulation, NKEF-
A is the albumen of host itself, and the potential toxic side effect produced is imported in the absence of allogenic material.
(4) illustrate
Fig. 1 is the PCR testing results of the cDNA molecular clonings of NKEF-A recombinant proteins.M：DNA Marker, the 1st~6 swimming
Road is pcr amplification product in embodiment 1, obtains expected amplified band.
Fig. 2 is the SDS-PAGE analysis results of NKEF-A recombinant proteins；M：Standard protein molecular weight Marker, swimming lane 1 is
Control is not induced, and swimming lane 2 is NKEF-A recombinant protein induced expression results, it is contemplated that size 22.3KDa.
Fig. 3 is the result that NKEF-A recombinant proteins promote the Antigen-activated APC of KLH.On：Real time PCR PBS,
MHC-II and CD80/86 change result after KLH, KLH+NKEF-A are stimulated respectively.Under：Flow cytometry PBS, KLH,
APC activation results after KLH+NKEF-A is stimulated respectively.
Fig. 4 is the result that NKEF-A recombinant proteins promote the Antigen-activated T lymphocytes of KLH.On：Real time PCR
Lck and CD154 change result after PBS, KLH, KLH+NKEF-A are stimulated respectively.Under：Flow cytometry PBS, KLH, KLH
The activation results of T cell after+NKEF-A is stimulated respectively.
Fig. 5 is the result that NKEF-A recombinant proteins promote the Antigen-activated bone-marrow-derived lymphocytes of KLH.On：Real time PCR
CD40 and mIgM change result after PBS, KLH, KLH+NKEF-A are stimulated respectively.Under：Flow cytometry PBS, KLH, KLH
The activation results of B cell after+NKEF-A is stimulated respectively.
Fig. 6 is the result that NKEF-A recombinant proteins promote antibody (IgM) to produce.
Fig. 7 is survival rate change curve of the NKEF-A recombinant proteins to bacterial vaccine immunoprotection.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1, the cDNA molecular clonings for encoding NKEF-A albumen
Explanation according to Trizol kits (TaKaRa) extracts RNA from filefish spleen, using 3'-Full RACE Core
RNA reverse transcriptions are cDNA by Set kits (TaKaRa), using this cDNA as template, use forward primer F1：5’-
CCATGGCTGCAGGCAAAGCTC-3 ' and reverse primer R1：5 '-CTCGAGGTGCTTGGAGAAGAACTC-3 ' enter performing PCR (body
1) system is shown in Table expands, and obtains the coding nucleotide sequence of NKEF-A recombinant proteins.PCR conditions are：94 DEG C of pre-degeneration 4min, 94 DEG C
It is denatured 30s, 56 DEG C of annealing 30s, 72 DEG C of extensions 30s, 72 DEG C of extension 10min, totally 35 circulations, 1.2% agarose of PCR primer
Gel electrophoresis identifies that as a result display has been obtained and the expected NKEF-A cDNA bands (Fig. 1) matched.
The NKEF-A cDNA fragments of above-mentioned amplification are reclaimed using PCR primer QIAquick Gel Extraction Kit (Axygen), fragment will be reclaimed
Connect (system is shown in Table 2) to arrive in pUCm-T plasmid vectors (TaKaRa), 42 DEG C of heat-shock transformed E.coli DH5 α competent cells,
Be incubated at containing Amp (ampicillin, the μ g/ml of final concentration 50), IPTG (isopropylthiogalactoside, final concentration 200mM) and
On X-gal (beta galactosidase, the μ g/ml of final concentration 40) LB flat boards, 37 DEG C it is incubated overnight, picking white positive bacteria
Fall, extract plasmid, identified by PCR and digestion (system is shown in Table 3), and send company (Shanghai English fine horse) to carry out sequence analysis.As a result
Show, NKEF-A cDNA sequence total length 602bp, encode 200 amino acid residues, coincide with expected, recombinant plasmid is named as
pT/NKEF-A。
The PCR reaction systems of the NKEF-A cDNA of table 1 amplifications
The μ l T-A of table 2 10 clone system
The μ l double digestion systems of table 3 20
Embodiment 2, NKEF-A recombinant proteins prokaryotic expression plasmid are built
Utilize NcoI the and XhoI restriction enzyme sites introduced respectively in PCR primer described in embodiment 1, digestion pT/NKEF-A weights
Group plasmid, endonuclease bamhi is identified with 1.2% agarose gel electrophoresis, and reclaims purpose fragment using the method for embodiment 1, and restructuring connects
It is connected in the expression plasmid pET28 (a) crossed through NcoI and XhoI double digestions, Transformed E .coli DH5 α, is coated on containing Kan (cards
That mycin, the μ g/ml of final concentration 50) LB flat boards on, 37 DEG C it is incubated overnight, screen recon, PCR and digestion identification, obtain
NKEF-A prokaryotic expression plasmids are obtained, pET28 (a)/NKEF-A is named as.
It is prepared by embodiment 3, NKEF-A recombinant proteins
The prokaryotic expression plasmid pET28 (a) that embodiment 2 is obtained/NKEF-A is transferred in Rosetta (BL21) engineering bacteria,
After recombinant screen, picking positive colony is simultaneously inoculated in LB fluid nutrient mediums of the 5ml containing Kan (the μ g/ml of final concentration 50), is put
37 DEG C of shaking tables, 250rpm culture 5h, then by nutrient solution with volume ratio 1:100 ratio, which is added, contains Kan (the μ g/ml of final concentration 50)
LB fluid nutrient mediums in spread cultivation, 37 DEG C, 250rpm is cultivated to bacterial concentration OD600When between 0.6~1.0, add
IPTG, final concentration of 0.1mM, are then transferred to 20 DEG C of low temperature shaking tables, 100rpm incubated overnights, inducing soluble recombinant protein table
Reach.Next day, bacterium solution is collected, 5000g, centrifugation 15min abandon supernatant, collect thalline, thalline is resuspended with appropriate PBS, ultrasonication is extremely
Bacterium solution is as clear as crystal.All clasmatosis liquid are collected, 4 DEG C, 10000g centrifugation 10min collect supernatant, utilize SDS-PAGE
Protein expression result is identified, using Ni-NTA posts (Shanghai life work) purification of recombinant proteins, NKEF-A recombinant protein (amino acid is obtained
Sequence is SEQ ID NO:Shown in 1, nucleotides sequence is classified as SEQ ID NO:Shown in 2).As a result as shown in Figure 2, in luring for being taken
Under the conditions of leading, destination protein is obtained, swimming lane 1 is standard protein molecular weight, and swimming lane 2 is that NKEF-A recombinant proteins do not induce control,
Swimming lane 3 is NKEF-A recombinant protein induced expression results, molecular weight 22.3KDa.
The facilitation of embodiment 4, NKEF-A recombinant proteins to adaptability humoral immunity
4.1NKEF-A recombinant proteins are analyzed APC activating effects
3 groups of experimental animals (zebra fish, 30 tails/group) are set：Antigen assay group, NKEF-A activating effects experimental group and PBS
Control group；PBS control group, 10 μ l PBS is injected per tail fish, it is the anti-of 1 μ g/ μ l that antigen assay group, which injects 10 μ l concentration per tail fish,
Former albumen KLH (Sigma), 10 μ l KLH and NKEF-A of the every tail fish injection of NKEF-A activating effects experimental group (embodiment 3 is obtained,
Similarly hereinafter) mixed liquor, wherein containing 10 μ g KLH antigens and 0.1 μ g NKEF-A recombinant proteins.After 72h, blood sinus donor source zebra fish
Blood, puts in the D-Hank ' s liquid containing heparin (50U/ml, Shanghai life work).Spleen and head-kidney are taken simultaneously, in containing heparin (50U/
Ml shredded in D-Hank ' s liquid), 200 eye mesh screens are crossed simultaneously with blood sample, filtered solution is gently added in lymphocyte point
Chaotropic (Shanghai life work) upper strata, room temperature 2500rpm centrifugation 25min, draws white nepheloid layer leucocyte, is transferred to another 10ml
In centrifuge tube, plus 6ml no-rod tractor D-Hank ' s liquid, 4 DEG C, 1200rpm centrifugation 10min abandon supernatant, collect cell precipitation, transfer
To 1.5ml EP pipes.
Precipitated with 1ml L-15 nutrient solutions (Corning) suspension cell, the anti-zebra fish MHC- of mouse is added into cell suspension
II polyclonal antibodies and rabbit-anti-zebra fish CD80/86 polyclonal antibodies, 4 DEG C of incubation 30min；4 DEG C, 1250rpm centrifugation 5min,
Supernatant is abandoned, 1ml L-15 nutrient solutions is added and cleans 3 times, anti-mouse-IgG immunomagnetic beadses (Shanghai life work), 4 DEG C of incubation 30min is added；
Upper magnetic bead sorting frame (the new sesame in Ningbo) adsorbs 5min, and unadsorbed cell solution is put in another EP pipe, adds anti-rabbit-IgG
Immunomagnetic beads (Shanghai life work), 4 DEG C of incubation 30min；The PBS that precooling is added in the immunomagnetic beads of absorption once, divides again
Add precooling PBS twice after choosing, discard PBS, add 1ml Trizol and extract RNA, the method according to embodiment 1 is inverse by RNA
Be transcribed into cDNA, using this cDNA as template, carry out quantitative fluorescent PCR (condition is shown in Table 4), analysis APC anakmetomeres MHC-II and
CD80/86 transcriptional expression.
The primer sequence of quantitative fluorescent PCR is：
MHC-II-F：5’-AAGGA TTTGATGGAGAGGAGC-3’；
MHC-II-R：5’-GATGGATGTCACAGGAGGGTC-3’；
CD80/86-F：5’-ATATGAATGTGTTGTTT-3’；
CD80/86-R：5’-AGGTGTAGTTGATG GTCTGG-3’.
Table 4：Real-time fluorescence quantitative PCR system (10 μ l)
With the anti-zebra fish MHC-II antibody of mouse and rabbit-anti-zebra fish CD80/86 antibody mediated immunity fluorescence labeling APC, magazine
In 4 DEG C of standing 1h, 4 DEG C, 1250rpm centrifugation 10min cleaning cells, then (connection section is biological, 1000 times with PE- sheep anti-mouse igg antibodies
Dilution), FITC- goat anti-rabbit igg antibodies (connection section biological, 500 times dilution) mark, 4 DEG C of standing 2h in magazine；4 DEG C, 1250rpm
10min cleaning cells are centrifuged, the fluorescence signal of 10000 cells, Cell are collected in FACScan flow cytometers (BD) detection
Quest and Mod Fit LT software analysis cell activation situations.As a result see Fig. 3, stimulated using PBS as control, KLH antigens are independent
When stimulating zebra fish, MHC-II raises 2 times, and CD80/86 is not almost raised, when KLH and NKEF-A co-injections, on MHC-II
Tune increases to 6 times, and CD80/86 up-regulations increase to 5 times.Equally, during KLH and NKEF-A coprocessing, the APC ratios of activation are by KLH
Produce 6.99 ± 0.79%% are individually stimulated to be increased to 15.41 ± 1.98%.
4.2NKEF-A recombinant proteins are analyzed the activating effect of T lymphocytes
T cell is sorted using anti-CD 4 antibodies, RNA is extracted, reverses as cDNA templates, method be the same as Example 4.1；T cell is lived
Change is evaluated with marker molecule Lck and CD154 expression, method be the same as Example 4.1.
Fluorescence quantification PCR primer sequence used is：
Lck-F：5’-AGATGAATG GTGTGACCAGTGTA-3’；
Lck-R：5’-GATCCTGTAGTGCTTGATGATGT-3’；
CD154-F：5’-CGAATGGCAACAGGGCACAAGAATG-3’；
CD154-R：5’-TCTAAACAC TCCTGCTGATGATGCC-3’；
CD4 the and CD154 proteantigens expression of T cell is detected that method is with implementation using immunofluorescence label
Example 4.1.As a result Fig. 4 is seen, using blank PBS injections group as control, during the independent stimulation test fish of KLH antigens, Lck is about raised
3 times, CD154 then only slight up-regulations, and when with KLH and NKEF-A co-injections, Lck up-regulation increases by 8 times, CD154 up-regulations
4 times of increase.Equally, during KLH and NKEF-A coprocessing, the APC cell proportions of activation individually stimulated by KLH generation 9.00% ±
1.55 are increased to 19.58 ± 2.37%.
4.3NKEF-A recombinant proteins are analyzed the activating effect of bone-marrow-derived lymphocyte
B cell is sorted using anti-BCR (mIgM) antibody, RNA is extracted, reverses as cDNA templates, method be the same as Example 4.1；B
Cell activation is evaluated with marker molecule BCR (mIgM) and CD40 expression, method be the same as Example 4.1.
Fluorescence quantification PCR primer sequence used is：
mIgM-F：5’-TGAGCACAATAAGCGGAAAG-3’；
mIgM-R：5’-GCCAAGTCACA AACACCT-3’；
CD40-F：5’-GTCACACTGATCTGCCTTGTG-3’；
CD40-R：5’-TATC CGCTCTGATACTCTCCA-3’；
The BCR (mIgM) and CD40 proteantigens expression of B cell are detected that method is same using immunofluorescence label
Embodiment 4.1.As a result see Fig. 5, using blank PBS injections group as control, during the independent stimulation test fish of KLH antigens, CD40 and
MIgM only has slight up-regulation, when KLH and NKEF-A co-injections, 4 times of CD40 up-regulations increase, 3 times of mIgM expression increase.Equally,
When KLH and NKEF-A coprocessing, the B cell ratio of activation individually stimulates the 17.97 ± 3.22% of generation to be increased to by KLH
24.64 ± 4.40%.
The promotion effect analysis that 4.4NKEF-A recombinant proteins are produced to antibody
5 groups of experimental animals (zebra fish, 25 tails/group) are set, are injected intraperitoneally respectively：①KLH；②KLH+0.01μ
gNKEF-A；③KLH+0.05μgNKEF-A；④KLH+0.1μg NKEF-A；5. KLH+FCA (Sigma), wherein KLH albumen resist
Former dosage is 10 μ g, after injection twice in the 1st day and the 14th day, in the 28th day booster immunization once, takes a blood sample within the 35th day, collects
Serum.The IgM antibody growing amount in serum is detected using ELISA method.As a result seeing that Fig. 6, KLH are individually injected stimulates what is produced
IgM antibody titre is relatively low, and when adding NKEF-A (0.01 μ g and 0.05 μ g) of low dosage, its auxiliaring effect is not obvious, works as NKEF-
When A dosage reaches 0.1 μ g, the IgM antibody titre that auxiliary is produced significantly rises.Compared with classical Freund's adjuvant,
NKEF-A recombinant proteins have met or exceeded the auxiliaring effect of Freund's adjuvant.
Pushing Function Analysis of the 4.5NKEF-A recombinant proteins to bacterial vaccine immunoprotection
Using 0.5% (V/V) Formalin inactivation method, prepare Aeromonas hydrophila (Aeromonas hydrophila,
A.h, BSK-10 plants, fresh water research institute of Zhejiang Province provides) killed whole-cell vaccine.Formalin treatment conditions are room temperature inactivation 12
Hour, the A.h bacterium solutions after inactivation abandoned supernatant through 5000rpm, centrifugation 5 minutes, and PBS suspension thallines precipitation is repeated 3 times, is
A.h whole-bacterial-vaccines, -20 DEG C of packing are preserved.150 tail Adult Zebrafishs are taken, 3 groups, 50 tails/group, respectively by dorsal fin skin are randomly divided into
Lower injection PBS (control group), A.h vaccines (5.0 × 106CFU/ tails), A.h vaccine+NKEF-A recombinant proteins (5.0 × 106CFU+
0.1 μ g/ tails) carry out immunity inoculation.Test fish after immune is raised 35 days in 28 DEG C, and then per tail, experiment fish distinguishes abdominal cavity note again
Penetrate 5.0 × 106Malicious infection is attacked in CFU Aeromonas hydrophilas, progress, and once, Continuous Observation 24 hours, record is dead for observation in every 3 hours
Die experiment fish number.Experiment is repeated 3 times, and counts 24 hours protective rates.Protective rate=(the 1- experimental groups death rate/control group is dead
Rate) × 100%.As a result Fig. 7 and table 5 are seen, the death rate of the non-immunized controls group after infected by Aeromonas hydrophila attacks poison is 76.8
± 4.6%；The death rate of A.h vaccine immunity groups is 54.7 ± 3.3%, and protective rate is 28.8 ± 0.4%；And A.h vaccines are combined
The death rate of NKEF-A recombinant protein immune groups is 35.1 ± 2.8%, and protective rate is 54.4 ± 1.3%.With A.h vaccine immunity groups
Compare, the death rate of A.h vaccine union and recombination NKEF-A protein immunization groups significantly reduces (P<0.05), protective rate is significantly improved
(P<0.05), show that NKEF-A recombinant proteins have the effect for promoting bacterial vaccine immunoprotection activity.
The facilitation that table 5.NKEF-A recombinant proteins are protected to vaccine immunity
In the present invention, NKEF-A recombinant proteins can promote the generation of antibody by activating APC and T, bone-marrow-derived lymphocyte,
So as to strengthen the immune response of body fight primary stimuli.Prepared by this recombinant protein convenient, can be with water miscible form and antigen
Mixing, easy to operate, effect substantially, has broad application prospects in research and production.It should be noted that experiment is arranged above
What is lifted is only some instantiations of the present invention, it is clear that the present invention also has many deformations, one of ordinary skill in the art's energy
All deformations for directly exporting or associating from present disclosure, are considered as protection scope of the present invention.
Claims (3)
1. a kind of application of fish NKEF-A recombinant proteins in treatment fish septicemia preparation is prepared, it is characterised in that the fish
The amino acid sequence of class NKEF-A recombinant proteins is SEQ ID NO:Shown in 1.
2. application as claimed in claim 1, it is characterised in that：The preparation is by Aeromonas hydrophila whole-cell vaccines and has
SEQ ID NO:The recombinant protein of amino acid sequence shown in 1 is constituted.
3. application as claimed in claim 2, it is characterised in that：It is described that there is SEQ ID NO:The weight of amino acid sequence shown in 1
Histone consumption is 0.1~0.25 μ g/ tails, and the Aeromonas hydrophila whole-cell vaccines consumption is 5 × 105~5 × 106CFU/
Tail.
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| US5250295A (en) * | 1991-11-04 | 1993-10-05 | The Regents Of The Univ. Of California | Natural killer cell enhancing factor |
| CN1324837A (en) * | 2000-05-24 | 2001-12-05 | 上海博德基因开发有限公司 | New polypeptide human natural mortality cell enhancing factor B13.64 and polynuceotides for encoding same |
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