CN103060347B - Recombinant carp PKC delta gene and recombinant carp PKC delta protein as well as preparation method, detection method and application thereof - Google Patents

Abstract

The invention discloses a recombinant carp PKC delta gene and recombinant carp PKC delta protein as well as a preparation method, a detection method and the application of the recombinant carp PKC delta gene and the recombinant carp PKC delta protein. The recombinant carp PKC delta gene has the sequence shown by SEQ ID No. 1. The recombinant carp PKC delta protein has the sequence shown by SEQ ID No. 2. The gene clone and recombinant expression which are related to the recombinant carp PKC delta protein have very important functions; as the functions of the recombinant carp PKC delta gene and the recombinant carp PKC delta protein are clearly researched, the oxidation resistance of cells can be improved, the oxidative damage to the cells can be prevented, the growth of the cells can be regulated and controlled, and the like; and the antibody prepared by the recombinant carp PKC delta protein can be used for detecting the expression conditions of the PKC delta gene in the different types of cells, different cell periods and different individual development stages.

Description

Restructuring carp PKC δ gene, albumen and preparation and detection method and application

Technical field

The invention belongs to biological technical field, be specifically related to a kind of restructuring carp PKC δ gene, albumen and preparation and detection method and application.

Background technology

(Protein kinase C, PKC) is relevant with many cells' biological effects for protein kinase C, in the transmission of cell information, has vital role.Due to P K C be carcinogenic promoting agent Buddhist ripple ester at intracellular high-affinity receptor, thereby the effect in tumor development attracts people's attention, and found in kinds of tumors tissue that PKC content is higher than normally.

Up to the present, 10 kinds of PKC subclass (8-3) in mammalian tissues, have been determined, be divided into tri-groups of A, B, C, A group is called typical case or traditional PKC(classicalor conventional PKC, CPKC), comprise α, β I, β II and γ subclass, wherein β I and β II have the homology of height, be that different montages by same mRNA form, A group membership molecular weight is at 76-78kDa.B group is novel PKC(atypical PKC, aPKC), comprise δ, ε, η (L) and θ subclass, molecular weight is at 77-83kDa.C group is atypia PKC(atypicalPKC, aPKC), by ζ and λ subclass, formed, molecular weight is 67kDa.Wherein, PKC δ regulation and control people and mouse liver cell growth and anti-oxidant in play an important role.

So far, about PKC δ, in the research of mammalian cell growth, propagation and anti-oxidant middle performance regulating effect, in the mammiferous cells such as people, rat, mouse, carry out, and obtained many achievements, but there are no the report of the cDNA nucleotide sequence of carp PKC δ gene and the aminoacid sequence corresponding with it.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of restructuring carp PKC δ gene, albumen and preparation and detection method and application.

The present invention addresses the above problem adopted technical scheme: a kind of restructuring carp PKC δ gene is one of following sequence: the 1) gene order shown in SEQ ID No:1 in sequence table; 2) by SEQ ID NO: the gene order shown in 1 is through replacement, disappearance or the interpolation of one or several base and the identical gene order of aminoacid sequence of expressing with the gene order shown in SEQ ID No:1.

A restructuring carp PKC δ aminoacid sequence is one of following sequence: 1) SEQ ID NO: shown in 2 by the expressed aminoacid sequence of carp PKC Δ gene of recombinating described in claim 1; 2) by SEQ ID NO: aminoacid sequence shown in 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and with there is the NO with SEQ ID: 2 the identical activity of aminoacid sequence by SEQ ID NO: the protein that 2 sequences are derivative.

A preparation method for above-mentioned restructuring carp PKC δ gene, comprises the steps:

1) the total RNA the total RNA of its intestinal tissue of take that gather carp carry out reverse transcription operation as template, obtain cDNA the first chain;

2) cDNA first chain of take in step 1) is template, utilizes primer P1, P2 to carry out pcr amplification, obtains SEQ ID NO: the sequence shown in 1, and described primer P1 has SEQ ID NO: the sequence shown in 3, primer P2 has SEQ ID NO: the sequence shown in 4; Described step 2) in, take Pl, P2 as primer, utilize archaeal dna polymerase to carry out in 50 μ l pcr amplification systems of PCR reaction each component and ratio is: cDNA the first chain, 2.5 μ l; The P1 of 50 μ M, 0.25 μ l; The P2 of 50 μ M, 0.25 μ l; 10 * LA PCR Buffer II (Mg ²⁺plus), 5.0 μ l; The dNTP Mixture of each 2.5 mM, 8.0 μ l; ddH ₂o, 33.5 μ l; The archaeal dna polymerase of 5 U/ μ l, 0.5 μ l; Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30 sec, 72 ℃ are extended 2 min, totally 40 circulations; Last 72 ℃ are extended 10 min, and reaction completes.

A detection method for the restructuring carp PKC δ gene of above-mentioned preparation, comprises the steps:

1) the pcr amplification employing 2 * Taq PCR MasterMix that the PCR of P1 and P2 is reclaimed to product carries out, and in 50 μ l pcr amplification systems, each component and ratio thereof are: the PKC δ of 50 times of dilutions reclaims product, 1.0 μ l; The P3 of 50 μ M, 0.25 μ l; The P4 of 50 μ M, 0.25 μ l; 2 * Taq PCR MasterMix, 25.0 μ l; ddH ₂o, 23.5 μ l; Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 5 min, and reaction completes; Described P3 has SEQ ID NO: the sequence shown in 5, and primer P4 has SEQ ID NO: the sequence shown in 6;

2) get 5 μ l PCR products with 1.0% sepharose at 100V, under the condition of 10 min, carry out electrophoresis detection, gel imaging system observation analysis result.

Particularly, in this detection method, the PKC δ of described 50 times of dilutions reclaims the product prepared restructuring carp PKC δ gene cDNA sequence in the preparation method of carp PKC δ gene cDNA encoding region nucleotide sequence that refers to recombinate.

A detection method for the restructuring carp PKC δ gene of above-mentioned preparation, is characterized in that, comprises the steps:

1) adopt 2 * Taq PCR Master Mix to carry out the pcr amplification of P1 and P2 PCR recovery product, in 50 μ l pcr amplification systems, each component and ratio thereof are: the PKC δ of 50 times of dilutions reclaims product, 1.0 μ l; The P5 of 50 μ M, 0.25 μ l; The P6 of 50 μ M, 0.25 μ l; 2 * Taq PCR Master Mix, 25.0 μ l; ddH ₂o, 23.5 μ l; Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ (P5, P6) 30sec that anneals, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 5min, and reaction completes; Described P5 has SEQ ID NO: the sequence shown in 7, and primer P6 has SEQ ID NO: the sequence shown in 8;

2) get 5 μ l PCR products with 1.0% sepharose at 100V, under the condition of 10 min, carry out electrophoresis detection, gel imaging system observation analysis result.

The application of above-mentioned restructuring carp PKC δ gene in the expression that detects carp PKC δ gene.

Particularly, the expression of carp PKC δ gene comprises that carp PKC δ gene is at various types of cells and tissue, body early embryo, individual expression under different states.

In order to detect the expression of above-mentioned application, according to SEQ ID NO: the upstream primer of sequences Design shown in 1 PD1, downstream primer PD2, described PD1 has SEQ ID NO: the sequence shown in 9, primer PD2 has SEQ ID NO: the sequence shown in 10; Adopt fluorescent quantitative PCR, in 20 μ l pcr amplification systems, each component and ratio thereof are: cDNA, 2.0 μ l; The ND1 of 10 μ M, 1.0 μ l; The ND2 of 10 μ M, 1.0 μ l; iI(2 *), 10.0 μ l; DH ₂o, 6.0 μ l; Reaction cycle condition: 95 ℃ of denaturation 30sec; 95 ℃ of sex change 5sec, 60 ℃ of annealing 15sec, 72 ℃ are extended 15sec, totally 44 circulations; Last 72 ℃ are extended 2min, and reaction completes.

The gene clone that the present invention research and development is relevant to carp PKC δ albumen and recombinant expressedly there is extremely important effect, because the PKC δ gene of carp and the function of albumen are known in research, can be used in and improve cell cultures survival rate, raising cell resistance of oxidation, with aspects such as regulating cell growths, the antibody of being prepared by restructuring PKC δ albumen can detect PKC δ gene at the expression of different types of cell, different cell cycle and individual different developmental phases.

The coding head of district 2061bp of the restructuring PKC δ gene that the present invention obtains, and determined its nucleotide sequence and the aminoacid sequence of inferring thus.By comparing known zebra fish, people, rat, the nucleotide sequence of the PKC δ gene of mouse, find conserved regions, then according to the sequence of PKC δ gene (NM_214708.1) the cDNA coding region of zebrafish embryo, under the prerequisite of amino acid coding, design upstream primer (5 '-from initiator codon ATG) not breaking, downstream primer 5 ' end originates in the natural terminator codon TGA of zebra fish PKC δ gene, (upstream primer is called P1 to have designed a pair of primer for the carp PKCδ gene cDNA encoding district fragment that increases by RT-PCR method, downstream primer is called P2), and apply this primer amplification has been gone out to specific fragment, after order-checking, obtained the cDNA coding region 2061bp fragment of carp PKC δ gene, sequential analysis shows that with sequence (NM_214708.1) homology of zebra fish be 88%, and kept correct coding.This cDNA fragment is called " cPKC δ ".

So the nucleotide sequence of the PKC δ of the different plant species that the object of the invention is to know by oneself design primer, then by RT-PCR method obtain the recombinating cDNA coding region fragment of carp PKC δ gene.To nucleotide sequence and the aminoacid sequence (sequence refers to sequence table) of deduction thus of coding region 2061bp of cDNA of the gene of coding carp PKC δ albumen is provided after this sequencing fragment.

In sum, the invention has the beneficial effects as follows: the gene clone that restructuring of the present invention carp PKC δ albumen is relevant and recombinant expressedly there is extremely important effect, because the function of research clear restructuring carp PKC δ gene and albumen, can be used in and improve cell cultures survival rate, raising cell resistance of oxidation, prevent cellular oxidation damage, with aspects such as regulating cell growths, the antibody of being prepared by restructuring PKC δ albumen can detect PKC δ gene at the expression of different types of cell, different cell cycle and individual different developmental phases.

Accompanying drawing explanation

Fig. 1 is the electrophorogram of restructuring carp PKC δ gene order;

Fig. 2 be take the electrophorogram that P3, P4 carry out PCR evaluation as primer;

Fig. 3 be take the electrophorogram that P5, P6 carry out PCR evaluation as primer;

Fig. 4 is the detection schematic diagram of the different intestinal segment signaling molecule of carp PKC δ mRNA relative expression quantity;

Fig. 5 is the detection schematic diagram of inositol on the impact of young jian carp anterior intestine PKC δ phosphorylation;

Fig. 6 is the detection schematic diagram of inositol on the impact of intestines PKC δ phosphorylation in young jian carp;

Fig. 7 is the detection schematic diagram of inositol on the impact of young jian carp hindgut PKC δ phosphorylation.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is done to detailed description further, but embodiments of the present invention are not limited to this.

Embodiment 1:

The restructuring carp PKC δ gene of the present embodiment is one of following sequence: the 1) gene order shown in SEQ ID No:1 in sequence table; 2) by SEQ ID NO: the gene order shown in 1 is through replacement, disappearance or the interpolation of one or several base and the identical gene order of aminoacid sequence of expressing with the gene order shown in SEQ ID No:1.As the C of 903 replace to G, the aminoacid sequence of sequence encoding is identical.

The restructuring carp PKC δ aminoacid sequence of the present embodiment is one of following sequence: 1) SEQ ID NO: shown in 2 by the expressed aminoacid sequence of carp PKC Δ gene of recombinating described in claim 1; 2) by SEQ ID NO: aminoacid sequence shown in 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and with there is the NO with SEQ ID: 2 the identical activity of aminoacid sequence by SEQ ID NO: the protein that 2 sequences are derivative.As the K of 323 replace to L, the biological activity of protein can not change.

Embodiment 2:

In order to obtain the restructuring carp PKC δ gene order described in embodiment 1, can adopt the method for the present embodiment, specific as follows:

First according to the standard-required that extracts total RNA gather carp (Common carp, cyprinus carpio) various organization sample.The tissue block such as kidney, enteron aisle, muscle, spleen and liver pancreas are got after slaughtering carp immediately in scene, put into enter immediately liquid nitrogen after freeze pipe and preserve, and take back laboratory and be placed on-80 ℃ of Refrigerator stores standby.Then utilize the total RNA extraction reagent box (TaKaRa RNAiso Reagent) of TaKaRa and extract according to the schedule of operation of working instructions total RNA that carp enteron aisle, muscle, kidney, liver pancreas and spleen etc. are organized, it is last that according to the Mammals PKC δ gene of bibliographical information, the expression in various organization extracts result with total RNA, selected take the total RNA of intestinal tissue and carry out reverse transcription operation as template, obtain cDNA the first chain.Take this cDNA first chain is template again, utilizes Auele Specific Primer P1, P2 to carry out pcr amplification, obtains object fragment.Afterwards object fragment separated after electrophoresis is cut to glue and reclaim, measure the concentration of double-stranded cDNA, with nested PCR method, amplified production is identified.The amplified production of P1 and P2 primer pair is by the pcr amplification of P3 and P4, P5 and P6, and electrophoresis is identified obtains expecting big or small DNA fragmentation, preliminary identification the exactness of P1 and P2 amplified production.The sample of the amplified production of P1 and P2 primer pair is served to the order-checking of Hai Shenggong biotechnology company limited.As a result of, obtained the nucleotide sequence in the carp cPKC δ gene cDNA encoding district of 2061bp.

The specific PCR primer P1 of the present invention and the P2 that show feature above-mentioned, can utilize RT-PCR method to amplify the cDNA coding region fragment of the cPKC δ gene of carp.The nucleotide sequence of the carp PKC δ gene cDNA obtaining according to the present invention can further detect the tissue expression specificity of PKC δ gene, the clone that also can further realize carp PKC δ full length gene cDNA by the method for RT-PCR or hybridization.CDNA of the present invention is recombinated on expression vector and may give expression to complete PKC δ albumen, and then can be used for antibody producing, detect expression of carp various types of cells and tissue, body early embryo, individual PKC δ gene under different states etc.

The clone of the cDNA coding region 2061bp fragment of carp PKC δ gene

In order to clone the cDNA that comprises 2061bp coding region from the histiocytic PKC δ of carp enteron aisle gene, according to the disclosed nucleotide sequence of zebra fish (NM_214708.1), first the PCR primer pair of the cDNA that allows amplification 2061bp is synthesized in design, that is:

Upstream primer 5'-ATGGCCCCTTTCCTGAGGATTGCT-3'(P1)

Downstream primer 5'-GACCACATTGTGTCTCACTTTTGCA-3'(P2).

Then design synthesized other two pairs for differentiating the primer of P1 and P2 amplified production, that is:

Upstream primer P3,5'-AAATCCACCAAGCGACCTGA-3',

Downstream primer P4,5'-TACCCACAATCCCTAACCGG-3';

Upstream primer P5,5'-ACCTCTACTCCACTTTTCAA-3',

Downstream primer P6,5'-ATATTACCCACAATCCCTAA-3';

In order to utilize the cDNA of RT-PCR method clone PKC δ gene, from the total RNA of carp enteron aisle tissue extraction, utilize TaKaRa total RNA extraction reagent box (TaKaRa RNAiso Reagent) and extract carp enteron aisle total tissue RNA according to the schedule of operation of working instructions, carrying out electrophoresis detection and ultraviolet determination RNA concentration, to be placed on-80 ℃ of Refrigerator stores standby.Then, utilize the M-MLV ThermoScript II of TaKaRa and carry out reverse transcription reaction according to the schedule of operation of working instructions, obtaining cDNA the first chain.

CDNA the first chain obtained above of take is template, and Pl, P2 are primer, utilizes TaKaRa LA Taq archaeal dna polymerase to carry out PCR reaction.50 μ l pcr amplification system: cDNA, 2.5 μ l; P1(50 μ M), 0.25 μ l; P2(50 μ M), 0.25 μ l; 10 * LA PCR Buffer II (Mg ²⁺plus), 5.0 μ l; DNTP Mixture (each 2.5 mM), 8.0 μ l; ddH ₂o, 33.5 μ l; taKaRa LA Taq(5 U/ μ l), 0.5 μ l.Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 2 min, totally 40 circulations; Last 72 ℃ are extended 8 min, and reaction completes.Get 5 μ l PCR products and carry out electrophoresis detection (100V, 10 min), gel imaging system observation analysis result with 1.0% sepharose.As a result of, obtained specificity object fragment (the SEQ ID NO: sequence shown in 1) of 2061bp.

The pcr amplification that P1 and P2 PCR reclaim product adopts 2 * Taq PCR Master Mix(KT201) carry out 50 μ l pcr amplification systems: PKC δ reclaims product (50 * dilution), 1.0 μ l; P3(50 μ M), 0.25 μ l and P4(50 μ M) or P5(50 μ M), 0.25 μ l and P6(50 μ M), 0.25 μ l; 2 * Taq PCR Master Mix, 25.0 μ l; ddH ₂o, 23.5 μ l.Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 5min, and reaction completes.Get 5 μ l PCR products and with 1.0% sepharose, carry out electrophoresis detection (100V, 10min), gel imaging system observation analysis result.As a result of, obtained respectively approximately 900 and the amplified production of approximately 550 bp.Its size conforms to the amplified production 556bp of P5P6 primer pair design with the amplified production 922bp of P3P4 primer pair design, shows the specificity of amplified production.

P1P2 amplified production through twice evaluation is sent to order-checking for the Sheng Gong biotechnology company limited of Shanghai.As a result of, obtained the nucleotide sequence of cDNA coding region of the carp PKC δ gene of 2061bp.

The nucleotide sequence of the cDNA of carp PKC δ gene

SEQ ID NO:1 has shown the cDNA clone's that this embodiment obtains nucleotide sequence, and SEQ ID NO:2 is the aminoacid sequence of inferring thus.The nucleotide sequence of PKC δ gene, it has comprised the open reading frame of the 2061bp of cDNA coding region, 686 amino-acid residues of this open reading frame coding, molecular weight is 78498.36Da.

The comparison of the nucleotide sequence of restructuring carp PKC δ gene cDNA that the present invention obtains and the nucleotide sequence of the PKC δ gene cDNA of zebra fish (NM_214708.1), people (NM_001090993.1), mouse (NM_0111) and rat (NM_133307.1).Result shows: the homology of carp PKC δ and zebra fish, people, Mouse and rat is respectively 90.1%, 70.7%, 71.0% and 70.3%.

As clearly demonstrate and as above explanation, the invention provides the cDNA of the 2061bp that utilizes the primer in RT-PCR method clone carp PKCδ gene cDNA encoding district and the PKC δ protein of coding carp and the aminoacid sequence of deduction thus.This cDNA has comprised the nucleotide sequence of 2061bp, the protein that its coding contains 686 amino-acid residues, and after further transformation, it can be subcloned into as expressed on the protokaryons such as pET or pcDNA3.1 or carrier for expression of eukaryon.The cDNA fragment of restructuring may give expression to complete PKC δ protein polypeptide section, and then can be used for antibody producing, detects expression of carp various types of cells and tissue, body early embryo, individual PKC δ gene under different states etc.

Embodiment 3:

The present embodiment is that the restructuring carp PKC δ albumen of embodiment 1 and embodiment 2 records is in the application of the different intestinal segment PKC of mensuration carp δ genetic expression.

This example is the expression at each intestinal segment of carp with fluorescent quantitative PCR technique research PKC δ gene.Choose the healthy carp sampling of 6 tails that body weight approaches.Test fish enteron aisle is divided into 7 intestinal segments of nearly equal length according to the method for (1997) such as Villanueva, measure respectively each intestinal segment PKC δ mRNA abundance.

Sample liquid nitrogen grinding, extracts total RNA.Fluorescent quantitation adopts Prime Script with carp enteron aisle cDNA the first chain ^tMthe precious biotechnology (Dalian) of RT reagent Kit(company limited, DRR037AS) synthetic, by the method operation of test kit specification sheets.

PKC δ cDNA fluorescent quantitation amplification: the cDNA coding region sequence of the restructuring carp PKC δ gene that clone obtains according to the present invention, is designed to a pair of PKC δ specificity quantitative amplification primer:

Upstream primer PD1,5'-AAATCCACCAAGCGACCT-3';

Downstream primer PD2,5'-CGAACCCTCCCACAGACG-3';

Fluorescent quantitative PCR adopts (precious biotechnology (Dalian) company limited, DRR083A) carries out RT-PCR Kit II (Perfect Real Time), 20 μ l pcr amplification system: cDNA, 2.0 μ l; PD1(10 μ M), 1.0 μ l; PD2(10 μ M), 1.0 μ l; iI(2 *), 10.0 μ l; DH ₂o, 6.0 μ l.Reaction cycle condition: 95 ℃ of denaturation 30sec; 95 ℃ of sex change 5sec, 55 ℃ of annealing 15sec, 72 ℃ are extended 15sec, totally 44 circulations; Last 72 ℃ are extended 2min, and reaction completes.

Result shows: each intestinal segment of carp all has PKC δ mrna expression, remarkable other each intestinal segments of the 1st and the 4th intestinal segment ( p< 0.05), the 6th intestinal segment is minimum, all the other intestinal segment differences not significantly ( p> 0.05).

Embodiment 4:

The present embodiment is that the restructuring carp PKC δ albumen that embodiment 1 and embodiment 2 record is being measured the application of inositol on the impact of carp enteron aisle PKC δ protein phosphorylation.

The phosphorylation antibody that this example screens according to PKC δ protein sequence, the impact of detection limit inositol on the different intestinal segment PKC of carp δ protein phosphorylation.Result shows: inositol significantly improved PKC δ in anterior intestine and middle intestines phosphorylation level ( p< 0.05); Inositol on the not impact of hindgut PKC δ phosphorylation level ( p> 0.05).

By detecting carp PKC δ gene at various types of cells and tissue, body early embryo, individual expression under different states, can the intermediate data result detecting further be analyzed and be studied, phosphorylation and correlation circumstance to the expression of carp PKC δ gene, its expressing protein, make further research, and then can be used for antibody producing.

As mentioned above, can implement preferably the present invention.

<110> Sichuan Agricultural University

<120> restructuring carp PKC δ gene, albumen and preparation and detection method and application

<130> restructuring carp PKC δ gene, albumen and preparation and detection method and application

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 2061

<212> DNA

<213> carp

<400> 1

atggcccctt tcctgaggat tgcttttaat gcctatgatc ttggcacatt ctcaccaatg 60

gctgaggcac cattctgtgc cgtcaaaatg aaagaagctc tcagcactga gcgaggaaaa 120

actttgatcc agaagaagcc caccatgtat cctgcttggc gatccacatt tgatgcacat 180

atttatgagg gacgtgtcat acaaatacta ctcatgagaa ctgcagagga acctttagca 240

gaagtaacag tgggtgtgtc tgttttggca gaacgatgca agaaggcaaa tggacgtgca 300

gagttttggg ttgatttgca accttcagga aaggttatga tgtctgtaca gttctttttg 360

gaggatgcag atacagactt tcatcattca gtagtgtcag aagaggaggc accaaatctc 420

aatcgtcgtc gtggagctat taagcaggcg aagattcact tcatcaaaaa ccatgagttc 480

attgccacct tcttcagaca gcccaccttc tgctctgtct gcagagattt tgtttggggt 540

cttaacaagc aaggttacaa gtgtagacaa tgcaatgcag ccatccacaa gaagtgtatt 600

gataaaatta tcggaaggtg cacgggcact gcagccaata gtcgagacac tatgtttcag 660

aaggagcgtt ttaagattga catgccacat cgcttaagac tcaactcaca ctacaactac 720

atgagcccca ccttttgtga ccattgtggc agtctgctgt ggggtcttgt caaacagggc 780

ctcaaatgcg aggactgctc catgaatgtt catcataagt gccagactaa agtggccaac 840

ctgtgtggca tcaaccagaa gctactggca gaggctctta cccaagtctc aactaaatcc 900

accaagcgac ctgaaagcaa ctcacagagc aacacacagg atgttggggt ttaccaggat 960

tttaataaaa gtcctgatgt tagtgatgcg gctccttatg gtcgtctgtg ggagggttcg 1020

agtcctcatc ctccctctcg aatcacccac caaacgcgta tcactgctga gcatttcatt 1080

ttacacaaag tcctgggcaa gggaagcttt ggcaaggtgc tgttggctga gctaaggggc 1140

actggtgaat ggtttgcagt gaaggcgtta aagaaggatg tggtgttgat ggatgatgat 1200

gtggagtgca ccatggtaga aaaacgagtg ctggctctcg cctgggaaaa tccgttcctt 1260

acacacctct actccacttt tcaaaccaag gagcacttat tctttgtgat ggagtatctg 1320

aatggaggag acttgatgtt tcatatacag gaaaaggggc gttttgatct gtacagagcc 1380

acgttctatg cagctgaaat agtgtgtggc ctgcagtttc tccattccaa aggcgttatt 1440

tacagggatc tcaaactgga taatgtgatg ttagatggag atggtcacat aaaaatagct 1500

gattttggaa tgtgtaagga gaatgttttt ggggaaaatc gggccacaac tttctgtgga 1560

actccagatt atatcgctcc tgagattctt ctgggtcagc agtattcatt ctcagtagac 1620

tggtggtcgt ttggcatgct ggtgtatgag atgctgattg gtcagtctcc attccacggt 1680

gacgacgagg acgagctgtt tgaatcgatc cgaatggaca cacctcatta tccccgctgg 1740

atcacagcgg atactcgaga catgctagaa aagctgtttg agcgcgaccc atcacgccgg 1800

ttagggattg tgggtaatat aagagggcac ctgttcttca agactgtcaa ctggtctgct 1860

ttagaaagga aagaagttga gcctcccttt aaaccaaaag tgaaagctcc aaatgactgc 1920

agtaactttg accgggagtt tttgagtgag aaaccccgtc tctcacactg cgagaaaggc 1980

ctgattgatt ccatggacca gagcgttttt gctgggttct cattcattaa tccaataatg 2040

gagcatcttg tgcaaaagtg a 2061

<210> 2

<211> 686

<212> PRT

<213> carp

<400> 2

Met Ala Pro Phe Leu Arg Ile Ala Phe Asn Ala Tyr Asp Leu Gly Thr

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Phe Ser Pro Met Ala Glu Ala Pro Phe Cys Ala Val Lys Met Lys Glu

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Ala Leu Ser Thr Glu Arg Gly Lys Thr Leu Ile Gln Lys Lys Pro Thr

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Met Tyr Pro Ala Trp Arg Ser Thr Phe Asp Ala His Ile Tyr Glu Gly

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Arg Val Ile Gln Ile Leu Leu Met Arg Thr Ala Glu Glu Pro Leu Ala

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Glu Val Thr Val Gly Val Ser Val Leu Ala Glu Arg Cys Lys Lys Ala

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Asn Gly Arg Ala Glu Phe Trp Val Asp Leu Gln Pro Ser Gly Lys Val

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Met Met Ser Val Gln Phe Phe Leu Glu Asp Ala Asp Thr Asp Phe His

115 120 125

His Ser Val Val Ser Glu Glu Glu Ala Pro Asn Leu Asn Arg Arg Arg

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Gly Ala Ile Lys Gln Ala Lys Ile His Phe Ile Lys Asn His Glu Phe

145 150 155 160

Ile Ala Thr Phe Phe Arg Gln Pro Thr Phe Cys Ser Val Cys Arg Asp

165 170 175

Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Lys Cys Arg Gln Cys Asn

180 185 190

Ala Ala Ile His Lys Lys Cys Ile Asp Lys Ile Ile Gly Arg Cys Thr

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Gly Thr Ala Ala Asn Ser Arg Asp Thr Met Phe Gln Lys Glu Arg Phe

210 215 220

Lys Ile Asp Met Pro His Arg Leu Arg Leu Asn Ser His Tyr Asn Tyr

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Met Ser Pro Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Leu

245 250 255

Val Lys Gln Gly Leu Lys Cys Glu Asp Cys Ser Met Asn Val His His

260 265 270

Lys Cys Gln Thr Lys Val Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu

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Leu Ala Glu Ala Leu Thr Gln Val Ser Thr Lys Ser Thr Lys Arg Pro

290 295 300

Glu Ser Asn Ser Gln Ser Asn Thr Gln Asp Val Gly Val Tyr Gln Asp

305 310 315 320

Phe Asn Lys Ser Pro Asp Val Ser Asp Ala Ala Pro Tyr Gly Arg Leu

325 330 335

Trp Glu Gly Ser Ser Pro His Pro Pro Ser Arg Ile Thr His Gln Thr

340 345 350

Arg Ile Thr Ala Glu His Phe Ile Leu His Lys Val Leu Gly Lys Gly

355 360 365

Ser Phe Gly Lys Val Leu Leu Ala Glu Leu Arg Gly Thr Gly Glu Trp

370 375 380

Phe Ala Val Lys Ala Leu Lys Lys Asp Val Val Leu Met Asp Asp Asp

385 390 395 400

Val Glu Cys Thr Met Val Glu Lys Arg Val Leu Ala Leu Ala Trp Glu

405 410 415

Asn Pro Phe Leu Thr His Leu Tyr Ser Thr Phe Gln Thr Lys Glu His

420 425 430

Leu Phe Phe Val Met Glu Tyr Leu Asn Gly Gly Asp Leu Met Phe His

435 440 445

Ile Gln Glu Lys Gly Arg Phe Asp Leu Tyr Arg Ala Thr Phe Tyr Ala

450 455 460

Ala Glu Ile Val Cys Gly Leu Gln Phe Leu His Ser Lys Gly Val Ile

465 470 475 480

Tyr Arg Asp Leu Lys Leu Asp Asn Val Met Leu Asp Gly Asp Gly His

485 490 495

Ile Lys Ile Ala Asp Phe Gly Met Cys Lys Glu Asn Val Phe Gly Glu

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Asn Arg Ala Thr Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu

515 520 525

Ile Leu Leu Gly Gln Gln Tyr Ser Phe Ser Val Asp Trp Trp Ser Phe

530 535 540

Gly Met Leu Val Tyr Glu Met Leu Ile Gly Gln Ser Pro Phe His Gly

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Asp Asp Glu Asp Glu Leu Phe Glu Ser Ile Arg Met Asp Thr Pro His

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Tyr Pro Arg Trp Ile Thr Ala Asp Thr Arg Asp Met Leu Glu Lys Leu

580 585 590

Phe Glu Arg Asp Pro Ser Arg Arg Leu Gly Ile Val Gly Asn Ile Arg

595 600 605

Gly His Leu Phe Phe Lys Thr Val Asn Trp Ser Ala Leu Glu Arg Lys

610 615 620

Glu Val Glu Pro Pro Phe Lys Pro Lys Val Lys Ala Pro Asn Asp Cys

625 630 635 640

Ser Asn Phe Asp Arg Glu Phe Leu Ser Glu Lys Pro Arg Leu Ser His

645 650 655

Cys Glu Lys Gly Leu Ile Asp Ser Met Asp Gln Ser Val Phe Ala Gly

660 665 670

Phe Ser Phe Ile Asn Pro Ile Met Glu His Leu Val Gln Lys

675 680 685

<210> 3

<211> 24

<212> DNA

<213> artificial sequence

<400> 3

atggcccctt tcctgaggat tgct 24

<210> 4

<211> 25

<212> DNA

<213> artificial sequence

<400> 4

gaccacattg tgtctcactt ttgca 25

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

aaatccacca agcgacctga 20

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<400> 6

tacccacaat ccctaaccgg 20

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<400> 7

acctctactc cacttttcaa 20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

atattaccca caatccctaa 20

<210> 9

<211> 18

<212> DNA

<213> artificial sequence

<400> 9

aaatccacca agcgacct 18

<210> 10

<211> 18

<212> DNA

<213> artificial sequence

<400> 10

cgaaccctcc cacagacg 18

Claims (4)

1. the non-diagnostic use of carp PKC δ gene in the expression that detects carp PKC δ gene of recombinating, the expression of carp PKC δ gene comprises that carp PKC δ gene is at various types of cells and tissue, body early embryo, individual expression under different states; One of restructuring carp PKC δ gene is following sequence: the 1) gene order shown in SEQ ID No:1 in sequence table; 2) by the replacement of one or several base of gene order process shown in SEQ ID NO:1 and the gene order identical with the aminoacid sequence of the gene order expression shown in SEQ ID No:1.

2. the carp PKC δ protein of recombinating is being measured the application of inositol on the impact of carp enteron aisle PKC δ protein phosphorylation, restructuring carp PKC δ protein be shown in SEQ ID NO:2 by the expressed aminoacid sequence of carp PKC δ gene of recombinating described in claim 1.

3. application according to claim 1, is characterized in that, the preparation method of described restructuring carp PKC δ gene comprises the steps:

1) the total RNA the total RNA of its intestinal tissue of take that gather carp carry out reverse transcription operation as template, obtain cDNA the first chain;

2) take step 1) in cDNA the first chain be template, utilize primer P1, P2 to carry out pcr amplification, obtain the sequence shown in SEQ ID NO:1, described primer P1 is the sequence shown in SEQ ID NO:3, primer P2 is the sequence shown in SEQ ID NO:4; Described step 2) in, take Pl, P2 as primer, utilize archaeal dna polymerase to carry out in 50 μ l pcr amplification systems of PCR reaction each component and ratio is: cDNA the first chain, 2.5 μ l; The P1 of 50 μ M, 0.25 μ l; The P2 of 50 μ M, 0.25 μ l; Mg ²⁺10 * LA PCR Buffer II of Plus specification, 5.0 μ l; The dNTP Mixture of each2.5mM, 8.0 μ l; DdH2O, 33.5 μ l; The archaeal dna polymerase of 5U/ μ l, 0.5 μ l; Reaction cycle condition: 94 ℃ of denaturation 1min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 2min, totally 40 circulations; Last 72 ℃ are extended 10min, and reaction completes.

4. application according to claim 1, it is characterized in that, in detection, according to sequences Design upstream primer PD1, downstream primer PD2 shown in SEQ ID NO:1, described PD1 is the sequence shown in SEQ ID NO:9, and primer PD2 is the sequence shown in SEQ ID NO:10; Adopt fluorescent quantitative PCR, in 20 μ l pcr amplification systems, each component and ratio thereof are: cDNA, 2.0 μ l; The PD1 of 10 μ M, 1.0 μ l; The PD2 of 10 μ M, 1.0 μ l; premix Ex Taq ^tM iI, 10.0 μ l; DH ₂o, 6.0 μ l; Reaction cycle condition: 95 ℃ of denaturation 30sec; 95 ℃ of sex change 5sec, 60 ℃ of annealing 15sec, 72 ℃ are extended 15sec, totally 44 circulations; Last 72 ℃ are extended 2min, and reaction completes.