CN104479004A - Black carp IFN-gamma gene and application - Google Patents
Black carp IFN-gamma gene and application Download PDFInfo
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Abstract
The invention discloses a black carp IFN-gamma gene and an application. The sequence of a separated black carp IFN-gamma gene is shown in SEQ ID No. 1, and the coded protein is shown in SEQ ID No. 2. The expression changes of the IFN-gamma gene after being induced by Poly I: C is detected by applying fluorogenic quantitative PCR; the detection result finds that the Poly I: C is capable of remarkably inducing the expression of the IFN-gamma gene in kidney, spleen, gill, head-kidney and skin tissues; and the black carp IFN-gamma gene is successfully expressed in vitro by transfecting recombinant plasmids in grass carp CIK cells. The open reading frame sequence of the black carp IFN-gamma gene is connected to an eukaryotic expression vector pcDNA3.1(+) to be prepared into a DNA vaccine which is injected in black carp bodies, so that the infection prevention capacity of the black carp bodies for viruses can be remarkable enhanced.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of black carp IFN-γ gene and application.
Technical background
Interferon, rabbit (IFN) is that a class can induce vertebrate cells to enter the secretor type cytokine of antiviral state, according to the difference of gene, protein structure and functional property, is divided into I type, II type and III Interferon, rabbit.Different with III Interferon, rabbit from I type, vertebrate interferon type Ⅱ only finds INF-γ up to now.IFN-γ produces primarily of specific cells such as NK cell, activated T lymphocytes, scavenger cell and neuronal cells.In the natural immunity, intracellular pathogen infection causes mononuclear phagocyte and antigen presenting cell secretion IL-12 and IL-18, IL-12 and IL-18 stimulates NK emiocytosis IFN-γ, then IFN-γ inducing macrophage produces toxicant as reactive oxygen intermediate and reactive nitrogen intermediate, removes intra-cellular pathogens; IFN-γ also synthesizes a large amount of antiviral protein as 2 '-5 by inducing host cell ' oligo-adenylate synthetase, dsRNA dependent kinases (PKR), G associated proteins and adenosine deaminase, set up antiviral state.IFN-γ also regulatory T-cell propagation and differentiation, mediated leucocytes gathering, enhancement antigen offers, in the reaction of cell-mediated adaptive immunity, play prior effect.Recent years, IFN-gamma gene sequences is cloned in several fish, comprises zebra fish, globe fish, rainbow trout, Atlantic salmon, channel catfish, goldfish, carp etc.
Black carp (Mylopharyngodon piceus) is the economic fish of the main freshwater aquiculture of China, the present invention carries out Cloned culturing to black carp IFN-γ gene, and express in grass carp CIK cell, be intended to weave construction, genetic evolution feature, the expression regulation rule for studying black carp IFN γ gene and in antiviral immunity, the regulating and controlling effect of other cytokines laid a good foundation.
Summary of the invention
The invention discloses a kind of black carp IFN-γ gene, its sequence is for described in SEQ ID NO.1, and the aminoacid sequence of coding is for shown in SEQ ID NO.2, and in the natural immunity, black carp IFN-γ can express by adjustment portion immunogene.
Another object of the present invention there are provided the application of a kind of black carp IFN-γ gene in preparation raising fresh-water fishes immunizing power medicine.Be preferred for black carp, this is prepared as DNA vaccination, for black carp, the immunizing power of black carp can be significantly improved.
In order to achieve the above object, the present invention takes following technical measures:
A kind of black carp IFN-γ gene, is prepared by following steps:
Extract total black carp by Trizol (Invitrogen) specification sheets and organize RNA.After being used by the total serum IgE of each tissue Dnase I (Takara) to process respectively, Reverse Transcription box SMART PCR cDNA Synthesis Kit (Clontech) is utilized to carry out reverse transcription synthesis cDNA first chain.
According to known teleostei IFN-γ sequences Design degenerated primer JB1-F and JB1-R.After amplification obtains intermediate segment, forward (RACE1-3F is designed respectively again by SMARTer RACE cDNA Amplification kit (Clontech), RACE2-3F) and oppositely (RACE1-5R, RACE2-5R) nested primer, through two-wheeled PCR react amplification its 3 ' and 5 ' end fragment.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 65 ~ 72 DEG C of 45s, run 5 circulations; 94 DEG C of 30s, 65 ~ 72 DEG C of 45s, run 5 circulations; 94 DEG C of 30s, 65 ~ 72 DEG C of 30s, 72 DEG C of 45s, run 27 circulations; 72 DEG C of 10min.First round pcr amplification product sterilized water is got 1 μ L diluent takes turns PCR reaction template as second after diluting 50 times, reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 60 ~ 70 DEG C of 30s, 72 DEG C of 45s, run 35 circulations; 72 DEG C of 10min.。Object product, after 1.2% agarose gel electrophoresis detects, reclaims purifying, after sequencing result splicing, namely obtain black carp IFN-γ gene, its sequence is for shown in SEQID NO.1, and the protein of coding is for shown in SEQ ID NO.2, open reading frame is 549bp, 182 amino acid of encoding.
Black carp IFN-γ gene is improving the application in fresh-water fishes immunizing power medicine in preparation, its application process is as follows:
The open reading frame sequence of black carp IFN-γ gene is connected on eukaryotic expression vector pcDNA3.1 (+), be transformed in bacillus coli DH 5 alpha recipient cell, PCR screening positive clone, plasmid is extracted after enlarged culturing, by this plasmid intramuscular injection in black carp, the antiviral ability of black carp can be significantly improved.
Compared with prior art, the present invention has the following advantages:
1. the present invention discloses the cDNA full length sequence of black carp IFN-γ first, and carries out sequential analysis to it.
2. found in black carp tissue through Poly I:C stimulate after, the expression in IFN-γ different tissues.
3. in grass carp CIK cell, express black carp IFN-γ by carrier for expression of eukaryon.
4. find can improve Mx gene and MyD88 gene expression amount in black carp spleen at black carp expression in vivo IFN-γ gene, for the expression regulation rule of research black carp IFN-γ in immunne response and anti-infectious immunity and mechanism of action have established test and theoretical basis.
5., by black carp intramuscular injection recombinant plasmid pcDNA3.1 (+)-IFN-γ, can effectively infect by prevention and therapy GCRV.
Accompanying drawing explanation
Fig. 1 is the analysis of IFN amino acid sequence homology.
Fig. 2 is that black carp IFN-γ compares with other vertebrates IFN-γ aminoacid sequences.
Fig. 3 is the evolutionary relationship that phylogenetic tree analyzes teleostei IFN-γ molecule.
Fig. 4 is that fluorescent quantitation detects black carp IFN-γ at the post-stimulatory expression of PolyI:C.
Fig. 5 is the expression of different time after indirect immunofluorescence technique detection Transfected Recombinant Plasmid grass carp CIK cell.
Embodiment
Technical scheme of the present invention, as special standby explanation, is the ordinary skill in the art.
Embodiment 1:
A kind of black carp IFN-γ gene, is prepared by following steps:
1. extract total serum IgE and synthesis the first chain cDNA
Black carp weight is about 1Kg, purchased from plant, supports 2 weeks temporarily before experiment.Extract total black carp by Trizol (Invitrogen) specification sheets and organize RNA.After being used by the total serum IgE of each tissue Dnase I (Takara) to process respectively, Reverse Transcription box SMART PCR cDNA Synthesis Kit (Clontech) is utilized to carry out reverse transcription synthesis cDNA first chain.
2. increase black carp IFN-γ cDNA and genome sequence
According to known teleostei IFN-γ sequences Design degenerated primer (table 1).After amplification obtains intermediate segment, then press SMARTerRACE cDNA Amplification kit (Clontech) and design forward and reverse nested primer respectively, through two-wheeled PCR reaction increase its 3 ' and 5 ' end fragment.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 72 DEG C of 45s, run 5 circulations; 94 DEG C of 30s, 70 DEG C of 45s, run 5 circulations; 94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 45s, run 27 circulations; 72 DEG C of 10min.First round pcr amplification product sterilized water is got 1 μ L diluent takes turns PCR reaction template as second after diluting 50 times, reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 45s, run 35 circulations; 72 DEG C of 10min.Object product, after 1.2% agarose gel electrophoresis detects, reclaims purifying, and after sequencing result splicing, final acquisition black carp IFN-γ gene, its sequence is for shown in SEQ ID NO.1.This full length gene is 895bp, and its open reading frame is 549bp, 182 amino acid of encoding, its 5 ' and 3 ' non-coding region comprise 138bp, 208bp respectively.
Table 1 the primer sequence of the present invention
3. sequential analysis
The BLAST software of the homogenic NCBI of being searched through (http://www.ncbi.nlm.nih.gov/blast) carries out; The deduction of aminoacid sequence is translated by the Translate software of EXPASY website (http://web.expasy.org/translate/).The prediction of signal peptide is predicted by SignalP3.0 (http://www.cbs.dtu.dk/services/SignalP/).Secondary protein structure is analyzed by PSIRED (http://bioinf.cs.ucl.ac.uk/psipred/).The multiple ratio of aminoacid sequence is to use CLUSTAL W 2.0 (http://www.ebi.ac.uk/Tools/clustalw2/index.html
).The homology analysis of aminoacid sequence uses DNAstar Megalign program.Adopt the adjacent method (Neighbor-Joining) of MEGA4.0 software to carry out phylogenetic analysis, 1000 bootstraps are set and assess.
The sequence similarity of amino acid alignment result display black carp IFN-γ and higher vertebrate IFN-γ is lower, is only 14.5% ~ 18.0%.Be 26.7% ~ 91.0% with other teleostei IFN-γ similaritys, wherein with the similarity the highest (Fig. 1) of grass carp (Ctenopharyngodon idella) IFN-γ.Existence 1 signal peptide sequence (MTAQHMMAIFWGVCLLILRRMTYA) is held at black carp IFN-γ molecule N, and in C-terminal, there is characteristic sequence ([I/V]-QX-[K/Q]-A-X2-E-[L/F]-X2-[I/V]) and the nuclear targeting motifs (Nuclear localization site, NLS) (Fig. 2) of IFN-γ family.By teleostei IFN-γ protein sequence with MEGA4.0 software building NJ systematic evolution tree.The topological framework of systematic evolution tree shows: it is one (Fig. 3) that black carp IFN-γ and grass carp (Ctenopharyngodon idella) IFN-γ gathers.
Comprise 7 αhelix in secondary structure analysis result display black carp IFN-γ sequence, its arrangement is similar with the IFN-γ of higher vertebrate.This conservative secondary structure contributes to being formed and stablizes its dimeric forms, so with its receptors bind, its N end have 1 signal peptide, also there is IFN-γ characteristic sequence in C-terminal and nuclear targeting motifs (NLS).
Embodiment 2:
Poly I:C is on the impact of black carp IFN-γ expression amount
Fish (750 ± 5 grams/fish) will be tested and be divided into 2 groups, often organize 3 tails.The every endnote in control group abdominal cavity penetrates PBS 500 μ L, and the every endnote in stimulating group abdominal cavity penetrates 500 μ L (2.5mg) Poly I:C.Its brain, the heart, kidney, spleen, the gill, head-kidney and skin histology is got after stimulating 24h.
Extract after each total tissue RNA is reversed into cDNA, analyzed by the black carp tissue of the two calibration curve method of real time fluorescent quantitative to injection Poly I:C and PBS.Be reference gene (primer is ACTIN-F and ACTIN-R) according to obtained quantitative primer JC-F and JC-R of black carp IFN-γ primers, black carp β-actin.The amplified production of two pairs of primers is connected to pMD-19T carrier, and extracts plasmid.Measure plasmid concentration, calculate plasmid copy number.Plasmid, after 10 times of gradient dilutions, carries out fluorescent quantitative PCR as standard form, production standard curve.Quantitative real time PCR Instrument (Rotor-Gene 6000Qiagen) increases, and condition is: 94 DEG C of 2min; 94 DEG C of 15s, 63 DEG C of 15s, 72 DEG C of 20s, 40 circulations, and draw solubility curve.All samples reaction all repeats 3 times.Calculate the copy number of IFN-γ in each tissue sample and the copy number of β-actin gene according to typical curve, with the expression amount of β-actin gene, destination gene expression amount is normalized.
As shown in Figure 4, in kidney, spleen, the gill, head-kidney and skin histology, Poly I:C significantly induces IFN-γ up-regulated expression.In head-kidney, raising change multiple the highest, is 25 times; Next is spleen, skin, the gill and kidney, and the expression amount of IFN-γ is respectively 22,18,13 and 9 times of control group; Noticeable change (Fig. 4) is not had in brain and heart tissue.Quantitative result of the present invention display, after Poly I:C induces, the remarkable up-regulated expression of IFN-γ in the fish such as head-kidney, spleen Main Immune Organs, show the cell derived of vertebrates IFN-γ and function comparatively conservative in the process of evolving.
Embodiment 3:
Black carp IFN-γ gene eukaryotic expression vector builds, and its process is as follows:
Utilize primer M.IFN γ-F/M.IFN γ-R to amplify black carp IFN-γ open reading frame with EcoR I and Hind III restriction enzyme site, remove black carp IFN-γ termination codon during design of primers, and add that 6 × his label carries out amalgamation and expression at 3 ' end.By double digestion, object fragment is connected on eukaryotic expression vector pcDNA3.1 (+), be transformed in bacillus coli DH 5 alpha recipient cell, PCR screening positive clone, plasmid is extracted after enlarged culturing, cut after qualification through enzyme, called after: pcDNA3.1 (+)-IFN-γ, serves the order-checking of Hai Shenggong biotechnology company limited.
With recombinant plasmid pcDNA3.1 (+)-IFN-γ transfection grass carp CIK cell, cultivate 24h, 48h, 72h, 96h and 120h for 25 DEG C and take out one bottle of cell respectively, utilize his antibody (Abcam) as primary antibodie, carry out indirect immuno fluorescent detection.Result shows, has the redness of fresh light to occur after the grass carp CIK cell 24h of transfection recombinant plasmid under green glow excites, and along with the increase of cell cultures time, the increasing number of red fluorescence, 72h red fluorescence quantity is maximum; The grass carp CIK cell of transfection recombinant plasmid is not had then not have red fluorescence to occur (Fig. 5).
Embodiment 4:
Black carp (750 ± 5 grams/fish) intramuscular injection 100 microlitre is contained the PBS of 10 microgram recombinant plasmid pcDNA3.1 (+)-IFN-γ, and establish empty plasmid and PBS to inject control group.
Within after injection the 14th day, extract black carp spleen tissue RNA, found by quantitative fluorescence analysis, the immunogene expression amounts such as Mx (primer Mx-F/Mx-R), MyD88 (primer MyD88F/MyD88R) in injection recombinant plasmid black carp spleen are all apparently higher than control group.
Embodiment 5:
Black carp (750 ± 5 grams/fish) is divided into three groups, often organizes 50 tail fishes.Intramuscular injection 100 microlitre contains the PBS of 10 microgram recombinant plasmid pcDNA3.1 (+)-IFN-γ, and sets empty plasmid pcDNA3.1 (+) and PBS injection as control group.
Latter 14th day of injection, abdominal injection 500 microlitre 1 × 10
8tCID
50the GCRV (CCTCC NO:V201217) of/ml carries out attacking poison.It is 80% that result display compares recombinant plasmid experimental group group relative protection ratio with control group (PBS group, empty plasmid group), and empty plasmid injection group and control group have 43 tail fishes dead.
SEQUENCE LISTING
<110> Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120> black carp IFN-γ gene and application
<130> black carp IFN-γ gene and application
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 895
<212> DNA
<213> black carp
<400> 1
gggggggggg gtattcagac cattgtcggg acacgagaag agtcgggtgc tgtgagacag 60
cttaaaaagt caacttcggc ttttaaaaca cagaactaca agtcaaactt cagcgagaga 120
cttatactaa caaggactat gactgcacaa cacatgatgg ccattttctg gggagtatgc 180
ttgttgattt taagacggat gacatatgcc gaggccagcg tccctgagaa cctggacaag 240
agcattgatg agctgaaagc acactatata aaagatgacc ttgagctaca caatgcacat 300
cctgtcttcc tgcgggtcct gaaagactta aaggtgaatt ttgaggaaag tgaacagaat 360
ctattgatga gcatcataat ggagacatac agtaggatat tcactcgcat gcagaacgag 420
agcctggatg aagctacaaa agacagattg gcacatgttc aacagcattt gaaaaaactg 480
caagaaaact acttcccagg caagagtgca gagctcaaga catatgcaga aaccctatgg 540
gcgattaagg aaaatgaccc aatcgtccag cgcaaagcac tgtttgagtt caagcgcgtc 600
tacagagaag caacacagct gaaaaacctg aagaataaag accgccggag gcgacaggcc 660
aaaagcatca ggaggcagaa gtcttagtag atcttgatca tcatttaaag aatgttggaa 720
aatcagtgtt atttattcta tttataaagt tgtgcacata ttgtgcacaa gagataactg 780
gtcaaactga cagtatttat ttgtttttat ttatttgaag aaatcactgt aaaccacttg 840
ctagttacat agcaaatgtt gtaaggtcta tgactaaata aaattatttt ttaat 895
<210> 2
<211> 182
<212> PRT
<213> black carp
<400> 2
Met Thr Ala Gln His Met Met Ala Ile Phe Trp Gly Val Cys Leu Leu
1 5 10 15
Ile Leu Arg Arg Met Thr Tyr Ala Glu Ala Ser Val Pro Glu Asn Leu
20 25 30
Asp Lys Ser Ile Asp Glu Leu Lys Ala His Tyr Ile Lys Asp Asp Leu
35 40 45
Glu Leu His Asn Ala His Pro Val Phe Leu Arg Val Leu Lys Asp Leu
50 55 60
Lys Val Asn Phe Glu Glu Ser Glu Gln Asn Leu Leu Met Ser Ile Ile
65 70 75 80
Met Glu Thr Tyr Ser Arg Ile Phe Thr Arg Met Gln Asn Glu Ser Leu
85 90 95
Asp Glu Ala Thr Lys Asp Arg Leu Ala His Val Gln Gln His Leu Lys
100 105 110
Lys Leu Gln Glu Asn Tyr Phe Pro Gly Lys Ser Ala Glu Leu Lys Thr
115 120 125
Tyr Ala Glu Thr Leu Trp Ala Ile Lys Glu Asn Asp Pro Ile Val Gln
130 135 140
Arg Lys Ala Leu Phe Glu Phe Lys Arg Val Tyr Arg Glu Ala Thr Gln
145 150 155 160
Leu Lys Asn Leu Lys Asn Lys Asp Arg Arg Arg Arg Gln Ala Lys Ser
165 170 175
Ile Arg Arg Gln Lys Ser
180
Claims (5)
1. the black carp IFN-γ albumen be separated, its sequence is for shown in SEQ ID NO.2.
2. the nucleotide sequence that albumen described in claim 1 is corresponding.
3. nucleotide sequence according to claim 2, its sequence is for shown in SEQ ID NO.1.
4. albumen according to claim 1 or gene according to claim 2 improve the application in fresh-water fishes immunizing power medicine in preparation.
5. application according to claim 4, described fresh-water fishes are black carp.
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Cited By (6)
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| CN105018512A (en) * | 2015-08-18 | 2015-11-04 | 浙江省淡水水产研究所 | Prokaryotic expression recombinant plasmid pET-IFN gamma, interferon and application |
| CN108707195A (en) * | 2018-05-31 | 2018-10-26 | 苏州大学 | 3 gene of Mandarin fish IFN-α, recombinant protein, preparation method and application |
| CN109627316A (en) * | 2018-12-27 | 2019-04-16 | 华中农业大学 | Recombinant protein and the application of 2 gene of grass carp IFN-γ and its coding |
| CN113476599A (en) * | 2021-07-05 | 2021-10-08 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Vaccine immunopotentiator recombinant IFN gamma, IL-1 beta and TNF alpha, and preparation method and application thereof |
| CN113667005A (en) * | 2021-09-13 | 2021-11-19 | 海南热带海洋学院 | Pacific codfish interferon-gamma protein, gene, recombinant plasmid, recombinant yeast engineering bacteria and application thereof |
| CN113717268A (en) * | 2021-09-27 | 2021-11-30 | 北京市水产科学研究所（国家淡水渔业工程技术研究中心） | Application of koi serum amyloid A5 or encoding gene thereof in regulation and control of koi against pathogenic bacteria infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109627316A (en) * | 2018-12-27 | 2019-04-16 | 华中农业大学 | Recombinant protein and the application of 2 gene of grass carp IFN-γ and its coding |
| CN113476599A (en) * | 2021-07-05 | 2021-10-08 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Vaccine immunopotentiator recombinant IFN gamma, IL-1 beta and TNF alpha, and preparation method and application thereof |
| CN113476599B (en) * | 2021-07-05 | 2023-08-22 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Vaccine immunopotentiator recombinant IFN gamma, IL-1 beta and TNF alpha, and preparation method and application thereof |
| CN113667005A (en) * | 2021-09-13 | 2021-11-19 | 海南热带海洋学院 | Pacific codfish interferon-gamma protein, gene, recombinant plasmid, recombinant yeast engineering bacteria and application thereof |
| CN113717268A (en) * | 2021-09-27 | 2021-11-30 | 北京市水产科学研究所（国家淡水渔业工程技术研究中心） | Application of koi serum amyloid A5 or encoding gene thereof in regulation and control of koi against pathogenic bacteria infection |
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