CN1238382C - Molt-inhibiting hormone-1 protein of mitten crab - Google Patents
Molt-inhibiting hormone-1 protein of mitten crab Download PDFInfo
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Abstract
The present invention belongs to the technical field of genetic engineering. The gene length of the molting inhibiting hormone 1(Ers-MIH1) of mitten crabs is 3506 bp, and the molt inhibiting hormone comprises three extrons, 2 introns, the 5'-terminal upstream control region of 412 bp and the 3'-terminal UTR of 917 bp. Polypeptide derived according to Ers-MIH 1 gene sequence is composed of 75 amino acid mature peptides and 35 amino acid signal peptides. The mature peptide has the function of inhibiting the mitten crab from molting. Consequently, the polypeptide has the important functions of regulation and control for the growth of the mitten crabs. The Gen Bank searching number of an Ers-MIH 1 gene is AY310313. The present invention adopts the Ers-MIH 1 gene to clone and prepare fusion protein, and the fusion protein is used for immunizing an animal to prepare a polyclonal antibody. Meanwhile, antibody or gene expressed control is used for solving the problem of a small body which is caused by abnormal molting and occurs during cultivation.
Description
One, technical field
The invention belongs to gene engineering technology field.
Two, background technology
Mitten crab (Eriocheir japonica sinensis) has another name called river crab, steamed crab, eriocheir sinensis, is the important economic crab of China.Casting off a skin is the important step of river crab growth, it is that the river crab growth is necessary that ectoskeletal periodicity is casted off a skin, but some biennial river crab is because of the not accomplished stiff crab that forms that casts off a skin in breeding process, and individuality is less than normal, market value is on the low side, brings bigger financial loss to the producer.(molt-inhibiting hormone, major function MIH) is that the growth to crustacean plays critical regulating effect by suppressing the synthetic of Y-organ moulting hormone to MIH.
Three, summary of the invention
The sequence of the described mitten crab MIH 1 of the inventor (Ers-MIH 1) gene is that the contriver obtains by a series of experiment in genetic resources institute of life science institute of Nanjing Normal University.Experimental procedure and result are:
1, according to the aminoacid sequence of other crab class MIHs design degenerated primer F1 (5 '-AGAGTWATHAAYGAYGARTGTCC-3 ') and R48 (5 '-CACACACCACASGAAGTCYTC-3 '), utilization RT-PCR technology has obtained 1 treaty 160bp from the intermolt optic stalk of mitten crab adult electrophoretic band (Fig. 1) carries out sequencing then.
2, according to the 1 sequences Design primer Ers FP that obtains (5 '-AAACCTGATCGGGAATCGTGAC-3 '), utilization 3 ' RACE technology amplifies the cDNA band (Fig. 2) of 1 treaty 1kb.It is cDNA sequence (the GenBank searching number: AY309062) of Ers-MIH 1 gene of 1 145bp that the order-checking back obtains length, this sequence is made up of the coding region (67 amino acid of encoding) of 203bp and 3 ' end non-translational region of 942bp, and tailing signal AATAAA is positioned at apart from 897bp place, terminator codon (TAA) downstream.
3, the position that distributes with reference to the intron of Ban Wen Charybdis (Charybdis feriatus) and edible ecliptic crab (Cancer pagurus) MIH gene, Partial cDNA Sequence according to acquired Ers-MIH 1 gene, be designed for the primer ErsRP (5 '-AGTTATTGCCCGAGGATG-3 ') of amplification intron 2, be that template is carried out PCR with primer ErsFP and ErsRP with the mitten crab genomic dna then, amplification obtains intron 2 (Fig. 3).
4, according to the Partial cDNA Sequence of acquired Ers-MIH 1 gene, be designed for nested primer IFP1 (5 '-CCAACATCTTCCGCATCGAC-3 '), the IRP1 (5 '-TCACAGATCCAGTCCACCTTC-3 ') of inverse PCR amplification introne 1 and 5 ' end upstream regulatory region and IRP2 (5 '-CTTGTAGATGTCACGATTCCC-3).With the Pst I enzyme magnificent Eriocheir genomic dna that hits, carry out then from connecting reaction.To connect reaction product certainly is that template is carried out the inverse PCR amplification, obtains the fragment (Fig. 4) about 1 about 2kb.This fragment cloning order-checking back is analyzed by Blastx, and the result shows the partial sequence that has obtained 5 of Ers-MIH 1 gene ' end upstream regulatory region, exons 1, introne 1 and exon 2.
5, sequence, the sequence of intron 2 and the sequencing result of inverse PCR product that 3 ' RACE is obtained splices, and the result has obtained Ers-MIH 1 gene order of 3 506bp.(National Centerfor Biotechnology Information NCBI) formally accepts the newcomer that this gene is GenBank, and searching number is AY310313 in American National biotechnology information center.
6, according to Ers-MIH 1 genomic dna sequence that obtains be designed for amplification Ers-MIH 1 mature peptide cDNA sequence primer ErsM1 (5 '-ccggattcATGGGAATCATCAACGCCGAG-3) and ErsM2 (5 '-cgctcgagTTATTGCCCGAGGATGCT-3 '), with this primer amplification is gone out the cDNA (Fig. 5) of Ers-MIH 1 mature peptide, sequencing result shows that the sequence of corresponding section in the cDNA sequence of acquisition and Ers-MIH 1 genomic dna sequence is in full accord.
gcggagttct?gtccaccgcc?aagctaataa?tatgtcgcta?aagacgcatt?ttccaaacct?60
tctcgaggag?gaggaggtag?aagaagagga?gacggggagg?aggaggagga?ggaggaagag?120
ggtagcgcgc?ggtcatagga?acccccccct?ccgccttccc?cgcccccgcc?gtgacgtaag?180
tggcaggcag?gtttataagg?gcggctcgtc?agacagtcag?ggcattcagc?ctttggaatc?240
accagggact?gttcccctgt?tgaagcccct?gagcaccaga?gagacgcttc?ctccggctcc?300
tgcgtccacc?tcctactccc?tccgcagcgc?cgcccctcag?tctcaccact?cccactccgc?360
cctcactccc?ttcttcggcg?cctccgtcca?cggctccgtc?ctctaagcca?ccatggtgtc?420
ccgcgctcaa?tccagatttt?cctctcaggt?tagtttgagg?acgcacgtgt?tgggtagtca?480
gcgttatcgt?gtaatatgta?gggtgtgtgt?gtgtgtgtgt?gtgtgtgtgg?gttttcatct?540
tgtttcttgc?tatttttttc?gttttgccca?tgtttttttt?cttcgttggg?gtacaaagaa?600
aatctcgctc?attttttagg?taaattatag?aacttggctc?tactatattt?tgcaacgatt?660
tccgggcatg?tcatctgagc?cccaagtact?gacagacaga?agtagtgaga?catttgtatt?720
gcggcgctgg?ggatgaaatt?ctagggtcac?tgcaagacga?tctccaggga?aaacattgcc?780
cgggataaga?tacaaaagtt?acagcattga?gtaaagacgc?tttccaatac?attcataagc?840
atcgtgtgcc?attattcgaa?cagcagggag?cagcgagtag?cgggcttttt?ttattattgt?900
gttttttttg?tgcccttgag?ctgtcccctt?tgttgtaaaa?aaaaaaaacg?ttacccagac?960
ctgtgactgg?aagagcctgg?tggatgcaga?taacctttct?ttctatatcc?ttcctgacta?1020
cgacttgagc?gtggtcgtat?ttgtttcctc?tgtatttctt?cacccttgag?ctgcttcctt?1080
gtgtgaaaga?aagagggtgt?gagaagggtt?aagcgaggcc?ctggaacact?aaagaagctg?1140
aggctgatgt?atggagagga?atagaagggc?gaagggcaag?catgaaggac?gctgtcaggt?1200
ctgtgaatgg?cctagactcg?tgcatttaga?aggtaggaga?gaaagagtta?gctgagtccc?1260
tggcccatgt?acgaagctgc?catatctgtt?gatgggagag?gctggtgaac?gcagagggta?1320
gagggaaaga?gttagctgag?gccctggcac?atgtaggaag?atgccgtatc?tgttgatggg?1380
agaggctggt?gaacgcagag?ggtagaggga?aagagttagc?tgaggccctg?gcacatgtag?1440
gaagctgcca?tatctgttga?tgggagaggc?tggtgaacac?agagggtaga?gggaaagagt?1500
tagctgaggc?cctggcccat?gtaggaagct?gccatatctg?ttgatgggag?aggctggtga?1560
acgcagagga?taaagggaac?ggctttgtta?ggtgaggccc?aggagcatgt?atcacgccgg?1620
ctcacgacac?ggtgctttac?agaggacgtg?gctggtggcg?gcggtggtgc?tggccgtcct?1680
gtgtagcttc?ggtgtccagc?gggccgcggc?gggaatcatc?aacgccgagt?gtccaaacat?1740
gatcgggaat?cgtgacatct?acaagaaggt?ggactggatc?tgtgaggact?gcgccaacat?1800
cttccgcatc?gacggtctgg?gcatgctctg?caggtacgca?agagagagag?agagagagag?1860
agagagagag?agagagagag?agagagagag?agagtaaaat?gaataaaaaa?ggaaaaaaag?1920
atctccttat?actcttcctg?ctcctccccc?agcttctctt?ctctctgctc?atcctcttct?1980
tcctcctcct?tctcctcctc?ttccttcact?tataccaatg?ttccatgttc?ataactgacc?2040
ctcatcatca?tcatcttcct?cctcctactc?ctcctcctcc?tcctcttcct?gctcctcccc?2100
cagcttctct?tctctctgct?catcctcttc?ttcctcctcc?ttcacttata?ccaatgttcc?2160
tttttcatat?tcctcctcct?cctcctcctc?ttcctcctcc?tactcctcct?cctcctcctc?2220
ctcttgctcc?tcccccagct?tctcttctct?ctgctcatcc?tcttcttcct?cttcctcctc?2280
ctcctccttc?acacttatac?caatgttcca?tgttcataac?cgaccctcct?cttcctcctc?2340
ctcctcctcc?tctccaccac?cctcttcacc?gatcacctaa?accctcatga?tccccctcca?2400
cctcttcatc?gttgttacca?ccaccaccat?catcatcatc?accagtttcc?ccatgcctaa?2460
cgctcccaca?cttcaccttt?caggaagaac?tgctttagga?acatcgactt?cctgtggtgt?2520
gtgtacgcct?cggaacggca?cgcacagaag?gacgacctca?cgcgttacgt?cagcatcctc?2580
gggcaataac?tcctgggtgc?cccacctgcc?gctccctccc?tccctccccg?gggtatctgc?2640
cgctgctgga?gatgccccga?gggtgaagcc?gatgtccggg?aagcgccttg?aagtgaagag?2700
tgaagtgcgt?ctctccagca?gctgtccctc?cctctctggc?tgctcttcgt?ccgtggtgta?2760
agaatccagc?ggctaaagcc?ttggttcagt?ctctcgcgag?gagctttgat?tggcctccgt?2820
tcacctccat?tcagtcgctt?gtctctgttt?agctccgttc?acgccttcgt?ctattgttca?2880
gccgttcacc?ttccgttcac?ctcgtttacc?cacaaagctc?cacttgtcag?tgctttcctg?2940
ggtcctccag?aaaggtcttg?catatcaggg?acactatttt?agcattggga?attggaggta?3000
acagagtata?ggaaaagaca?attacatttt?tgtttgtgta?catggacgag?tattcgaatg?3060
tagaaggctg?ctgtagtagt?tgtaatagta?gctgtagtat?ataggaggag?gaggaggagc?3120
agaaggagga?gcaggagtgg?aaaaggaaga?agagaggagg?aagaaggtat?ttgaaagtag?3180
aaataagttg?aaagacatct?cactgaaaca?gtattctcaa?aactctagtc?aactctaaag?3240
ctgtttgagg?gttttctctt?tatgaatgtg?tatgtgtgat?cgtactcttg?tttatttgca?3300
ttcttggtta?gtgttgaatc?attcctttgt?gtgtgagtgt?gtgtatgtca?acttcctttg?3360
ttattttgtt?tcactttgtg?ttatcctctc?tcgttttgtc?ctaatttatt?gaggttgtag?3420
tatttattga?aatctgagcc?gctgtgttct?tccacgtctt?agaattaatt?gtggctgata?3480
aaaaaaataa?agagaaacga?aagagc
The genomic dna sequence of mitten crab MIH 1
Met?Val?Ser?Arg?Ala?Gln?Ser?Arg?Phe?Ser?Ser?Gln?Arg?Thr?Trp?Leu?Val?Ala?Ala?Val
1 6 11 16
Val?Leu?Ala?Val?Leu?Cys?Ser?Phe?Gly?Val?Gln?Arg?Ala?Ala?Ala?Gly?Ile?Ile?Asn?Ala
21 26 31 36
Glu?Cys?Pro?Asn?Met?Ile?Gly?Asn?Arg?Asp?Ile?Tyr?Lys?Lys?Val?Asp?Trp?Ile?Cys?Glu
41 46 51 56
Asp?Cys?Ala?Asn?Ile?Phe?Arg?Ile?Asp?Gly?Leu?Gly?Met?Leu?Cys?Arg?Lys?Asn?Cys?Phe
61 66 71 76
Arg?Asn?Ile?Asp?Phe?Leu?Trp?Cys?Val?TYr?Ala?Ser?Glu?Arg?His?Ala?Gln?Lys?Asp?Asp
81 86 91 96
Leu?Thr?Arg?Tyr?Val?Ser?Ile?Leu?Gly?Gln
101 106
The aminoacid sequence of mitten crab MIH 1 gene coded protein
Purpose of the present invention:
In view of mitten crab MIH 1 (Ers-MIH 1) gene is that the inventor can suppress the gene that it is casted off a skin what mitten crab was cloned first, promptly this gene has the function that potential is regulated the mitten crab growth, the present invention intends by the research to Ers-MIH 1 gene expression regulation mechanism and function, seek the solution river crab from the genetic engineering technique angle and cause the method for individual this problem less than normal because of casting off a skin unusually, for the development culture of Chinese mitten crab is already served.The research of Ers-MIH 1 gene can be used for:
1, prepare this gene Fusion albumen: this fusion rotein can be used for the cast off a skin research of mechanism of mitten crab.
2, the preparation of preparation antibody: Ers-MIH 1 gene coded protein antibody, can be used for the detection method according to the immunology principle design such as immunohistochemical staining, ELISA, immuno-electron microscope, co-immunoprecipitation, also can be used for location and the quantitative examination of this gene coded protein in histocyte.This antibody might be used to reduce the activity of MIH in the mitten crab body, thereby promotes to cast off a skin and grow.
3, can or reduce this expression of gene by the Protocols in Molecular Biology inhibition, thereby promote casting off a skin and growing of mitten crab.
Technical solution of the present invention:
1, the preparation of mitten crab MIH 1 (Ers-MIH 1) fusion rotein
(1) be template with the total RNA of mitten crab optic stalk that extracts, with synthetic the 1st chain cDNA of Superscript First-StrandSynthesis System for RT-PCR test kit reverse transcription.
(2) be the cDNA (Fig. 5) that template amplification goes out Ers-MIH 1 mature peptide with primer ErsM1 (5 '-ccggattcATGGGAATCATCAACGCCGAG-3 ') and ErsM2 (5 '-cgctcgagTTATTGCCCGAGGATGCT-3 ') with above-mentioned the 1st chain cDNA, reaction conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min.
(3) behind the above-mentioned amplified production rubber tapping purifying, be connected in pMD18-T carrier (Dalian is precious biological), construction recombination plasmid pMD18-T-MIH 1 transforms DH5 α competent cell.The positive colony that filters out is inserted fragments sequence measure, the plasmid of the correct positive colony that will check order then and expression vector pGEX-4T-1 (available from Amersham Parmaciaa company) use Bam H I and Xho I at 37 ℃ of double digestions a few hours (Fig. 6) simultaneously.Agarose gel electrophoresis connects with the T4DNA ligase enzyme after reclaiming endonuclease bamhi and pGEX-4T-1 carrier.To connect product transformed into escherichia coli DH5 α, and make up recombinant expression plasmid pGEX-4T-NIH 1 and carry out double digestion evaluation (Fig. 6).
(4) recombinant plasmid pGEX-4T-MIH 1 is transformed the BL21 bacterial strain after, induce some hrs with IPTG, add that the sample damping fluid boils smudge cells after centrifugal, carry out the SDS-PAGE electrophoresis (Fig. 7) of 12% concentration.
(5) utilize the GST label to carry out the affinity chromatography separation and purification of fusion rotein.
2, mitten crab MIH 1 (Ers-MIH 1) gene coded protein specific polyclonal antibody
Fusion rotein is combined with carrier proteins KLH (Keyhol Limpet Hymocyanin), through the dialysis after again with fully/Freund (Complete/Incomplete Freund ' s Adjuvant) balanced mix, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, totally three times, after 3 months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity, measure antibody titers in each immunize rabbit serum.Screen high titre serum, further do competition and suppress the specificity that immunity test is determined antibody with antigen.Determine its extension rate that is used for immunohistochemical staining 1: 200～500 according to ELISA result.
3, reduce the activity of MIH by antigen antibody reaction
By injection or infusion method the specific antibody of mitten crab MIH 1 (Ers-MIH 1) gene coded protein is sent in the mitten crab body, suppress or reduce the activity of MIH, reach the purpose that promotes that mitten crab casts off a skin and grows, and be determined by experiment the suitable dose of antibody effect.
4, the inhibition of mitten crab MIH 1 (Ers-MIH 1) genetic expression
The double stranded rna molecule special according to the synthetic Ers-MIH of the dna sequence dna of Ers-MIH 11, by the method for ingesting, injecting and soaking double stranded rna molecule is imported in the mitten crab body promptly by RNA interference (RNA interference, RNAi) technology is sealed Ers-MIH 1 expression of gene or is sealed or inhibition or reduction Ers-MIH 1 expression of gene by technology such as Antisense RNA Technique and knock out, thereby promotes casting off a skin and growing of mitten crab.
Four, description of drawings
The electrophorogram of Fig. 1, degenerated primer amplified production (M:DNA mark; 1～6: the amplified production of degenerated primer)
Fig. 2,3 ' RACE PCR result (M:DNA mark; 1:3 ' RACE PCR product)
Pcr amplification result (the M:DNA mark of Fig. 3, Ers-MIH 1 gene intron 2; 1: the pcr amplification product of intron 2)
Fig. 4, inverse PCR amplification (M:DNA mark; 1: the inverse PCR amplified production)
CDNA amplification (the M:DNA mark of Fig. 5, Ers-MIH 1 gene mature peptide; The cDNA amplification of 1～3:Ers-MIH, 1 gene mature peptide)
Fig. 6, recombinant plasmid pMD18-T-MIH 1 and pGEX-4T-MIH 1 restricted enzyme cutting analysis (M:DNA molecular weight marker; 1-4: candidate's plasmid pMD18-T-MIH 1 is through Bam H I and Xho I double digestion; 5: candidate's plasmid pGEX-4T-MIH 1 is through Bam H I and Xho I double digestion)
The sex change polyacrylamide gel electrophoresis of e. coli total protein is analyzed (M: molecular weight protein marker in Fig. 7, the fusion rotein abduction delivering process; 1-2:BL21/pGEX-4T-1 induced respectively 1 hour and 6 hours through IPTG; 3-7:BL21/pGEX-4T-MIH1 induced respectively 1 hour through IPTG, and 2 hours, 3 hours, 4 hours, 6 hours, arrow was depicted as the fusion rotein rMIH of expression)
Five, embodiment
(1) preparation of mitten crab MIH 1 (Ers-MIH 1) fusion rotein
1, be template with the total RNA of mitten crab optic stalk that extracts, with synthetic the 1st chain cDNA of Superscript First-Strand SynthesisSystem for RT-PCR test kit reverse transcription.
2, be the cDNA that template amplification goes out Ers-MIH 1 mature peptide with primer ErsM1 (5 '-ccggattcATGGGAATCATCAACGCCGAG-3 ') and ErsM2 (5 '-cgctcgagTTATTGCCCGAGGATGCT-3 ') with above-mentioned the 1st chain cDNA, reaction conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min.
3, behind the above-mentioned amplified production rubber tapping purifying, be connected in pMD18-T carrier (Dalian is precious biological), construction recombination plasmid pMD18-T-MIH 1 transforms DH5 α competent cell.The positive colony that filters out is inserted fragments sequence measure, the plasmid of the correct positive colony that will check order then and expression vector pGEX-4T-1 (available from Amersham Parmaciaa company) use BamH I and Xho I 37 ℃ of double digestions 6 hours simultaneously.Agarose gel electrophoresis connects with the T4 dna ligase after reclaiming endonuclease bamhi and pGEX-4T-1 carrier.To connect product transformed into escherichia coli DH5 α, and make up recombinant expression plasmid pGEX-4T-MIH 1 and carry out the double digestion evaluation.
4, recombinant plasmid pGEX-4T-MIH 1 is transformed the BL21 bacterial strain after, induced 3 hours with IPTG, add that the sample damping fluid boils smudge cells after centrifugal, carry out the SDS-PAGE electrophoresis of 12% concentration.
5, utilize the GST label to carry out the affinity chromatography separation and purification of fusion rotein, obtain the fusion rotein about 10mg.
(2) mitten crab MIH 1 (Ers-MIH 1) gene coded protein specific polyclonal antibody
Fusion rotein is combined with carrier proteins KLH (Keyhole Limpet Hymocyanin), through the dialysis after again with complete Freund (Complete/Incomplete Freund ' s Adjuvant) balanced mix, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, totally three times, after 3 months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity, measure antibody titers in each immunize rabbit serum, screen high titre serum (1: 10000), be used for next step operation.
(3) reduce the activity of MIH by antigen antibody reaction
By infusion method the antibody of mitten crab MIH 1 (Ers-MIH 1) gene coded protein is sent in the mitten crab body, in and the activity of the MIH of expression in vivo, promote that mitten crab casts off a skin and grows.
(4) inhibition of mitten crab MIH 1 (Ers-MIH 1) genetic expression
The double stranded rna molecule special according to the synthetic Ers-MIH of the dna sequence dna of Ers-MIH 11, method by injection imports double stranded rna molecule in the mitten crab body, interfere (RNA interference by RNA, RNAi) technology reduces Ers-MIH 1 expression of gene (reducing the activity at least 70% of MIH), promotes casting off a skin and growing of mitten crab.
＜110〉Nanjing Normal University
＜120〉mitten crab MIH 1 gene
<160>1
<210>1
<211>3506bp
＜212〉genomic dna
＜213〉mitten crab (Eriocheir japonica sinensis)
<400>1
gcggagttct?gtccaccgcc?aagctaataa?tatgtcgcta?aagacgcatt?ttccaaacct?60
tctcgaggag?gaggaggtag?aagaagagga?gacggggagg?aggaggagga?ggaggaagag?120
ggtagcgcgc?ggtcatagga?acccccccct?ccgccttccc?cgcccccgcc?gtgacgtaag?180
tggcaggcag?gtttataagg?gcggctcgtc?agacagtcag?ggcattcagc?ctttggaatc?240
accagggact?gttcccctgt?tgaagcccct?gagcaccaga?gagacgcttc?ctccggctcc?300
tgcgtccacc?tcctactccc?tccgcagcgc?cgcccctcag?tctcaccact?cccactccgc?360
cctcactccc?ttcttcggcg?cctccgtcca?cggctccgtc?ctctaagcca?ccatggtgtc?420
ccgcgctcaa?tccagatttt?cctctcaggt?tagtttgagg?acgcacgtgt?tgggtagtca?480
gcgttatcgt?gtaatatgta?gggtgtgtgt?gtgtgtgtgt?gtgtgtgtgg?gttttcatct?540
tgtttcttgc?tatttttttc?gttttgccca?tgtttttttt?cttcgttggg?gtacaaagaa?600
aatctcgctc?attttttagg?taaattatag?aacttggctc?tactatattt?tgcaacgatt?660
tccgggcatg?tcatctgagc?cccaagtact?gacagacaga?agtagtgaga?catttgtatt?720
gcggcgctgg?ggatgaaatt?ctagggtcac?tgcaagacga?tctccaggga?aaacattgcc?780
cgggataaga?tacaaaagtt?acagcattga?gtaaagacgc?tttccaatac?attcataagc?840
atcgtgtgcc?attattcgaa?cagcagggag?cagcgagtag?cgggcttttt?ttattattgt?900
gttttttttg?tgcccttgag?ctgtcccctt?tgttgtaaaa?aaaaaaaacg?ttacccagac?960
ctgtgactgg?aagagcctgg?tggatgcaga?taacctttct?ttctatatcc?ttcctgacta?1020
cgacttgagc?gtggtcgtat?ttgtttcctc?tgtatttctt?cacccttgag?ctgcttcctt?1080
gtgtgaaaga?aagagggtgt?gagaagggtt?aagcgaggcc?ctggaacact?aaagaagctg?1140
aggctgatgt?atggagagga?atagaagggc?gaagggcaag?catgaaggac?gctgtcaggt?1200
ctgtgaatgg?cctagactcg?tgcatttaga?aggtaggaga?gaaagagtta?gctgagtccc?1260
tggcccatgt?acgaagctgc?catatctgtt?gatgggagag?gctggtgaac?gcagagggta?1320
gagggaaaga?gttagctgag?gccctggcac?atgtaggaag?atgccgtatc?tgttgatggg?1380
agaggctggt?gaacgcagag?ggtagaggga?aagagttagc?tgaggccctg?gcacatgtag?1440
gaagctgcca?tatctgttga?tgggagaggc?tggtgaacac?agagggtaga?gggaaagagt?1500
tagctgaggc?cctggcccat?gtaggaagct?gccatatctg?ttgatgggag?aggctggtga?1560
acgcagagga?taaagggaac?ggctttgtta?ggtgaggccc?aggagcatgt?atcacgccgg?1620
ctcacgacac?ggtgctttac?agaggacgtg?gctggtggcg?gcggtggtgc?tggccgtcct?1680
gtgtagcttc?ggtgtccagc?gggccgcggc?gggaatcatc?aacgccgagt?gtccaaacat?1740
gatcgggaat?cgtgacatct?acaagaaggt?ggactggatc?tgtgaggact?gcgccaacat?1800
cttccgcatc?gacggtctgg?gcatgctctg?caggtacgca?agagagagag?agagagagag?1860
agagagagag?agagagagag?agagagagag?agagtaaaat?gaataaaaaa?ggaaaaaaag?1920
atctccttat?actcttcctg?ctcctccccc?agcttctctt?ctctctgctc?atcctcttct?1980
tcctcctcct?tctcctcctc?ttccttcact?tataccaatg?ttccatgttc?ataactgacc?2040
ctcatcatca?tcatcttcct?cctcctactc?ctcctcctcc?tcctcttcct?gctcctcccc?2100
cagcttctct?tctctctgct?catcctcttc?ttcctcctcc?ttcacttata?ccaatgttcc?2160
tttttcatat?tcctcctcct?cctcctcctc?ttcctcctcc?tactcctcct?cctcctcctc?2220
ctcttgctcc?tcccccagct?tctcttctct?ctgctcatcc?tcttcttcct?cttcctcctc?2280
ctcctccttc?acacttatac?caatgttcca?tgttcataac?cgaccctcct?cttcctcctc?2340
ctcctcctcc?tctccaccac?cctcttcacc?gatcacctaa?accctcatga?tccccctcca?2400
cctcttcatc?gttgttacca?ccaccaccat?catcatcatc?accagtttcc?ccatgcctaa?2460
cgctcccaca?cttcaccttt?caggaagaac?tgctttagga?acatcgactt?cctgtggtgt?2520
gtgtacgcct?cggaacggca?cgcacagaag?gacgacctca?cgcgttacgt?cagcatcctc?2580
gggcaataac?tcctgggtgc?cccacctgcc?gctccctccc?tccctccccg?gggtatctgc?2640
cgctgctgga?gatgccccga?gggtgaagcc?gatgtccggg?aagcgccttg?aagtgaagag?2700
tgaagtgcgt?ctctccagca?gctgtccctc?cctctctggc?tgctcttcgt?ccgtggtgta?2760
agaatccagc?ggctaaagcc?ttggttcagt?ctctcgcgag?gagctttgat?tggcctccgt?2820
tcacctccat?tcagtcgctt?gtctctgttt?agctccgttc?acgccttcgt?ctattgttca?2880
gccgttcacc?ttccgttcac?ctcgtttacc?cacaaagctc?cacttgtcag?tgctttcctg?2940
ggtcctccag?aaaggtcttg?catatcaggg?acactatttt?agcattggga?attggaggta?3000
acagagtata?ggaaaagaca?attacatttt?tgtttgtgta?catggacgag?tattcgaatg?3060
tagaaggctg?ctgtagtagt?tgtaatagta?gctgtagtat?ataggaggag?gaggaggagc?3120
agaaggagga?gcaggagtgg?aaaaggaaga?agagaggagg?aagaaggtat?ttgaaagtag?3180
aaataagttg?aaagacatct?cactgaaaca?gtattctcaa?aactctagtc?aactctaaag?3240
ctgtttgagg?gttttctctt?tatgaatgtg?tatgtgtgat?cgtactcttg?tttatttgca?3300
ttcttggtta?gtgttgaatc?attcctttgt?gtgtgagtgt?gtgtatgtca?acttcctttg?3360
ttattttgtt?tcactttgtg?ttatcctctc?tcgttttgtc?ctaatttatt?gaggttgtag?3420
tatttattga?aatctgagcc?gctgtgttct?tccacgtctt?agaattaatt?gtggctgata?3480
aaaaaaataa?agagaaacga?aagagc
The genomic dna sequence of mitten crab MIH 1
Met?Val?Ser?Arg?Ala?Gln?Ser?Arg?Phe?Ser?Ser?Gln?Arg?Thr?Trp?Leu?Val?Ala?Ala?V
al
1 6 11 16
Val?Leu?Ala?Val?Leu?Cys?Ser?Phe?Gly?Val?Gln?Arg?Ala?Ala?Ala?Gly?Ile?Ile?Asn?A
la
21 26 31 36
Glu?Cys?Pro?Asn?Met?Ile?Gly?Asn?Arg?Asp?Ile?Tyr?Lys?Lys?Val?Asp?Trp?Ile?Cys?G
lu
41 46 51 56
Asp?Cys?Ala?Asn?Ile?Phe?Arg?Ile?Asp?Gly?Leu?Gly?Met?Leu?Cys?Arg?Lys?Asn?Cys?P
he
61 66 71 76
Arg?Asn?Ile?Asp?Phe?Leu?Trp?Cys?Val?Tyr?Ala?Ser?Glu?Arg?His?Ala?Gln?Lys?Asp?A
sp
81 86 91 96
Leu?Thr?Arg?Tyr?Val?Ser?Ile?Leu?Gly?Gln
101 106
The aminoacid sequence of mitten crab MIH 1 gene coded protein
Claims (4)
1, a kind of fusion rotein of mitten crab MIH 1 gene clone preparation, it is characterized in that being prepared into GST-Ers-MIH 1-pGEX-4T-1 expression plasmid corresponding to the nucleotide sequence and the plasmid pGEX-4T-1 of exon, in the LB of no NaCL, cultivate OD for 30～35 ℃ with mitten crab MIH 1 gene
600Be 0.5, induce through IPTG that smudge cells obtains the fusion rotein of purifying after GST affinity chromatography column purification, this fusion rotein is Ers-MIH 1 a gene extron amino acids coding, and sequence is:
Met?Val?Ser?Arg?Ala?Gln?Ser?Arg?Phe?Ser?Ser?Gln?Arg?Thr?Trp?Leu?Val?Ala?Ala?Val
1 6 11 16
Val?Leu?Ala?Val?Leu?Cys?Ser?Phe?Gly?Val?Gln?Arg?Ala?Ala?Ala?Gly?Ile?Ile?Asn?Ala
21 26 31 36
Glu?Cys?Pro?Asn?Met?Ile?Gly?Asn?Arg?Asp?Ile?Tyr?Lys?Lys?Val?Asp?Trp?Ile?Cys?Glu
41 46 51 56
Asp?Cys?Ala?Asn?Ile?Phe?Arg?Ile?Asp?Gly?Leu?Gly?Met?Leu?Cys?Arg?Lys?Asn?Cys?Phe
61 66 71 76
Arg?Asn?Ile?Asp?Phe?Leu?Trp?Cys?Val?Tyr?Ala?Ser?Glu?Arg?His?Ala?Gln?Lys?Asp?Asp
81 86 91 96
Leu?Thr?Arg?Tyr?Val?Ser?Ile?Leu?Gly?Gln。
101 106
2, the polyclonal antibody of fusion rotein preparation according to claim 1, after it is characterized in that fusion rotein and complete and Freund mixing, immune new zealand rabbit prepares polyclonal antibody.
3, the application of fusion rotein according to claim 1 is characterized in that this fusion rotein can be used as preparation and promotes mitten crab to cast off a skin and the biotechnological formulation of individual growth.
4, a kind of method that promotes mitten crab to cast off a skin and grow, it is characterized in that by RNA interference or sense-rna or gene knockout molecular biology method, suppress mitten crab MIH 1 expression of gene, promote casting off a skin and growth of mitten crab individuality.
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2003
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