CN107469076A

CN107469076A - Application of the albumen of IL 2 in animal vaccine adjuvant is prepared

Info

Publication number: CN107469076A
Application number: CN201710656372.3A
Authority: CN
Inventors: 曹永生; 卢彤岩; 李绍戊; 徐黎明; 王荻; 赵景壮; 刘红柏
Original assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Current assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2017-12-15
Anticipated expiration: 2037-08-03
Also published as: CN107469076B

Abstract

The invention discloses application of the albumen of IL 2 in animal vaccine adjuvant is prepared.The albumen of IL 2 is that amino acid sequence is protein in sequence table shown in sequence 2.Experiment proves; compared with injecting the rainbow trout of infectious hematopoietic necrosis's poison G-protein; the IgT genes of rainbow trout of injection infectious hematopoietic necrosis's poison G-protein and the albumen of IL 2, Mx genes, Viperin genes, CD4 genes, the expression of CD8 genes and TNF α genes significantly improve; the potency of neutralizing antibody is significantly improved, and the propagation of lymphocyte is significantly increased and the protection of infectious hematopoietic necrosis's poison G-protein is effectively lifted.Therefore, the albumen of IL 2 has important application value in animal vaccine adjuvant is prepared.

Description

Application of the IL-2 albumen in animal vaccine adjuvant is prepared

Technical field

The present invention relates to cultivation field, and in particular to application of the IL-2 albumen in animal vaccine adjuvant is prepared.

Background technology

The important sources that fish and aquatic products are still the food of the current whole world mankind and nutrition obtains.At present, rainbow trout is The important composition of majority state commercialization cold water fish, but as the increasing of cultivation density and the deterioration of breeding environment, rainbow trout are supported Grow and threatened often by contagious disease.Vaccine is proved to be the effective means of preventing and treating rainbow trout pathogenic microorganisms infringement.

Many countries are in view of the use of the misgivings limitation live vaccine of bio-safety, and inactivated vaccine or recombinant vaccine Expected protecting effect can not be provided in some cases.In addition, injecting immune is the major way of current vaccines for fish inoculation, Which not only takes, effort, is even more unsuitable for juvenile fish and is immunized.For economically, oral immunity is that vaccines for fish connects The desirable route of kind.But individually the immune response of oral delivery antigen stimulation is usually weaker.Adjuvant is can be anti-by strengthening The ability and continuation of former inducing specific immunity reaction improves the validity of vaccine.Many experiments show to help in vaccines for fish The use of agent can optimize to the immune effect of vaccine from different perspectives.

The content of the invention

The technical problems to be solved by the invention are how to improve the validity of animal vaccine.

In order to solve the above technical problems, the present invention protects the application of IL-2 albumen first, the application of IL-2 albumen can be At least one of a1) to a6)：A1 animal vaccine adjuvant) is prepared；A2 the expression of gene involved in immunity in animal body) is strengthened It is horizontal；A3 the potency of neutralizing antibody in animal body) is improved；A4 the propagation of animal body medium size lymphocyte) is promoted；A5) lifted The protection of animal vaccine；A6) prepare and promote product of the animal body to the immune response of antigen.

Any of in above-mentioned application, the IL-2 albumen can be b1) to b6)：

B1) amino acid sequence is the protein shown in sequence 2 in sequence table；

B2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label obtain；

B3) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or The protein with identical function that missing and/or addition obtain；

B4) amino acid sequence is the protein shown in sequence 4 in sequence table；

B5) the fused protein that the N-terminal of the protein in sequence table shown in sequence 4 or/and C-terminal connection label obtain；

B6) by the amino acid sequence shown in sequence in sequence table 4 by one or several amino acid residues substitution and/or The protein with identical function that missing and/or addition obtain.

Above, the 1st to 20 removal from N-terminal by the IL-2 albumen shown in sequence in sequence table 4, that is, obtain sequence table IL-2 albumen shown in middle sequence 2.Those skilled in the art are generally acknowledged that this 20 amino acid sequence encoded signal peptides of removal, The specific function of IL-2 albumen is not influenceed.

Encode the nucleic acid molecules of the IL-2 albumen, or, the expression cassette containing the nucleic acid molecules for encoding the IL-2 albumen, Recombinant vector or recombinant bacterium, application, be a1) to a6) at least one of：A1 animal vaccine adjuvant) is prepared；A2) enhancing is dynamic The expression of gene involved in immunity in thing body；A3 the potency of neutralizing antibody in animal body) is improved；A4 animal machine) is promoted The propagation of body medium size lymphocyte；A5 the protection of animal vaccine) is lifted；A6) prepare and promote animal body should to the immune of antigen The product answered.

In above-mentioned application, the recombinant vector can be recombinant plasmid pIL-2.

The building process of the recombinant plasmid pIL-2 is as follows：(1) total serum IgE of rainbow trout is extracted, reverse transcription, obtains cDNA the One chain；(2) using first chain of cDNA as template, with 5 '-AAGCTTTTATGAACTTAGACGCTTTGC-3 ' and 5 '- GAATTCGCCACCATGAACCCAATTCCCAGACTCCT-3 ' is that primer enters performing PCR amplification, obtains pcr amplification product；(3) will Pcr amplification product connects with pMD18T carriers, obtains middle interstitial granules；(4) take middle interstitial granules, with restriction enzyme EcoRI and XhoI carries out double digestion, reclaims about 390bp digestion products；(5) pcDNA3.1 carriers are taken, with restriction enzyme EcoRI and XhoI carries out double digestion, reclaims about 5.4kb carrier framework；(6) digestion products and carrier framework are attached, recombinated Plasmid pIL-2.In recombinant plasmid pIL-2 containing sequence in ordered list 1 shown in DNA molecular.Recombinant plasmid pIL-2 expressed sequences IL-2 albumen in table shown in sequence 2.

Any of in above-mentioned application, the nucleic acid molecules can be c1) to c6)：

C1) DNA molecular of the code area as shown in sequence 1 in sequence table；

C2) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；

C3) DNA molecular of the code area as shown in sequence 3 in sequence table；

C4) nucleotide sequence is the DNA molecular shown in sequence 3 in sequence table；

C5) and c1) or c2) c3) or c4) nucleotide sequence that limits has 75% or more than 75% homogeneity, and volume The DNA molecular of the code IL-2 albumen；

C6) under strict conditions with c1) c2) or c3) or the c4) nucleotide sequence hybridization that limits, and encode the IL- The DNA molecular of 2 albumen.

In order to solve the above technical problems, present invention also offers a kind of product, it contains t1) or t2) or t3)：

T1) the IL-2 albumen；

T2 the nucleic acid molecules of the IL-2 albumen) are encoded；

T3 expression cassette, recombinant vector or recombinant bacterium) containing the nucleic acid molecules for encoding the IL-2 albumen.

The product can have following f1) to f6) at least one of function：F1) animal vaccine adjuvant；F2 animal) is strengthened The expression of gene involved in immunity in body；F3 the potency of neutralizing antibody in animal body) is improved；F4 animal body) is promoted The propagation of medium size lymphocyte；F5 the protection of animal vaccine) is lifted；F6 immune response of the animal body to antigen) is promoted.

The present invention also protects a kind of method of promotion animal body to the immune response of antigen, it may include following steps： The IL-2 albumen is expressed in animal body.

The present invention also protects a kind of method for the protection for lifting animal vaccine, it may include following steps：In animal body The middle expression IL-2 albumen.

Any of the above-described promotion animal body can specifically be presented as a2 to the immune response of antigen) to a4) at least It is a kind of：A2 the expression of gene involved in immunity in animal body) is strengthened；A3 the potency of neutralizing antibody in animal body) is improved； A4 the propagation of animal body medium size lymphocyte) is promoted.

Any of the above-described gene involved in immunity can be IgT genes, Mx genes, Viperin genes, CD4 genes, CD8 bases At least one of cause and TNF-α gene.

Any of the above-described vaccine can be nucleic acid vaccine.The nucleic acid vaccine concretely infectious hematopoietic necrosis Nucleic acid vaccine.

Any of the above-described antigen can be infectious hematopoietic necrosis's poison G-protein.

Any of the above-described infectious hematopoietic necrosis's poison G-protein can be d1) or d2) or d3)：

D1) amino acid sequence is the protein shown in sequence 6 in sequence table；

D2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 6 or/and C-terminal connection label obtain；

D3) by the amino acid sequence shown in sequence in sequence table 6 by one or several amino acid residues substitution and/or The protein with identical function that missing and/or addition obtain.

Any of the above-described animal can be (h1), (h2), (h3), (h4), (h5) or (h6)：(h1) non-human animal；(h2) Net-rope animal；(h3) salmon shape mesh animal；(h4) Gui Xing sections animal；(h5) Pacific Ocean salmon category animal；(h6) rainbow trout (Oncorhynchus mykiss)。

It is demonstrated experimentally that compared with injecting the rainbow trout of infectious hematopoietic necrosis's poison G-protein, infectious hematopoietic device is injected The IgT genes of the rainbow trout of official's necrosis virus G-protein and IL-2 albumen, Mx genes, Viperin genes, CD4 genes, CD8 genes and The expression of TNF-α gene is significantly improved, and the potency of neutralizing antibody significantly improves, and the propagation of lymphocyte is significantly increased and passed The protection of metachromia hematopoietic necrosis virus' G-protein is effectively lifted.Therefore, IL-2 albumen has in animal vaccine adjuvant is prepared There is important application value.

Brief description of the drawings

Fig. 1 is the identification of recombinant plasmid.

Fig. 2 is in the step 2 of embodiment 13 experimental result.

Fig. 3 is in the step 2 of embodiment 14 experimental result.

Fig. 4 is in the step 3 of embodiment 12 experimental result.

Fig. 5 is the experimental result of the step 3 of embodiment 2.

Fig. 6 is the experimental result of the step 4 of embodiment 2.

Fig. 7 is the experimental result of the step 6 of embodiment 2.

Fig. 8 is the experimental result of the step 7 of embodiment 2.

Embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even Average.

Rainbow trout (Oncorhynchus mykiss) in following embodiments is conventional rainbow trout fingerling, is raised in glass fiber reinforced plastic water In race's case, raising temperature is 14-16 DEG C.

Nylon wire is the product of Fisher Scientific companies；The sizing grid of nylon wire is 70 μm.RNA extraction examinations Agent box is the product of Promega companies.PMD18T carriers are the product of Takara companies.PET32a carriers are Novagen companies Product.Trans 2K DNA marker, Trans 15K DNA marker and M-MLV reverse transcription reagent box are Takara The product of company, catalog number are respectively 3427A, 3582A and 2640A.Ni-NTA Agarose are QIAGEN Products, Catalog number is 30210.Non- pre-dyed albumen Marker be Fermentas companies product, catalog number SM0431.Not Family name's Freund's complete adjuvant and incomplete Freund's adjuvant are the product of Sigma companies, and catalog number is respectively F5881 and F5506. BALB/c mouse is the product of the second affiliated hospital of Harbin Medical University Experimental Animal Center.PcDNA3.1 carriers and liposome Transfection reagent is the product of Invitrogen companies, and catalog number is respectively V79020 and L3000008.Anti- mouse FITC marks Note antibody, the anti-mouse antibody of horseradish peroxidase-labeled and the anti-rabbit secondary antibody of horseradish peroxidase-labeled are Abcam public affairs The product of department, catalog number are respectively ab6785, ab6728 and ab6721.PEE12.4 carriers are Lonza biologics public The product of department.Ficoll-Paque PLUS reagents are the product of GE Healthcare companies.WST-1 solution is green skies company Product, catalog number C0036.

IHNV-Sn1203 is recorded in following document：Xu Liming, Liu Hongbai, Yin Jiasheng, Lu Tong rock infectious hematopoietic devices The genotype of official's necrosis virus-Sn1203 strains and the bioinformatic analysis Chinese aquatic sciences .2014 of glycoprotein, 21 (1)： 180-188.IHNV-Sn1203 No. GenBank be KC660147.1.

IHNV G truncated proteins are recorded in following document：Tao Jian, Xu Liming, Liu Miao, Zhao Jingzhuan, Cao Yongsheng, Lu Tongyan, The prokaryotic expression and application fresh water fisherys of Yin Jiasheng, Liu Hong cypress infectious hematopoietic necrosis's poison epitope enrichment regions, 2015,45 (2), 49-55.

Coating buffer：PH9.6,0.05mol/L carbonate buffer solution.

PBST buffer solutions：By NaCl 8g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.24g and 0.5mLTween is dissolved in 1L deionized waters.

Antibody diluent：Add rabbit source rainbow trout IgM polyclonal antibodies into pH7.4,0.01mM PBS to its content For 0.4% (v/v).Rabbit source rainbow trout IgM polyclonal antibodies are recorded in following document：Zhao Jingzhuan, Xu Liming, Liu Miao, Cao Yongsheng, The expression of Yin Jiasheng, Liu Hongbai, Lu Tong rock rainbow trout IgM heavy chain constant region and the preparation of rabbit anti-serum, aquatic product journal, 2014,38 (8), 1175-1181.

Lysis Buffer solution：NaH containing 100mM₂PO₄Buffered with 8M urea pH8.0,10mM TrisHCl Liquid.

Substrate solution：By 100 μ L A liquid (0.0125g TMB are dissolved in into 80% (v/v) the DMSO aqueous solution) and 5mL B liquid (by hydrogen peroxide urea 0.05g, citric acid 1.057g and Na₂HPO₄·12H₂O 3.94g are dissolved in 100mL deionized waters, regulation 4.0) pH value is to mixing.

Embodiment 1, the preparation of adjuvant and its identification of biological activity

First, the clone of rainbow trout IL-2 genes

According to the nucleotide sequence (GenBank of rainbow trout IL-2 genes：NM_001164065.1), design and artificial synthesized draw Thing F1：5’-GGATCCAACCCAATTCCCAGACTCCT-3 ' (single underscore is restriction enzyme BamHI recognition site), Primer R1：5’-AAGCTT(single underscore is restriction enzyme Hind III identification position to TTATGAACTTAGACGCTTTGC-3 ' Point), primers F 2：5’-GAATTC (single underscore is restriction enzyme to ATGAACCCAATTCCCAGACTCCT-3 ' EcoRI recognition site, double underline are kozak sequences) and primer R2：5’-CTCGAGTCATGAACTTAGACGCTTTGC- 3 ' (single underscore is restriction enzyme XhoI recognition site).

1st, rainbow trout is put to death, strips spleen in sterile on ice rapidly, the MEM containing 10% (v/v) FBS in right amount is then added and cultivates Base, after syringe inner prop rolls, with nylon net filter, collect cell filtrate.

2nd, cell filtrate is taken, the phytohemagglutin phytolectin solution that concentration is 5 μ g/mL is added and (is dissolved in phytohemagglutin phytolectin PH7.4,0.01mM PBS obtain), 15 DEG C of quiescent culture 4h.

3rd, after completing step 1, nutrient solution is abandoned, cell is collected and using RNA extracts kits extraction total serum IgE, according to M-MLV Reverse transcriptase specification, reverse transcription is carried out with primer Oligo (dT) 18, obtains first chain of cDNA.

4th, using first chain of cDNA as template, performing PCR amplification is entered using primer pair first (being made up of primers F 1 and primer R1), Obtain 381bp pcr amplification product first.Using first chain of cDNA as template, using primer pair B (by primers F 2 and primer R2 groups Into) enter performing PCR amplification, obtain 390bp pcr amplification product second.

5th, pcr amplification product first is connected with pMD18T carriers, obtains recombinant plasmid first.By pcr amplification product second and PMD18T carriers connect, and obtain recombinant plasmid second.

Recombinant plasmid first and recombinant plasmid second are sequenced respectively.Sequencing result shows, recombinant plasmid first and restructuring matter Contain DNA molecular of the sequence 1 from 5 ' ends shown in the 1st to 366 in ordered list in grain second.By the institute of sequence in sequence table 1 The DNA molecular shown is named as rainbow trout IL-2 genes.

2nd, the preparation of polyclonal antibody

1st, recombinant plasmid pET32a-IL-2 structure

(1) recombinant plasmid first is taken, double digestion is carried out with restriction enzyme BamHI and Hind III, reclaims about 381bp enzyme Cut product first.

(2) pET32a carriers are taken, double digestion is carried out with restriction enzyme BamHI and Hind III, reclaims about 5.9kb load Body skeleton first.

(3) digestion products first and carrier framework first are attached, obtain recombinant plasmid pET32a-IL-2.

(4) recombinant plasmid pET32a-IL-2 is taken, enters performing PCR identification (being carried out using primer pair first), single endonuclease digestion identification respectively (being carried out using restriction enzyme BamHI) and double digestion identification (being carried out using restriction enzyme BamHI and Hind III).

Experimental result is shown in A in Fig. 1 (M1 is that Trans 2K DNA marker, M2 are Trans 15K DNA marker), swimming Road 1 identifies that swimming lane 2 is identified for single endonuclease digestion for PCR, and swimming lane 3 is identified for double digestion).

(5) recombinant plasmid pET32a-IL-2 is sequenced.

Identified according to PCR, digestion identification and sequencing result, carrying out structure to recombinant plasmid pET32a-IL-2 is described as follows： Small fragment between the recognition sequence of restriction enzyme BamHI and Hind III of pET32a carriers is replaced with into sequence 1 in sequence table Shown DNA molecular.In recombinant plasmid pET32a-IL-2, on the DNA molecular and carrier framework first shown in the sequence 1 of sequence table His-tag labels (being made up of 6 histidine residues) coded sequence fusion, formed fusion, expression there is His-tag The fusion protein of label.

2nd, the expression of fusion protein

(1) recombinant plasmid pET32a-IL-2 is imported into Escherichia coli Rosetta, obtains recombinant bacterium, the recombinant bacterium is named For Rosetta-pET32a-IL-2.

(2) Rosetta-pET32a-IL-2 monoclonals are taken, 5mL LB fluid nutrient mediums is seeded to and (contains 100 μ g/mL ammonia benzyls Penicillin (Amp)), 37 DEG C, 180rpm shaken cultivation 12h, obtain cultivating bacterium solution.

(3) culture bacterium solution is taken, is by volume 1:100, which are seeded to 500mL LB fluid nutrient mediums, (contains 100 μ g/mL Amp), 37 DEG C, 180rpm shaken cultivations to OD_600nmValue reaches 0.4~0.6, then adds IPTG and makes its concentration be 1mM, and 37 DEG C, 120rpm shaken cultivations 4h, 4 DEG C, 10000rpm centrifugation 10min, collect bacterial sediment.

(4) after completing step (3), bacterial sediment is taken, the Tris-HCl buffer solutions for adding pH8.0,100mM are resuspended, ultrasound It is broken that (ultrasonic power 600W, cyclic program are：Broken 4s, stops 6s, common 20min), then 4 DEG C, 10000rpm centrifugations 10min, obtain bacterial cell disruption supernatant and bacterial cell disruption precipitation.

Bacterial cell disruption supernatant and bacterial cell disruption precipitation are subjected to SDS-PAGE respectively.As a result show, fusion protein is main It is present in inclusion body, size is about 33.7kDa.

3rd, the purifying of fusion protein

(1) the bacterial cell disruption precipitation that (4) obtain is taken in step 2,6mL Lysis Buffer solution is added and is resuspended, then 4 DEG C stand until precipitation is completely dissolved, filtered through 0.45 μm of filter, obtain lysate.

(2) 2mL Ni-NTA Agarose are added into chromatographic column, are precipitated using gravitational method or low-speed centrifugation, so Supernatant is gently sucked afterwards, sequentially adds the cleaning of 6mL deionized waters and 6mL Lysis buffer solution (through 0.45 μm of filter Filtering) rinse, 3min is rotated back and forth, abandons liquid.

(3) after completing step (2), the lysate that step (1) obtains is added into the chromatographic column, 4 DEG C rotate back and forth (purpose is to make Ni to 60min²⁺Fully combined with fusion protein), abandon liquid.

(4) after completing step (3), it is repeated below step three times：5mL Wash buffer1 are added into the chromatographic column (NaH containing 100mM₂PO₄PH6.0,10mM TrisHCl buffer solutions with 8M urea), 3min is rotated back and forth, abandons liquid.

(5) after completing step (4), it is repeated below step three times：5mL Wash buffer2 are added into the chromatographic column (NaH containing 100mM₂PO₄PH5.3,10mM TrisHCl buffer solutions with 8M urea), 3min is rotated back and forth, abandons liquid.

(6) after completing step (5), it is repeated below step three times：2mL Elution are added into the chromatographic column Buffer (NaH containing 100mM₂PO₄PH4.5,10mM TrisHCl buffer solutions with 8M urea), 3min is rotated back and forth, is collected Liquid.

(7) OD for the liquid that detecting step (6) is collected_280nm, then 4 DEG C of dialysis, collect dialysis product.

(8) after completing step (7), dialysis product is subjected to SDS-PAGE.

Wash buffer1, Wash buffer2 and Elution buffer filter through 0.45 μm of filter.

Experimental result is shown in Fig. 2 (M is non-pre-dyed albumen Marker), and swimming lane 1 is dialysis product, and arrow is fusion protein).Knot Fruit shows that dialysis product is fusion protein solution.After testing, fusion protein concentration is about 0.325mg/ in fusion protein solution mL。

4th, the preparation of polyclonal antibody

(1) 1 parts by volume fusion protein solution (containing 200 μ g fusion proteins) and 1 parts by volume Freund's complete adjuvant are mixed, breast Change, obtain mixed liquor first.By 1 parts by volume fusion protein solution (containing 200 μ g fusion proteins) and 1 parts by volume incomplete Freund's adjuvant Mixing, emulsification, obtains mixed liquor second.

(2) mixed liquor first is taken, through the subcutaneous multi-point injection in the positions such as the back of the body, abdomen to 5 BALB/c mouse (every BALB/c mouses The volume for injecting mixed liquor first is identical).

(3) 14d of step (2) is completed, through the subcutaneous multi-point injection in the positions such as the back of the body, abdomen to 5 BALB/c mouses (every The volume of BALB/c mouse injection mixed liquor second is identical).

(4) 35d of step (2) is completed, respectively to the tail vein blood of BALB/c mouse, separates serum；Then use Indirect elisa method determines antiserum titre.Comprise the following steps that：

A, the fusion protein solution obtained with coating buffer dilution step 3, obtains protein solution；

B, microwell plate is taken, protein solution (containing 0.1 μ g fusion proteins) is added per hole, is sealed with sealed membrane, 4 DEG C, 50rpm bags By 12h, supernatant is then abandoned, is washed three times with PBST buffer solutions, each 3min, is patted dry on filter paper；

C, after completing step b, the PBST buffer solutions containing 5% (v/v) skimmed milk is added per hole, are sealed with sealed membrane, 37 DEG C Standing 1h, then abandons supernatant, is washed three times with PBST buffer solutions, each 3min, is patted dry on filter paper；

D, after completing step c, often the serum of the step of 2 times of doubling dilutions of hole addition (4) separation, is sealed, 37 with sealed membrane DEG C 1h is stood, then abandon supernatant, washed three times with PBST buffer solutions, each 3min, patted dry on filter paper；

5th, after completing step 4, the anti-mouse antibody of horseradish peroxidase-labeled is added per hole, is sealed with sealed membrane, 37 DEG C Standing 30min, then abandons supernatant, is washed three times with PBST buffer solutions, each 3min, is patted dry on filter paper；

7th, after completing step 6,100 μ L substrate solutions is added per hole, lucifuge colour developing 15min, it is dense that 70 μ L are then added per hole Spend the H for 1M₂SO₄The aqueous solution, measure OD value of each hole at 450nm.

Part of test results is shown in that (3 mice serums are the serum (i.e. immune serum) of step (4) separation to Fig. 3, are mixed Negative serum is the mixed liquor of 3 non-immune serums).As a result show, (mixed liquor is injected successively by immune mouse The mouse of first and mixed liquor second) serum final potency more than 1:25600, subsequent experimental requirements can be met completely.

(5) after completing step (4), it will be taken a blood sample by immune mouse by eyeball, then separate serum, freeze in -20 It is DEG C standby.The serum is the polyclonal antibody prepared.

3rd, the preparation of adjuvant and its identification of biological activity

1st, the preparation of adjuvant

Adjuvant provided by the invention is recombinant plasmid pIL-2.

(1) the recombinant plasmid second of 6 structures in step 1 is taken, double digestion is carried out with restriction enzyme EcoRI and XhoI, is returned Receive about 390bp digestion products second.

(2) pcDNA3.1 carriers are taken, double digestion is carried out with restriction enzyme EcoRI and XhoI, reclaims about 5.4kb load Body skeleton second.

(3) digestion products second and carrier framework second are attached, obtain recombinant plasmid pIL-2.

(4) recombinant plasmid pIL-2 is taken, enters performing PCR identification respectively and (is entered using primers F 2 and primer the R2 primer pair B formed OK), single endonuclease digestion identification (using restriction enzyme EcoRI carry out) and double digestion identification (using restriction enzyme EcoRI with XhoI is carried out).

Experimental result is shown in that (M1 is that Trans 2K DNA marker, M2 are Trans 15K DNA marker to B in Fig. 1, swimming Road 1 identifies that swimming lane 2 is identified for single endonuclease digestion for PCR, and swimming lane 3 is identified for double digestion).

(5) recombinant plasmid pIL-2 is sequenced.

Identified according to PCR, digestion identification and sequencing result, containing shown in sequence in ordered list 1 in recombinant plasmid pIL-2 DNA molecular.Protein (hereinafter referred to as IL-2 albumen or protein in recombinant plasmid pIL-2 expressed sequence tables shown in sequence 2 IL-2)。

2nd, the identification of the biological activity of adjuvant

Rainbow trout gonadal cell RTG-2 is that (network address is ATCC：https://www.atcc.org) product, production code member is CCL-55。

(1) 24 orifice plates are taken, 1.0 × 10 are added per hole⁵Individual rainbow trout gonadal cell RTG-2 and 0.3mL are newborn containing 10% (v/v) The MEM culture mediums of calf serum, 5%CO₂, 15 DEG C of culture to cell monolayers reach 40-60%.

(2) 1-3 μ g recombinant plasmid pIL-2 are added into the system for completing step (1), then using lipofectamine Transfected (with specific reference to the operating procedure of lipofectamine specification).

(3) system of step (2), 5%CO are taken into₂, 15 DEG C of culture 48h, it is then water-soluble with 4% (v/v) paraformaldehyde Liquid is fixed, then is washed 3 times with pH7.4,0.01mM PBS.

(4) take into the system of step (3), add 200 μ L polyclonal antibodies dilutions (by 49 parts by volume pH7.4, Polyclonal antibody prepared by 0.01mM PBS and 1 parts by volume step 2 mixes), 37 DEG C are incubated 1h, Ran Houyong PH7.4,0.01mM PBS wash 3 times.

(5) system of step (4) is taken into, adds the anti-mouse FITC labelled antibodies dilutions of 200 μ L (by 999 parts by volume PH7.4,0.01mM PBS and the anti-mouse FITC labelled antibodies of 1 parts by volume mix), 37 DEG C of incubation 30min, then Washed 3 times with pH7.4,0.01mM PBS.

(6) after completing step (5), it is placed under fluorescence inverted microscope and observes, records result.

According to above-mentioned steps, the recombinant plasmid pIL-2 in step (2) is replaced with into pcDNA3.1 carriers, other steps are equal It is constant, as control.

Experimental result is shown in Fig. 4 (A is recombinant plasmid pIL-2, and B is control).As a result show, recombinant plasmid pIL-2 can be in rainbow High-caliber expression in trout gonadal cell RTG-2, there is biological activity, next step experiment in vivo can be carried out.

The application of adjuvant prepared by embodiment 2, embodiment 1

First, the preparation of DNA vaccination (recombinant plasmid pEE12.4-G)

1st, the double chain DNA molecule in artificial synthesized sequence table shown in sequence 5 and using it as template, using primer pair third (by Primers F 3：5’-GAATTC(single underscore is restriction enzyme EcoRI identification to ATGGACACCATGATCACCACTCCG-3 ' Site) and primer R3：5’-GGATCC(single underscore is restriction enzyme BamHI to TCAGGACCGGTTTGCCAGGTGAT-3 ' Recognition site) composition) and enter performing PCR amplification, obtain 1539bp pcr amplification product third.

2nd, pcr amplification product third is taken, double digestion is carried out with restriction enzyme EcoRI and BamHI, recovery about 1539bp's Digestion products third.

3rd, pEE12.4 carriers are taken, double digestion is carried out with restriction enzyme EcoRI and BamHI, reclaims about 6.9kb load Body skeleton third.

4th, digestion products third and carrier framework third are attached, obtain recombinant plasmid pEE12.4-G.

Recombinant plasmid pEE12.4-G is sequenced.According to sequencing result, structure is carried out to recombinant plasmid pEE12.4-G It is described as follows：Small fragment between restriction enzyme EcoRI and the BamHI recognition sequence of pEE12.4 carriers is replaced with into sequence DNA molecular in table shown in sequence 5.Protein in recombinant plasmid pEE12.4-G expressed sequence tables shown in sequence 6 (infects Property hematopoietic necrosis virus' G-protein, hereinafter referred to as G-protein).

Recombinant plasmid pEE12.4-G is the DNA vaccination prepared.

2nd, it is immunized

It is (every that the rainbow trout that 180 urosome weights are 9.5-10.5g is randomly divided into combined immunization group, independent immune group and control group 60 tails of group), it is handled as follows respectively：

Combined immunization group：Intramuscular injection recombinant plasmid pEE12.4-G and recombinant plasmid pIL-2.Injection dosage is 2.5 μ g weights Group plasmid pEE12.4-G/ tails and 2.5 μ g recombinant plasmid pIL-2/ tails.

Independent immune group：Intramuscular injection recombinant plasmid pEE12.4-G.Injection dosage is 2.5 μ g recombinant plasmids pEE12.4- G/ tails.

Control group：Intramuscular injection pcDNA3.1 carriers.Injection dosage is 2.5 μ g pcDNA3.1 carriers/tail.

3rd, the influence that adjuvant prepared by embodiment 1 is expressed gene involved in immunity

1st, the 3d after step 2 is completed, each group puts to death 3 tail rainbow trouts, then strips spleen in sterile on ice rapidly.

2nd, spleen is taken respectively, and RNA extracts kits extraction total serum IgE is used after carrying out liquid nitrogen grinding, it is then anti-using M-MLV Transcript reagent box obtains cDNA.

3rd, using the cDNA that step 3 obtains as template, IgM genes, IgT genes, Mx in fluorescence quantitative PCR detection spleen are passed through Gene, Viperin genes, CD4 genes, the relative expression quantity of CD8 genes and TNF-α gene are (using β-Actin genes as internal reference Gene).

Identification IgM genes, IgT genes, Mx genes, Viperin genes, CD4 genes, CD8 genes, TNF-α gene and β- The nucleotide sequence of the primer of Actin genes refers to table 1.

Wherein quantitative fluorescent PCR loading system is 25 μ L, is drawn by 0.5 μ L sense primers (concentration is 10 μM), 0.5 μ L downstreams Thing (concentration is 10 μM), 12.5 μ L SYBR Premix Ex Taq (Tli RNaseH Plus) (2 × Conc.), 2 μ L templates are molten Liquid, 0.5 μ L ROX Reference Dye (50 × Conc.) and 9 μ L ddH₂O is formed.Sense primer is Primer in table 1 In contain " sense " primer, anti-sense primer be in table 1 in Primer contain " anti " primer.

Premix Ex Taq^TM(Tli RNaseH Plus) is the product of Takara companies, and catalog number is RR420A.SYBR Premix Ex Taq (Tli RNaseH Plus) (2 × Conc.) and ROX Reference Dye (50 × Conc.) it isPremix Ex Taq^TMComponent in (Tli RNaseH Plus).

Table 1

By gene involved in immunity in control group (IgM genes, IgT genes, Mx genes, Viperin genes, CD4 genes, CD8 Gene or TNF-α gene) relative expression quantity as 1, gene involved in immunity is relative in combined immunization group and independent immune group Expression quantity is shown in Fig. 5 (pEE12.4-G is independent immune group, and pEE12.4-G+pIL-2 is combined immunization group).Gene involved in immunity table It is compared up to using one-way analysis of variance, T is examined for the comparison between paired samples, P<0.05 is significant difference.

As a result show, IgT genes, Mx genes, Viperin genes, CD4 genes, CD8 genes and TNF- in combined immunization group The expression of α genes is all remarkably higher than independent immune group (P<0.05), the expression of IgM genes is also high in combined immunization group In independent immune group, but difference is not notable.

4th, adjuvant prepared by embodiment 1 is on influence caused by specific IgM antibodies

The 7th day after step 2 is completed, the 14th day, the 21st day, the 28th day and the 35th day respectively, gather the tail rainbow of each group 3 The blood of trout simultaneously separates serum, and the level of specific IgM antibodies in serum is then detected using ELISA method.Specific steps are such as Under：

1st, IHNV G truncated proteins are diluted with coating buffer, obtains the protein solution that concentration is 0.001 μ g/ μ L；

2nd, microwell plate is taken, 100 μ L protein solutions are added per hole, are sealed with sealed membrane, 4 DEG C, 50rpm coating 12h, is then abandoned Supernatant, washed three times with PBST buffer solutions, each 3min, patted dry on filter paper；

3rd, after completing step 2, the PBST buffer solutions that 100 μ L contain 5% (v/v) skimmed milk is added per hole, are sealed with sealed membrane, 37 DEG C of standing 1h, then abandon supernatant, are washed three times with PBST buffer solutions, each 3min, are patted dry on filter paper；

4th, after completing step 3,100 μ L serum dilutions are added per hole, and (into PBST buffer solutions, increase serum to its content is 1% (v/v), is sealed with sealed membrane, 37 DEG C of standing 1h, is then abandoned supernatant, is washed three times with PBST buffer solutions, each 3min, filter Patted dry on paper；

5th, after completing step 4,100 μ L antibody diluents is added per hole, are sealed with sealed membrane, 37 DEG C of standing 30min, then Supernatant is abandoned, is washed three times with PBST buffer solutions, each 3min, is patted dry on filter paper；

6th, after completing step 5, the anti-rabbit secondary antibody (working concentration 1 of 100 μ L horseradish peroxidase-labeleds is added per hole: 120000, diluted with pH7.4,0.01mM PBS), sealed with sealed membrane, 37 DEG C of standing 30min, then abandon supernatant, use PBST buffer solutions wash three times, each 3min, are patted dry on filter paper；

Experimental result see Fig. 6 (pEE12.4-G is independent immune group, and pEE12.4-G+pIL-2 is combined immunization group, PcDNA3.1 is control group).As a result show the 7th day after being immunized, there is specific IgM in independent immune group and combined immunization group Antibody produces, and is immunized and reaches within the 28th day highest level afterwards, the level of specific IgM antibodies in independent immune group and combined immunization group Without significant difference.

5th, the influence of adjuvant prepared by embodiment 1 to neutralize antibody titers

The 7th day after step 2 is completed, the 14th day, the 21st day, the 28th day and the 35th day respectively, gather the tail rainbow of each group 3 The blood of trout simultaneously separates serum, then (is recorded in using PNT methods in following document：SS Ristow, JD Avila, SE Lapatra, K Lauda.Detection and characterization of rainbow trout antibody Against Infectious hematopoietic necrosis virus, Diseases of Aquatic Organisms, 1993,15 (2), 109-114.) detect neutralize antibody titers in serum.Comprise the following steps that：

1st, serum is placed in 56 DEG C of water-bath inactivation 30min, cooling, obtains serum to be checked；

2nd, Tissue Culture Plate is taken, serum to be checked is done 2 times with MEM culture mediums and is serially diluted, is 50 μ L per boreliquid capacity (i.e. the 1st hole adds 50 μ L serum to be checked；2nd hole first adds 50 μ L serum to be checked and 50 μ L MEM culture mediums, fully mixed It is even, then draw 50 μ L and add the 3rd hole；50 μ L MEM culture mediums are added to the 3rd hole, fully mixes, then draws 50 μ L Add the 4th hole；The like)；

3rd, after completing step 2,50 μ L IHNV-Sn1203 (potency is 100TCID50/50 μ L) are added per hole, it is fully mixed It is even, it is subsequently placed in 15 DEG C of incubators and stands 1h；

4th, after completing step 3, the liquid in the every hole of the Tissue Culture Plate is transferred to the correspondence of another Tissue Culture Plate Kong Zhong, it is placed in 15 DEG C of incubators and stands 1h, abandon supernatant.

5th, after completing step 4, the MEM culture mediums that 500 μ L contain 1% (V/V) methylcellulose is added per hole, treat negative control After there is obvious plaque, supernatant is abandoned, is dyed with crystal violet, records each hole plaque formation quantity, plaque reduces 50% most High dilution is neutralize antibody titers.

In above-mentioned steps, the serum to be checked in step 2 is replaced with into healthy rainbow trout serum, other steps are constant, are Negative control.

In above-mentioned steps, the serum to be checked in step 2 is replaced with into IHNV-Sn1203 infection rainbow trout serum, other steps It is constant, as positive control.

In above-mentioned steps, the serum to be checked in step 2 is replaced with into MEM culture mediums, other steps are constant, are virus Control.

In above-mentioned steps, the IHNV-Sn1203 in step 3 is replaced with into MEM culture mediums, other steps are constant, are Normal control.

Experimental result is shown in Table 2 (potency>1:20 neutralizing antibody is considered positive).As a result show, the 14th day after being immunized, connection Closing immune group can induce positive neutralizing antibody generation (1:40), now the no positive neutralizing antibody of independent immune group produces；Combine and exempt from The neutralizing antibody of epidemic disease group reaches peak on 28th day after immune, though this result is later than independent immune group 7 days, after immune Neutralizing antibody in 35th day combined immunization group can still maintain 1:160, and individually the neutralizing antibody in immune group now then under It is down to 1:40, control group is not detected by positive neutralizing antibody in each week after immune.Therefore, the adjuvant that prepared by embodiment 1 can be with Influence neutralize antibody titers.

Table 2

	7 days after immune	14 days after immune	21 days after immune	28 days after immune	35 days after immune
						Combined immunization group	1:20	1:40	1:80	1:160	1:160
Independent immune group	1:20	1:20	1:160	1:80	1:40
						Control group	<1:20	<1:20	<1:20	<1:20	<1:20

6th, adjuvant prepared by embodiment 1 is on lymphopoietic influence

G-protein is recorded in following document：Tao Jian, Xu Liming, Liu Miao, Zhao Jingzhuan, Cao Yongsheng, Lu Tongyan, Yin Jiasheng, Liu The prokaryotic expression and application fresh water fisherys of red cypress infectious hematopoietic necrosis poison epitope enrichment region, 2015,45 (2), 49-55.

The 14th day after step 2 is completed and the 28th day respectively, combined immunization group and independent immune group are respectively put to death into 3 tails Rainbow trout, spleen is stripped in sterile on ice rapidly, lymphopoiesis index is then determined using mtt assay.Comprise the following steps that：

1st, spleen is rolled with syringe inner prop, through nylon net filter, is then separated using Ficoll-Paque PLUS reagents Lymphocyte, 5000rpm centrifugation 3min, is washed twice with sterile pH7.4,0.01mM PBS.

2nd, after completing step 1, the MEM culture mediums resuspension containing 10% (v/v) serum is added into the lymphocyte, is obtained Concentration is 5 × 10⁶Individual/mL cell suspending liquid.

3rd, 96 porocyte culture plates are taken, it is molten for 2 μ g/mL G-protein that 100 μ L cell suspending liquids and 50 μ L concentration are added per hole G-protein (is dissolved in pH7.4,0.01mM PBS to obtain) by liquid, 5%CO₂, 15 DEG C of culture 60h, 10 are then added per hole μ L WST-1 solution continues to cultivate 2h, measures the OD values at 450nm, that is, stimulates the OD in hole_450nm。

4th, according to step 3 the step of, it is that 5 μ g/mL PHA are water-soluble that the G-protein solution that concentration is 2 μ g/mL is replaced with into concentration Liquid, other steps are constant, obtain the OD of control wells_450nm。

Calculate stimulus index.The OD in stimulus index=stimulation hole_450nmThe OD of/control wells_450nm。

Experimental result is shown in Fig. 7 (pEE12.4-G is independent immune group, and pEE12.4-G+pIL-2 is combined immunization group).As a result Show, the 14th day after being immunized, stimulus index of the combined immunization group under PHA stimulation is significantly higher than independent immune group (P< 0.05)；28th day after immune, stimulus index of the combined immunization group in the case where PHA or G-protein are stimulated is significantly higher than independent immune group (P <0.05).Therefore, the adjuvant that prepared by embodiment 1 can influence the propagation of lymphocyte.

7th, the influence of adjuvant prepared by embodiment 1 to the protection of DNA vaccination (i.e. recombinant plasmid pEE12.4-G)

The 28d after step 2 is completed, each group is taken 30 tail rainbow trouts, by the way of intraperitoneal injection, entered with IHNV-Sn1203 Row attacks malicious (dosage is 300pfu/ tails), then observes fish body health situation day by day, records the The dead quantity of fish, draws accumulation and deposits Motility rate curve.

Experimental result see Fig. 8 (pEE12.4-G is independent immune group, and pEE12.4-G+pIL-2 is combined immunization group, PcDNA3.1 is control group).As a result show, attack after poison 14 days, the survival rate (43.3%) of independent immune group is significantly higher than control Group (13.3%), and the protective rate of combined immunization group can reach 80%.Therefore, the adjuvant that prepared by embodiment 1 can be from multi-angle pair The immune response of recombinant plasmid pEE12.4-G inductions optimizes, and effectively lifts DNA vaccination (i.e. recombinant plasmid pEE12.4-G) Protection.

<110>Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie

<120>Application of the IL-2 albumen in animal vaccine adjuvant is prepared

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 369

<212> DNA

<213>Rainbow trout（Oncorhynchus mykiss）

<400> 1

aacccaattc ccagactcct agctggaatc gattatctag aagaaaatat tacatgtcca 60

gattcagtct tctatacacc aactgatgta gaggatagtt gcattgttgc agcattggcc 120

tgttccatta aggaactgga cactgtgaaa gtagaatgcc tcgataaagc ggtccatctg 180

gaaagtatgc aacaccacat cagcatgact gccacggccc tacaaaagac gattgataag 240

gagaacagca caacggacac ttcagaatgc atctgtgaag acaagcggtt ggaaaagtct 300

ttcaaggact tcattcagaa cataagacat ttaactcaag ctcatgctgc aaagcgtcta 360

agttcataa 369

<210> 2

<211> 122

<212> PRT

<213>Rainbow trout（Oncorhynchus mykiss）

<400> 2

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Ile Thr Cys Pro Asp Ser Val Phe Tyr Thr Pro Thr Asp Val Glu Asp

20 25 30

Ser Cys Ile Val Ala Ala Leu Ala Cys Ser Ile Lys Glu Leu Asp Thr

35 40 45

Val Lys Val Glu Cys Leu Asp Lys Ala Val His Leu Glu Ser Met Gln

50 55 60

His His Ile Ser Met Thr Ala Thr Ala Leu Gln Lys Thr Ile Asp Lys

65 70 75 80

Glu Asn Ser Thr Thr Asp Thr Ser Glu Cys Ile Cys Glu Asp Lys Arg

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Leu Glu Lys Ser Phe Lys Asp Phe Ile Gln Asn Ile Arg His Leu Thr

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Gln Ala His Ala Ala Lys Arg Leu Ser Ser

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<210> 3

<211> 429

<212> DNA

<213>Rainbow trout（Oncorhynchus mykiss）

<400> 3

atggaccgtc gttacaggat ttcctttttg acgctttttc tcgccggttg tctacaagga 60

aacccaattc ccagactcct agctggaatc gattatctag aagaaaatat tacatgtcca 120

gattcagtct tctatacacc aactgatgta gaggatagtt gcattgttgc agcattggcc 180

tgttccatta aggaactgga cactgtgaaa gtagaatgcc tcgataaagc ggtccatctg 240

gaaagtatgc aacaccacat cagcatgact gccacggccc tacaaaagac gattgataag 300

gagaacagca caacggacac ttcagaatgc atctgtgaag acaagcggtt ggaaaagtct 360

ttcaaggact tcattcagaa cataagacat ttaactcaag ctcatgctgc aaagcgtcta 420

agttcataa 429

<210> 4

<211> 142

<212> PRT

<213>Rainbow trout（Oncorhynchus mykiss）

<400> 4

Met Asp Arg Arg Tyr Arg Ile Ser Phe Leu Thr Leu Phe Leu Ala Gly

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Cys Leu Gln Gly Asn Pro Ile Pro Arg Leu Leu Ala Gly Ile Asp Tyr

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Leu Glu Glu Asn Ile Thr Cys Pro Asp Ser Val Phe Tyr Thr Pro Thr

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Asp Val Glu Asp Ser Cys Ile Val Ala Ala Leu Ala Cys Ser Ile Lys

50 55 60

Glu Leu Asp Thr Val Lys Val Glu Cys Leu Asp Lys Ala Val His Leu

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Glu Ser Met Gln His His Ile Ser Met Thr Ala Thr Ala Leu Gln Lys

85 90 95

Thr Ile Asp Lys Glu Asn Ser Thr Thr Asp Thr Ser Glu Cys Ile Cys

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Glu Asp Lys Arg Leu Glu Lys Ser Phe Lys Asp Phe Ile Gln Asn Ile

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Arg His Leu Thr Gln Ala His Ala Ala Lys Arg Leu Ser Ser

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<210> 5

<211> 1527

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 5

atggacacca tgatcaccac tccgctcatt ctcattctaa tcacctgtgg agcaaacagc 60

caaacagtcc cccccgacac cgcaagcgaa tcagaccaac ccacctggtc aaacccgctc 120

ttcacctacc ccgagggatg cactctggac aaactctcca aggtcaatgc ttctcaactg 180

agatgcccaa ggatcttcga tgatgagaac agggggctaa tttcttatcc cgcctccatc 240

cggtccctgg cggtcggaaa cgacctcggg gcgattcaca cccaagggaa ctacatccac 300

aaagtcctgt accgcaccat ctgctcaaca gggttcttcg ggggtcagac gatagagaag 360

gtacttgtag aaatgaaact ctcaacgaga gaagcagggg catatgacac cacaaccgcg 420

gccgctctgt acttcccagc tccccgatgc caatggtaca ccgacaacgt acaaaatgat 480

ctcatcttct actacacaac ccaaaagagt gttctgagag atccctacac cagagacttc 540

ctggactcag attttgttgg aggaaaatgc actaaatcac cctgccagac tcattggtcc 600

aacgtagttt ggattggtga tgcagggata ccagcctgtg acgccagccc agaaataaac 660

ggtcacctct ttgttgataa aatctccagt cgagccgtga aggcaacgag ctacggacac 720

cacccctggg gactgcatcg ggcctgtatg attgagttct gtgggaaaca gtggatacgg 780

acagatctcg gtgacctgat atctgtagga tacaattctg gagcaaaaac cctctccttc 840

ccgaagtgtg aggacgagac ggtggggatg aggggaaacc tggatgactt tgcctatcta 900

gacgacctgg tgaaggcctc agagagcaga gaggaatgtc ttgaggcgca tgccgagata 960

atatcaacaa acagtgtgac tccatacctc ctatccaagt tccgatctcc acatcccgga 1020

ataaatgacg tctacgctat gcacaaaggc tccatctatc atgggatgtg catgacggtc 1080

gctgtggacg aggtatccaa ggacaggacg acgtacaggg cccatcacgc caccaacttc 1140

actaaatggg aacgaccctt tggggatgag tgggaaggct ttcacggatt gcacggaaac 1200

aacatcacca ttattccaga cctggagaaa tacgtcgccc agtacaagat gagcatgatg 1260

gaaccgatgg gcatcaaatc cgtaccccat ccaagcatcc tggccttcta caatgagaca 1320

gaagtatcgg ggatctccat caggaaattg gactcgttcg accttcaatc actccactgg 1380

agtttctggc ccacaatctc cacactgggt gggattcccc tggttctcct ccttgctgtt 1440

gccgcgtgct gctgctggtc agggagactt cccactccct ccgcgccgca gagtatcccc 1500

atgtatcacc tggcaaaccg gtcctga 1527

<210> 6

<211> 508

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 6

Met Asp Thr Met Ile Thr Thr Pro Leu Ile Leu Ile Leu Ile Thr Cys

1 5 10 15

Gly Ala Asn Ser Gln Thr Val Pro Pro Asp Thr Ala Ser Glu Ser Asp

20 25 30

Gln Pro Thr Trp Ser Asn Pro Leu Phe Thr Tyr Pro Glu Gly Cys Thr

35 40 45

Leu Asp Lys Leu Ser Lys Val Asn Ala Ser Gln Leu Arg Cys Pro Arg

50 55 60

Ile Phe Asp Asp Glu Asn Arg Gly Leu Ile Ser Tyr Pro Ala Ser Ile

65 70 75 80

Arg Ser Leu Ala Val Gly Asn Asp Leu Gly Ala Ile His Thr Gln Gly

85 90 95

Asn Tyr Ile His Lys Val Leu Tyr Arg Thr Ile Cys Ser Thr Gly Phe

100 105 110

Phe Gly Gly Gln Thr Ile Glu Lys Val Leu Val Glu Met Lys Leu Ser

115 120 125

Thr Arg Glu Ala Gly Ala Tyr Asp Thr Thr Thr Ala Ala Ala Leu Tyr

130 135 140

Phe Pro Ala Pro Arg Cys Gln Trp Tyr Thr Asp Asn Val Gln Asn Asp

145 150 155 160

Leu Ile Phe Tyr Tyr Thr Thr Gln Lys Ser Val Leu Arg Asp Pro Tyr

165 170 175

Thr Arg Asp Phe Leu Asp Ser Asp Phe Val Gly Gly Lys Cys Thr Lys

180 185 190

Ser Pro Cys Gln Thr His Trp Ser Asn Val Val Trp Ile Gly Asp Ala

195 200 205

Gly Ile Pro Ala Cys Asp Ala Ser Pro Glu Ile Asn Gly His Leu Phe

210 215 220

Val Asp Lys Ile Ser Ser Arg Ala Val Lys Ala Thr Ser Tyr Gly His

225 230 235 240

His Pro Trp Gly Leu His Arg Ala Cys Met Ile Glu Phe Cys Gly Lys

245 250 255

Gln Trp Ile Arg Thr Asp Leu Gly Asp Leu Ile Ser Val Gly Tyr Asn

260 265 270

Ser Gly Ala Lys Thr Leu Ser Phe Pro Lys Cys Glu Asp Glu Thr Val

275 280 285

Gly Met Arg Gly Asn Leu Asp Asp Phe Ala Tyr Leu Asp Asp Leu Val

290 295 300

Lys Ala Ser Glu Ser Arg Glu Glu Cys Leu Glu Ala His Ala Glu Ile

305 310 315 320

Ile Ser Thr Asn Ser Val Thr Pro Tyr Leu Leu Ser Lys Phe Arg Ser

325 330 335

Pro His Pro Gly Ile Asn Asp Val Tyr Ala Met His Lys Gly Ser Ile

340 345 350

Tyr His Gly Met Cys Met Thr Val Ala Val Asp Glu Val Ser Lys Asp

355 360 365

Arg Thr Thr Tyr Arg Ala His His Ala Thr Asn Phe Thr Lys Trp Glu

370 375 380

Arg Pro Phe Gly Asp Glu Trp Glu Gly Phe His Gly Leu His Gly Asn

385 390 395 400

Asn Ile Thr Ile Ile Pro Asp Leu Glu Lys Tyr Val Ala Gln Tyr Lys

405 410 415

Met Ser Met Met Glu Pro Met Gly Ile Lys Ser Val Pro His Pro Ser

420 425 430

Ile Leu Ala Phe Tyr Asn Glu Thr Glu Val Ser Gly Ile Ser Ile Arg

435 440 445

Lys Leu Asp Ser Phe Asp Leu Gln Ser Leu His Trp Ser Phe Trp Pro

450 455 460

Thr Ile Ser Thr Leu Gly Gly Ile Pro Leu Val Leu Leu Leu Ala Val

465 470 475 480

Ala Ala Cys Cys Cys Trp Ser Gly Arg Leu Pro Thr Pro Ser Ala Pro

485 490 495

Gln Ser Ile Pro Met Tyr His Leu Ala Asn Arg Ser

500 505

Claims

At least one of the application of 1.IL-2 albumen, it is a1) to a6)：A1 animal vaccine adjuvant) is prepared；A2 animal machine) is strengthened The expression of gene involved in immunity in body；A3 the potency of neutralizing antibody in animal body) is improved；A4) promote in animal body The propagation of lymphocyte；A5 the protection of animal vaccine) is lifted；A6) prepare and promote animal body to the immune response of antigen Product；

Any of the IL-2 albumen is b1) to b6)：

B1) amino acid sequence is the protein shown in sequence 2 in sequence table；

B2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label obtain；

B3) by substitution of the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing And/or the protein with identical function that addition obtains；

B4) amino acid sequence is the protein shown in sequence 4 in sequence table；

B5) the fused protein that the N-terminal of the protein in sequence table shown in sequence 4 or/and C-terminal connection label obtain；

B6) by substitution of the amino acid sequence shown in sequence in sequence table 4 by one or several amino acid residues and/or missing And/or the protein with identical function that addition obtains.
2. encoding the nucleic acid molecules of IL-2 albumen described in claim 1, or, contain IL-2 eggs described in coding claim 1 Expression cassette, recombinant vector or the recombinant bacterium of white nucleic acid molecules, application, be a1) to a6) at least one of：A1) prepare dynamic Thing vaccine adjuvant；A2 the expression of gene involved in immunity in animal body) is strengthened；A3 neutralizing antibody in animal body) is improved Potency；A4 the propagation of animal body medium size lymphocyte) is promoted；A5 the protection of animal vaccine) is lifted；A6) prepare and promote to move The product of the former immune response of thing body fight.
3. application as claimed in claim 2, it is characterised in that：Any of the nucleic acid molecules are c1) to c6)：

C1) DNA molecular of the code area as shown in sequence 1 in sequence table；

C2) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；

C3) DNA molecular of the code area as shown in sequence 3 in sequence table；

C4) nucleotide sequence is the DNA molecular shown in sequence 3 in sequence table；

C5) and c1) or c2) c3) or c4) nucleotide sequence that limits has 75% or more than 75% homogeneity, and encodes and weigh Profit requires the DNA molecular of IL-2 albumen described in 1；

C6) under strict conditions with c1) c2) or c3) or c4) limit nucleotide sequence hybridization, and encode claim 1 in The DNA molecular of the IL-2 albumen.
4. a kind of product, it contains t1) or t2) or t3)：

T1) IL-2 albumen described in claim 1；

T2 the nucleic acid molecules of IL-2 albumen described in claim 1) are encoded；

T3 expression cassette, recombinant vector or recombinant bacterium) containing the nucleic acid molecules of IL-2 albumen described in coding claim 1；

The product has following f1) to f6) at least one of function：

F1) animal vaccine adjuvant；F2 the expression of gene involved in immunity in animal body) is strengthened；F3) improve in animal body The potency of neutralizing antibody；F4 the propagation of animal body medium size lymphocyte) is promoted；F5 the protection of animal vaccine) is lifted；F6) promote Enter the former immune response of animal body fight.
5. the product as described in application or claim 4 as described in claims 1 to 3 is any, it is characterised in that：It is described immune Related gene is at least one of IgT genes, Mx genes, Viperin genes, CD4 genes, CD8 genes and TNF-α gene.
6. the application as described in claim 1,2,3 or 5 are any or the product as described in claim 4 or 5, it is characterised in that：Institute It is nucleic acid vaccine to state vaccine.
7. the product described in application as claimed in claim 6 or claim 6, it is characterised in that：The nucleic acid vaccine is biography Metachromia Hematopoietic Necrosis's disease nucleic acid vaccine.
The product as described in 8. application or claim 4 to 7 as described in claim 1,2,3,5,6 or 7 are any are any, it is special Sign is：The antigen is infectious hematopoietic necrosis's poison G-protein.
9. a kind of promotion animal body comprises the following steps to the method for the immune response of antigen：Power is expressed in animal body Profit requires IL-2 albumen described in 1.
10. a kind of method for the protection for lifting animal vaccine, comprises the following steps：Claim 1 is expressed in animal body Described in IL-2 albumen.