CN103525824B - Short octopus serpin OoSerpin gene and recombinant protein thereof and application - Google Patents
Short octopus serpin OoSerpin gene and recombinant protein thereof and application Download PDFInfo
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- CN103525824B CN103525824B CN201310518995.6A CN201310518995A CN103525824B CN 103525824 B CN103525824 B CN 103525824B CN 201310518995 A CN201310518995 A CN 201310518995A CN 103525824 B CN103525824 B CN 103525824B
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Abstract
The present invention is in-vitro recombination expression and the application of short octopus Serpin type protease inhibitor gene OoSerpin, the present invention utilizes the short octopus transcript profile library of structure, obtain the cDNA full length sequence of short octopus Serpin type protease inhibitor gene OoSerpin, the nucleotide sequence of this gene is as shown in SEQ ID No.1; Utilize RT-PCR expression technology to have expressed its proteins encoded, this recombinant protein has good restraining effect for the activity of trypsinase and Chymotrypsin and colibacillary growth.The research of this gene and recombinant protein thereof, for the further function understood Serpin type protease inhibitor gene and play in innate immunity, is developed new diseases prevention and treatment medicine, food and animal feedstuff additive and is had great importance.
Description
Technical field
The present invention relates to technical field of molecular biology, from short octopus cDNA library, specifically obtain the full-length cDNA sequence of short octopus Serpin type serpin OoSerpin gene, in-vitro recombination expression is carried out to its gene coding region; Relate to the separation and purification of this recombinant protein and the restraining effect to trypsinase and chymotrypsin activity thereof, and the restraining effect to Escherichia coli Growth; Also relate to this recombinant protein as proteinase inhibitor in human disease treatment and the potential application that is added on as fungistat in animal-feed.
Background technology
Innate immune defense is the first line of defence of invertebrates opposing pathogen infection, when after pathogenic micro-organism invasion body, host mainly completes immune defense by following several mode: first, by the pattern recognition receptors of innate immune system to the identification of alien material; Subsequently, non-own recognition signal causes the extracellular cascade reaction activating serine protease and remove serpin, thus the signal be infected is enlarged into stronger " danger " signal or removes false alarm; Then activate corresponding signal transduction pathway, cause the transcriptional activity of goal gene; Finally, activation effect thing reactive system kills and removes pathogenic agent.Serpin, as a kind of important immune factor, by suppressing harmful hydrolysis activation of too much proteolytic enzyme to carry out immunity moderation reaction, has important effect in the immunne response of invertebrates.The serpin found at present has hundreds of, and they are divided into tens families, and the serpin in animal body is mainly distributed in Serpin, Kazal, Kunitz and α-macroglobulin four family.Wherein, Serpin
Type serpin has relatively large molecular weight (40-60kDa) and relative conservative space structure, and has active centre ring (Reactive centre loop, RCL) at C end.The RCL of Serpin can be inserted into the avtive spot of particular target enzyme, thus forms a stable complicated body or produce the change of conformation, to prevent harmful hydrolysis activation of serine protease in organism, with control agent endoproteinase cascade reaction.The specific space structure of Serpin can suppress trypsinase and Quimotrase effectively, plays a significant role resisting in pathogenic agent and bacteria growing inhibiting process.In recent years, although find multiple serpin in marine animal, but the research up to now for marine invertebrate Serpin type proteinase inhibitor and function thereof is very limited, have no the report about short octopus Serpin type proteinase inhibitor.
Summary of the invention
The invention provides the microsatellite locus of 10 short octopus, and corresponding polymorphic micro-satellite primer, for cloning short octopus Serpin type protease inhibitor gene, Protein reconstitution is carried out to its structural domain, analyze the activity of recombinant protein, for understanding invertebrates innate immunity in depth, develop natural marine drug and fodder additives lays the foundation.
The object of the invention is to be realized by following technical scheme: clone obtains a kind of short octopus Serpin type protease inhibitor gene, this gene has the nucleotide sequence in SEQ ID No. 1, this sequence 1735bp, 5 ' non-coding region 214bp, 3 ' non-coding region 282bp, there is polyadenylic acid tail, open reading frame 1239bp, 412 amino acid of encoding; Aminoacid sequence is as shown in SEQ ID No. 2, and wherein 1-25 amino acid is signal peptide sequence; Have the structural domain of 1 Serpin type serpin, this structural domain contains 11 αhelix and 14 β-pleated sheet structure structures, and proteins encoded molecular weight is 46.5kDa, and iso-electric point is 8.52.When this recombinant protein mixes with mass ratio 18:1 ratio respectively with trypsinase and Chymotrypsin, trypsinase and the chymotrypsin activity of 86.5% and 55.0% can be suppressed respectively; At Escherichia coli Growth Inhibition test to the 7.5th hour, Escherichia coli Growth per-cent is suppressed to be 40.6%.
The present invention fills up the blank about short octopus Serpin type proteinase inhibitor research, there is provided a kind of and utilize the method for this short octopus serpin Ooserpin genes produce recombinant protein: described method is from the short octopus transcript profile library built, modern molecular biology technique analysis is utilized to obtain the cDNA total length of Serpin type proteinase inhibitor sequence, its recombinant plasmid is built with the method for prokaryotic expression, after induction, purifying obtains recombinant protein, recombinant protein is through renaturation, detect its restraining effect to trypsinase and chymotrypsin activity, and the restraining effect to Escherichia coli Growth.
The method of aforesaid gene or albumen or Restruction albumen can be applicable to be prepared in trypsin inhibitor medicine.
The method of aforesaid gene or albumen or Restruction albumen can be applicable to be prepared in chymotrypsin inhibitor medicine.
The product that the method for aforesaid gene or albumen or Restruction albumen is obtained is applied in foodstuff additive as fungistat.
The product that the method for aforesaid gene or albumen or Restruction albumen is obtained is applied in animal feedstuff additive as fungistat.
Advantage of the present invention: clone obtains a kind of cDNA full length sequence of short octopus Serpin type protease inhibitor gene, this gene has the base sequence in SEQ ID No.1, restructuring gives expression to recombinant protein to carrier, and the albumen obtained has the aminoacid sequence in SEQ ID No.2.This recombinant protein can suppress the activity of trypsinase and Chymotrypsin, and has certain restraining effect to Escherichia coli Growth.The research of this gene and recombinant protein activity thereof, for the further effect understood Serpin type protease inhibitor gene and play in short octopus innate immunity, and the new human diseases protective agents of development and animal feedstuff additive are all had great importance.
Embodiments of the invention Binding experiment and accompanying drawing thereof further illustrate as follows:
SEQ ID No.1 is the nucleotide sequence of short octopus serpin OoSerpin gene;
SEQ ID No.2 is the aminoacid sequence of short octopus serpin OoSerpin gene coded protein.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of short octopus serpin rOoSerpin of recombinating, wherein: M is protein molecular weight standard thing Marker; 1 is the rOoSerpin do not induced; 2 is the rOoSerpin induced; 3 is the rOoSerpin of purifying.
Fig. 2 be the short octopus serpin rOoSerpin of restructuring to the restraining effect figure of trypsinase and chymotrypsin activity, wherein: represent that trypsinase remains the change curve of enzymic activity under gradient concentration rOoSerpin suppresses; Represent that Chymotrypsin remains the change curve of enzymic activity under gradient concentration rOoSerpin suppresses.
Fig. 3 recombinates short octopus serpin rOoSerpin to the restraining effect figure of Escherichia coli Growth, wherein: represent the bacterial concentration change curve of intestinal bacteria under the short octopus serpin rOoSerpin restraining effect of restructuring; Represent intestinal bacteria not by the bacterial concentration change curve under restructuring short octopus serpin rOoSerpin effect.
Embodiment
Short octopus serpin OoSerpin gene of the present invention has the base sequence of SEQ ID No. 1, its proteins encoded has the aminoacid sequence of SEQ ID No. 2, this recombinant protein can suppress the protein-active of trypsinase and Chymotrypsin, and has certain restraining effect to Escherichia coli Growth.
The method of short octopus Serpin type protease inhibitor gene clone of the present invention and Protein reconstitution thereof is as follows:
Embodiment 1:
The acquisition of short octopus Serpin type protease inhibitor gene cDNA total length:
Short octopus is stimulated with G+, G-bacterium dipping bath, get the tissues such as the gill, sexual gland, muscle, hemocyte, mantle, the scrotum and hepatopancreas, extract sample total serum IgE respectively, with DNase I DNA digestion after RNA balanced mix, with enrichment with magnetic bead mRNA, break into short-movie section after synthesize a chain cDNA, after resynthesis two chain cDNA, purifying recovery, sticky end reparation, add joint, finally carry out PCR amplification; Transcript profile order-checking is carried out in the library built.Sequencing result obtains after splicing, and by BLASTN, BLASTP, BLASTX, the softwares such as ClustalX carry out same edge to the existing sequence that current laboratory is set up and analyse and compare, and obtain the cDNA total length of OoSerpin.CDNA SEQ ID No. 1 is the nucleotide sequence of short octopus serpin OoSerpin gene.
SEQ ID No. 2 is the aminoacid sequence of short octopus serpin OoSerpin gene coded protein.
Embodiment 2:
The in-vitro recombination expression of short octopus Serpin type protease inhibitor gene:
(1) PCR technology is utilized, with rOoSerpin-F:GGATCCAGCAAGATGAAATCATCCAACC and rOoSerpin-R:CTCGAGTCAATCTGCTGGGTTAGTTACCTTG for restructuring primer, with sequence shown in SEQ ID No.1 for template increases, reaction conditions is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 59 DEG C of annealing 30s, 72 DEG C extend 60s, carry out 35 circulations; Last 72 DEG C extend 10min.PCR product detects with 1.5% agarose gel electrophoresis, recovery and the purifying that test kit (TaKaRa) carries out PCR product is reclaimed with glue, be cloned into the pEASY-E1 expression vector (Transgen) with His label, be transformed in Trans1-T1 phage resistant competent cell (Transgen) again, after LB culture medium flat plate is cultivated, picking colony, with T7 Promoter-F:TAATACGACTCACTATAGGG and rOoSerpin-R:
CTCGAGTCAATCTGCTGGGTTAGTTACCTTG is respectively forward and reverse primer screening, carries out sequencing to positive colony.The correct clone that checks order extracts plasmid with plasmid extraction kit (Promega), is transformed in intestinal bacteria Transetta (DE3) competent cell (Transgen).Positive colony is 220rpm shaking culture in the LB liquid nutrient medium of 50 mg/ml at ampicillin concentration, and culture condition is 37 DEG C.When to reach OD600 be 0.5-0.7 to substratum concentration, add the IPTG that final concentration is 1mmol/L, continue to cultivate 4h.Get 1ml bacterium liquid centrifugal, after supernatant discarded, add 80 μ l water and 20 μ l 5 × albumen sample-loading buffers, 100 DEG C are boiled 10min, slightly centrifugal, and after supernatant discarded, the SDS-PAGE with 15% detects expression product.After residue bacterium liquid is centrifugal, utilize His-tag fusion protein purification test kit MagExtractor His-Tag
NPK-700(TOYOBO), according to operation steps purifying protein.
(2) purifying protein that (1) step obtained is through urea-TBS glycerol buffer (pH8.0) dialysis renaturation containing the Sleep-promoting factor B of the reduced glutathion of 2mM, 0.2mM, 1mM EDTA, 50mM Tris-HCl, 50mM NaCl, 10% glycerine, 1% glycine, urea concentration is substituted into 5M, 4M, 3M, 2M, 1M gradually from initial 6M, and last dialysis is to not glycerol adding during dialyzate without urea.Purifying protein is 4 DEG C of dialysis 12h in each concentration gradient, and the SDS-PAGE with 15% detects.
Embodiment 3:
To recombinate the restraining effect of short octopus Serpin type proteinase inhibitor to trypsinase and chymotrypsin activity.
Embodiment 2(2) recombinant protein that obtains of step with 15% SDS-PAGE detect after, measure protein concentration with BCA determination of protein concentration test kit (the green skies), with the Tris damping fluid Function protein concentration of 50mM.Get 20 μ L gradient concentrations and be respectively the recombinant protein solution of 0,10,20,40,80,160,230,314 and 960 μ g/ml and 20 μ l contain trypsin 50 μ g/ml, or Chymotrypsin (25 μ g/ml Sigma), Sigma) Tris buffer (50mM, pH8.0) solution mixes gently in 96 hole enzyme plates, room temperature effect 10min.Respectively with N-benzoyl-Phe-Val-Arg-p-nitroanilide (200 μ g/ml, and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (100 μ g/ml Sigma), Sigma) substrate as trypsinase and Chymotrypsin is dissolved in 50mM Tris buffer (pH8.0), then in every hole, 160 μ l substrates are added, after room temperature effect 20min, detect with microplate reader and detect its light absorption value at 405nm place.Not add the 50mM Tris buffer (pH 8.0) of recombinant protein for negative control.Often organize Setup Experiments 3 repetition.
Result: when recombinant protein mixes with mass ratio 18:1 ratio with trypsinase and Chymotrypsin, trypsinase and the chymotrypsin activity of 86.5% and 55.0% can be suppressed respectively: be 10 with the recombinant protein of 50 μ g proteolytic enzyme effects, 20, 40, 80, 160, 230, during 314 and 960 μ g, tryptic residual enzyme vigor is respectively 86.85%, 82.85%, 80.85%, 78.82%, 74.54%, 62.86%, 45.27%, 34.96% and 13.49%, the residual enzyme vigor of Chymotrypsin is respectively 92.9%, 85.5%, 82.7%, 73.4%, 65.3%, 63.3%, 60.1%, 54.2% and 45.0%.
Embodiment 4:
Short octopus Serpin type proteinase inhibitor recombinant protein is to the restraining effect of Escherichia coli Growth:
The mono-clonal intestinal bacteria 37 DEG C choosing band ammonia benzyl resistance are cultured to OD600=0.4, get restructuring OoSerpin albumen (the final concentration 1000 μ g/ml) mixing be dissolved in the Tris-Hcl of 50mM of 50 μ l bacterium liquid and equivalent, be added in 96 aseptic porocyte culture plates with liquid-transfering gun, and not contain the 50mM Tris-Hcl of recombinant protein in contrast.After flat board shakes and hatches 30 minutes under 37 DEG C of conditions, getting 20 μ l bacterium liquid is respectively added in aseptic 96 hole enzyme plates, and in every hole, add 180 μ lLB substratum (containing 100 μ g/mL ammonia benzyls), hatch 12 hours and detected its light absorption value every 30 minutes at 600nm place for 37 DEG C.Often organize Setup Experiments 3 repetition.
Result: when the recombinant protein of 1000 μ g/ml and the intestinal bacteria balanced mix of OD600=0.4 are cultured to the 7.5th hour, suppresses Escherichia coli Growth per-cent to be 40.6%.
Summary result: the present invention has prepared the recombinant protein of short octopus Serpin type proteinase inhibitor, demonstrates recombinant protein to the inhibit activities of trypsinase and Chymotrypsin and restraining effect, analyzes the restraining effect of recombinant protein to Escherichia coli Growth.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, modifies for above-described embodiment, and it is all possible for adding and replacing, and does not all exceed protection scope of the present invention.
nucleotide or aminoacid sequence table
<110> Shandong Prov. Marine Aquatic Product Inst
The short octopus serpin of <120>
<130>
<140>
<141>
<150>
<151>
<160> 2
<210> 1
<211> 1735
<212> DNA
The short octopus of <213> (Octopus ocellatus)
<220>
<221> CDS
<222> (215)...(1453)
<400> 1
CGACGACAAA CGTAAGAAGT AATTCCAACT TCCTCTCACT AATAAAAGAC AAAAATCAAT 60
ACGTTTTTCA AAATAAACTT ATTCCGGACA ACCTGTTCGC TTCCCTCGGC TCTTTGTGAA 120
AATCTTTCTG CGAATATTGT TGCAAAAACC TTCCCCAACG CAAATCCGGA AAAAAAAATC 180
CTCAATCAAG ACTGTATTTG GATAAATCTT GAACATGTTA AAGAAACGAA AGGGACTCGC 240
CTTGCTGTTC GTTGTTGTTA CTCTACAAAC TCTAACAGAA ATTAATAGCA GCAAGATGAA 300
ATCATCCAAC CATCACCAAG AGATTAAAAA CCTGGCTGCA TCAATTGATT CTTTTTCAGC 360
CTCCCTTTAT CAAGAAATGG TCACAGGAAT CAATGAGAAT GTCTTCATAT CCCCATTCAG 420
CATTGCTACC ATTTTATCAA TGGTTTATTT TGGAGCAAGA CAAAACACAG CTGAGCAGTT 480
GAAAAATGTC TTGAACATAA CTTCAACACC AAATGCTGTG GGTTCGGCTT TTAGAAATTA 540
TTTTTTGACT CTGAAGAATT CAAATGATGA TAACATATGT AAATCAGTGA ATCGTTTGTA 600
TGTTGATGAC AAATTCCCTA TTTCGAAAAG TTACAAGCAT GATATTTCAC AATATTTCTT 660
TACGGATATT AAAAATGTAG ATTTCGTAAG AAACGCTGAA AGAATAAGAA TCAATGCCAA 720
CAAATGGGTT AAGAAGGAAA CCTATAACGA AATCACTGAT CTGTTACCAA GTAAAAGTCT 780
GTCCGCTGAT ACAGCATTAC TTCTCGTTAA TGCCATGTAC TTTCAAGGAA TATGGGCCGA 840
AAGGTTCCAA GAAAAAGTAA CATCACCCCG AAAGTTTCAT CTAGGCTCTG GCAAGTCTGT 900
TTCGTGTGAT TTCATGTTTT TGTCTGAAGA TTTTAATACA AAGATAAGCT ATAACTATAA 960
AGCTGTAGAG TTACCATACG TGGGAGAGAA ATTTAGTATG TTTGTAATTC TTCCTAATAA 1020
AATGGATGGC CTTGCTGCTT TAGAGAAGGA AATTAATGTG CAATTTCTCA AAGAGATTAT 1080
AAAGGGAAAA GGATTCGAAG AGTTATCTCT TGAATTATAT TTACCAAAGT TTCGCTTTCA 1140
AAGTGCTCTC GAGTTATCTG ATGGTTTACA AAATCTCGGT CTTTCTGATA TCTTTGATGC 1200
CAGCAAAGCC AATTTATCAG GAATAAGTAG TCAGAGGGAG TTGTATGTGA GTAAATTCTT 1260
TCATAAAACA TTTATTGATG TAAATGAAAA AGGAACCGAA GCTGTAGCTG CCAGCGGAGA 1320
AGTGGTAATT AGTCCAGTCT CAGCCCAGAC TGGAACAGCA TTTGATGTTA ATCACCCTTT 1380
CATATTTTTA ATTGTTGATA GAAGAATTAA CATGATTTAT TTCATCGGCA AGGTAACTAA 1440
CCCAGCAGAT TAAAAACATC GCCCTTATGG CTTCTAAAGC ACCTGTTGTT TTCTATAAAG 1500
CCAATAAAAT AAAATTAAAA TTAAAATTAA AATTAAATTA AATTAAAAAT CACCTCTTCT 1560
ATCAACTCGC CCAAAAATGG TACTACGATA GACCTTGCAA CATTACAGAG ACTTAGTTAG 1620
AGTCCTAACA AAGAAGATTT GATTGAAATT ATTATTCAAA AACTTTATTT GAAATACCAA 1680
ATAAAAAGTT TTTAAAATAA ATTGAAAGTT ATGTAAAAAA AAAAAAAAAA AAAAA 1735
<210> 2
<211> 412
<212> PRT
The short octopus of <213> (Octopus ocellatus)
<220>
<400> 2
Met Leu Lys Lys Arg Lys Gly Leu Ala Leu Leu Phe Val Val Val Thr
1 5 10 15
Leu Gln Thr Leu Thr Glu Ile Asn Ser Ser Lys Met Lys Ser Ser Asn
20 25 30
His His Gln Glu Ile Lys Asn Leu Ala Ala Ser Ile Asp Ser Phe Ser
35 40 45
Ala Ser Leu Tyr Gln Glu Met Val Thr Gly Ile Asn Glu Asn Val Phe
50 55 60
Ile Ser Pro Phe Ser Ile Ala Thr Ile Leu Ser Met Val Tyr Phe Gly
65 70 75 80
Ala Arg Gln Asn Thr Ala Glu Gln Leu Lys Asn Val Leu Asn Ile Thr
85 90 95
Ser Thr Pro Asn Ala Val Gly Ser Ala Phe Arg Asn Tyr Phe Leu Thr
100 105 110
Leu Lys Asn Ser Asn Asp Asp Asn Ile Cys Lys Ser Val Asn Arg Leu
115 120 125
Tyr Val Asp Asp Lys Phe Pro Ile Ser Lys Ser Tyr Lys His Asp Ile
130 135 140
Ser Gln Tyr Phe Phe Thr Asp Ile Lys Asn Val Asp Phe Val Arg Asn
145 150 155 160
Ala Glu Arg Ile Arg Ile Asn Ala Asn Lys Trp Val Lys Lys Glu Thr
165 170 175
Tyr Asn Glu Ile Thr Asp Leu Leu Pro Ser Lys Ser Leu Ser Ala Asp
180 185 190
Thr Ala Leu Leu Leu Val Asn Ala Met Tyr Phe Gln Gly Ile Trp Ala
195 200 205
Glu Arg Phe Gln Glu Lys Val Thr Ser Pro Arg Lys Phe His Leu Gly
210 215 220
Ser Gly Lys Ser Val Ser Cys Asp Phe Met Phe Leu Ser Glu Asp Phe
225 230 235 240
Asn Thr Lys Ile Ser Tyr Asn Tyr Lys Ala Val Glu Leu Pro Tyr Val
245 250 255
Gly Glu Lys Phe Ser Met Phe Val Ile Leu Pro Asn Lys Met Asp Gly
260 265 270
Leu Ala Ala Leu Glu Lys Glu Ile Asn Val Gln Phe Leu Lys Glu Ile
275 280 285
Ile Lys Gly Lys Gly Phe Glu Glu Leu Ser Leu Glu Leu Tyr Leu Pro
290 295 300
Lys Phe Arg Phe Gln Ser Ala Leu Glu Leu Ser Asp Gly Leu Gln Asn
305 310 315 320
Leu Gly Leu Ser Asp Ile Phe Asp Ala Ser Lys Ala Asn Leu Ser Gly
325 330 335
Ile Ser Ser Gln Arg Glu Leu Tyr Val Ser Lys Phe Phe His Lys Thr
340 345 350
Phe Ile Asp Val Asn Glu Lys Gly Thr Glu Ala Val Ala Ala Ser Gly
355 360 365
Glu Val Val Ile Ser Pro Val Ser Ala Gln Thr Gly Thr Ala Phe Asp
370 375 380
Val Asn His Pro Phe Ile Phe Leu Ile Val Asp Arg Arg Ile Asn Met
385 390 395 400
Ile Tyr Phe Ile Gly Lys Val Thr Asn Pro Ala Asp
405 410
Claims (7)
1. a short octopus serpin OoSerpin gene, its nucleotides sequence is classified as sequence shown in SEQ ID NO.1.
2. the proteins encoded of short octopus serpin OoSerpin gene described in claim 1, its aminoacid sequence is sequence shown in SEQ ID No. 2.
3. utilize the method for a kind of short octopus serpin OoSerpin genes produce recombinant protein described in claim 1: it is characterized in that, utilize round pcr, with rOoSerpin-F:GGATCCAGCAAGATGAAATCATCCAACC and rOoSerpin-R:CTCGAGTCAATCTGCTGGGTTAGTTACCTTG for restructuring primer, with sequence shown in SEQ ID No.1 for template increases, gained PCR primer reclaims test kit with glue and reclaims and purifying, be cloned into pEASY-E1 expression vector, be transformed in Trans1-T1 phage resistant competent cell again, after cultivation, picking colony, forward and reverse primer screening is respectively with T7 Promoter-F:TAATACGACTCACTATAGGG and rOoSerpin-R:CTCGAGTCAATCTGCTGGGTTAGTTACCTTG, positive colony is checked order, after the correct clone that checks order extracts plasmid, be transformed in intestinal bacteria Transetta (DE3) competent cell, after inducing culture, utilize His-tag fusion protein purification kits, the purifying protein obtained is the reduced glutathion containing 2mM of 8.0 through pH, the Sleep-promoting factor B of 0.2mM, 1mM EDTA, 50mM Tris-HCl, 50mM NaCl, 10% glycerine, urea-TBS glycerol buffer the dialysis renaturation of 1% glycine, the described recombinant protein of final acquisition.
4. the method for gene according to claim 1 or albumen according to claim 2 or Restruction albumen according to claim 3 is preparing the application in trypsin inhibitor medicine.
5. the method for gene according to claim 1 or albumen according to claim 2 or Restruction albumen according to claim 3 is preparing the application in chymotrypsin inhibitor medicine.
6. gene according to claim 1 or albumen according to claim 2 are preparing the application in foodstuff additive as fungistat.
7. gene according to claim 1 or albumen according to claim 2 are preparing the application in animal feedstuff additive as fungistat.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703458A (en) * | 2012-06-20 | 2012-10-03 | 山东省海洋水产研究所 | Solen grandis serpin Sgkunitz gene as well as recombinant protein and application thereof |
| CN102787131A (en) * | 2012-09-03 | 2012-11-21 | 山东省海洋水产研究所 | Solen grandis chymotrypsin gene SgChy and recombinant protein of solen grandis chymotrypsin gene SgChy |
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|---|---|---|---|---|
| CN102703458A (en) * | 2012-06-20 | 2012-10-03 | 山东省海洋水产研究所 | Solen grandis serpin Sgkunitz gene as well as recombinant protein and application thereof |
| CN102787131A (en) * | 2012-09-03 | 2012-11-21 | 山东省海洋水产研究所 | Solen grandis chymotrypsin gene SgChy and recombinant protein of solen grandis chymotrypsin gene SgChy |
Non-Patent Citations (2)
| Title |
|---|
| 中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal) 的重组表达及活性分析;黄明等;《水产学报》;20110930;第35卷(第9期);1310-1318 * |
| 白鲢骨骼肌丝氨酸蛋白酶抑制剂的提取、纯化及其特性;胡新颖等;《中国水产科学》;20060331;第13卷(第2期);316-320 * |
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