CN104480079A - Cyanea capillata superoxide dismutase and its coding gene and use - Google Patents
Cyanea capillata superoxide dismutase and its coding gene and use Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
Abstract
The invention belongs to the technical field of biological medicine. At present, there is no Cyanea capillata superoxide dismutase-related research report. The invention provides Cyanea capillata superoxide dismutase and a preparation method thereof. The Cyanea capillata superoxide dismutase has a protein composted of an amino acid sequence shown in the formula of SEQ ID NO: 2. The invention also provides a coding gene of the Cyanea capillata superoxide dismutase and the coding gene has a nucleotide sequence shown in the formula of SEQ ID NO: 1. The invention also provides use of the Cyanea capillata superoxide dismutase and its coding gene in preparation of antioxidant drugs, antiradiation drugs, a skin protective agent, a food antiseptic and ageing-resistant drugs.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of Cyanea capillata superoxide-dismutase and encoding gene thereof and application.
Background technology
Medusa Cnidaria is the very huge marine plankton of widely distributed, the biological total amount of a class.Existing large quantity research shows, is rich in various bioactivators in medusa, comprises medusocongestin albumen and some New function albumen that are active strongly, that have good DEVELOPMENT PROSPECT.Jellyfish class is swum in the dark seawater surface of 4-6 rice, under living in strong optical radiation environment for a long time.Oxidative damage is one of uv-radiation important mechanisms causing body injury, there are some researches show, planktonic organism is avoid or reduce the infringement that uv-radiation causes, and selects through long adaptation, create in its body of a great variety, structure is special, active strong anti-oxidation active substance.Flourishing antioxidant system plays an important role in Cell protection and organism are from light and photopigment equivalent damage, high light ultraviolet can be protected the radiation injury of organism, remove the free radical produced in oxidizing reaction.Studies have found that, Cyanea capillata tentacle extract has very strong antioxidation activity in vitro, strong more than conventional antioxidant vitamin C to the Scavenging activity of active chalcogen.Also from jellyfish Rhopilema esculentum, Stomolophus meleagris, the multiple albumen with obvious scavenging free radicals function is isolated respectively by the method such as ammonium sulfate precipitation, gel permeation chromatography.These results all show, containing active strong anti-oxidant activity component in medusa, are important sources that is anti-oxidant, anti-radiation active substances.However, at present to the not yet systematic understanding such as sequence information, expression, antioxidant activity level of the composition of antioxidant system in medusa, important antioxidant reductase (albumen).
Superoxide-dismutase (SOD) is the important antioxidase of a class be extensively present in prokaryotic organism and eukaryote, and these biologies can be protected from ultra-oxygen anion free radical (O
2 -) oxidative damage that causes, be organism indispensable important protective enzyme in its Metabolic activity.Superoxide-dismutase belongs to melts combine enzyme, can catalysis ultra-oxygen anion free radical generation disproportionation reaction specifically, Hydrogen Peroxide and water, and is considered to the main effect enzyme playing this function.Its reaction process is as follows:
2O
2 -+2H
+＝H
2O
2+O
2
The hydrogen peroxide produced in reaction can be removed by catalase and peroxidase further, is converted into water and oxygen molecule.The murder by poisoning of the ultra-oxygen anion free radical that organism is produced from normal metabolic processes by self aerobic cell of this reaction protection, thus make cell keep its normal vitality.In addition, superoxide-dismutase be also found to improve significantly body to the immunizing power of bacterium and virus infection and noxious chemical is exposed, the resistibility of the deleterious effect such as thermal stimulus.According to the difference playing metal prothetic group needed for active function, superoxide-dismutase can be divided into Four types: (1) needs the superoxide-dismutase Mn-SOD (Sod A) of manganese; (2) the superoxide-dismutase Fe-SOD (Sod B) of iron is needed; (3) the superoxide-dismutase Cu/Zn SOD (Sod C) of copper and zinc is needed; (4) the superoxide-dismutase Ni-SOD (Sod N) of nickel is needed.Superoxide-dismutase is described as " street cleaner " of rubbish in organism, is mainly used in the various diseases such as treatment oxygen intoxication, senile cataract, diabetes, cardiovascular disorder clinically; Superoxide-dismutase also can be used as antiradiation agent, for adjuvant radiotherapy, to reduce the side effect of high dose radiation; Superoxide-dismutase is also widely used in daily chemical industry, and the skin care product that with the addition of superoxide-dismutase have obvious resisting age of skin, go the functions such as foxiness.In view of above-mentioned effect, in the present invention, the superoxide-dismutase in Cyanea capillata source possesses significant Development volue, has huge application potential and wide market outlook anti-oxidant with antiradiation drug, makeup, food, agricultural and chemical industry.
The seminar at place of the present invention is devoted to extraction and the research of active ingredient in medusa always, obtain Chinese invention patent ZL201310189933.5, denomination of invention is " a kind of Cyanea capillata thioredoxin and encoding gene and application " thereof, application publication number: CN103232979A; Applied for Chinese patent CN201310188702.2, denomination of invention is " a kind of Cyanea capillata peroxiredoxin and encoding gene and application " thereof, application publication number: CN103255113A; CN201310258184.7, denomination of invention is " a kind of Cyanea capillata astacin sample metalloprotease CALP1 and encoding gene and expression method " thereof; CN201310629343.X, denomination of invention is " a kind of preparation method of jellyfish cardiovascular toxin crude extract ", application publication number: CN103613653A; CN201310628068.X, denomination of invention is " a kind of preparation method of jellyfish hematoxin crude extract ", application publication number: CN103626861A etc.
At present, there is not yet the relevant research report to jellyfish superoxide-dismutase both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of Cyanea capillata superoxide-dismutase and encoding gene thereof, another object of the present invention is to provide this Cyanea capillata superoxide-dismutase preparing the application in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives, antiaging agent etc.
The present invention by building Cyanea capillata tentacle tissue cDNA library, and measures the sequence of this library recombinant clone and annotates analysis, obtains the encoding gene of Cyanea capillata superoxide-dismutase.This Cyanea capillata superoxide-dismutase is the jellyfish Like superoxide dismutase with obvious anti-oxidant activity of Late Cambrian, be the important activity component of antioxidant system in medusa, this albumen has a good application prospect in studying at anti-oxidant, antiradiation drug.
Main technical schemes of the present invention is, by building Cyanea capillata tentacle tissue cDNA library, checking order and screen it, obtaining the encoding gene of Cyanea capillata superoxide-dismutase, and study its anti-oxidant activity.
A first aspect of the present invention, provides a kind of Cyanea capillata superoxide-dismutase, described a kind of Cyanea capillata superoxide-dismutase, has the protein of following (I) or (II):
The protein of the aminoacid sequence composition shown in (I) SEQ ID NO:2;
Amino acid sequence shown in (II) SEQ ID NO:2 is substituted, lacks and/or adds one or several amino acid and the protein derivative by (I) of same function.
Described a kind of Cyanea capillata superoxide-dismutase, the aminoacid sequence as shown in SEQ ID NO:2, this albumen is not containing signal peptide, and be non-secreted protein, be positioned tenuigenin, molecular weight is 16.0kDa, and iso-electric point is 6.2.
Present invention also offers a kind of encoding gene of Cyanea capillata superoxide-dismutase, the DNA molecular for following (I) or (II):
Nucleotide sequence shown in (I) SEQ ID NO:1;
The nucleotide sequence of nucleotide sequence homology more than 80% shown in (II) Yu SEQ ID NO:1.
Described a kind of Cyanea capillata superoxide dismutase gene, this nucleotide sequence total length 745bp, as shown in SEQ ID NO:1.
A second aspect of the present invention, provide a kind of preparation method of Cyanea capillata superoxide-dismutase, the method comprises the steps:
(1) Cyanea capillata tentacle tissue cDNA library is built;
(2) sequence of above-mentioned Cyanea capillata tentacle tissue cDNA library recombinant clone is measured and analyzed, obtain the nucleotide sequence of encoding superoxide dismutase;
(3) expression plasmid of Cyanea capillata superoxide-dismutase is built, recombinant bacterial strain;
(4) expression of Cyanea capillata superoxide-dismutase;
(5) purifying of restructuring Cyanea capillata superoxide-dismutase.
Cyanea capillata tentacle tissue cDNA library in described step (1) builds by the following method:
1) extracting Cyanea capillata tentacle total tissue RNA;
2) separating mRNA, synthesis Cyanea capillata tentacle tissue cDNA;
3) above-mentioned cDNA is inserted pUC19 plasmid vector, then be converted in bacillus coli DH 5 alpha, coat LB culture medium flat plate and carry out blue hickie screening, be namely built into Cyanea capillata tentacle tissue cDNA library.
The expression plasmid of the structure Cyanea capillata superoxide-dismutase in described step (3), recombinant bacterial strain, concrete steps are:
1) according to the restriction enzyme site of Cyanea capillata superoxide dismutase gene sequence and prokaryotic expression carrier pET24a, a pair PCR primer with specific cleavage site Nde I and Xho I is designed, as follows:
Upstream primer: 5 '-GGGAATTCCATATGGCTACATTGAAAGC-3 '
Downstream primer: 5 '-CGCTCGAGCGCATTGGCTATT-3 '
PCR reaction conditions is: 95 DEG C of insulation 5min; 95 DEG C of insulation 30sec, 55.5 DEG C of insulation 30sec, 68 DEG C of insulation 1min, 35 circulations; 68 DEG C of insulation 5min;
2) enzyme cut back to close after PCR primer and pET24a prokaryotic expression plasmid;
3) utilize two restriction enzyme sites to be connected to by encoding sequence on pET24a carrier, build recombinant expression plasmid, be then transformed into intestinal bacteria Rosetta (DE3) pLysS and obtain recombinant bacterial strain.
The expression condition of the Cyanea capillata superoxide-dismutase in described step (4) is: 12 DEG C, induction 10 hours under 0.5mMIPTG, 150rpm, under this inductive condition, recombinant protein can be expressed with soluble form.
Purifying in described step (5) is for adopting nickel ion affinity chromatograph post single step purification and get final product, and elution requirement is account for the binding buffer liquid of cumulative volume 50% and the elution buffer of 50% respectively; Wherein, binding buffer liquid is NaH
2pO
420mM; NaCl 500mM; Imidazoles 30mM; PH 7.4, elution buffer is NaH
2pO
420mM; NaCl 500mM; Imidazoles 500mM; PH 7.4.This preparation method is simple, with low cost.
A third aspect of the present invention, provides Cyanea capillata superoxide-dismutase and encoding gene is preparing the application in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives, antiaging agent.
The restructuring Cyanea capillata superoxide-dismutase that the present invention obtains has significant anti-oxidant activity.At detection recombinant protein in the experiment of ultra-oxygen anion free radical rejection ability, can be observed the rising along with restructuring Cyanea capillata copper-zinc superoxide dismutase protein concentration, in reaction system, the inhibiting rate of superoxide anion obviously increases, and when superoxide-dismutase reaches 500 μ g/mL level, superoxide anion is almost completely suppressed.This experiment demonstrates restructuring Cyanea capillata copper-zinc superoxide dismutase to the Scavenging activity of ultra-oxygen anion free radical.
Therefore, the Cyanea capillata superoxide-dismutase related in the present invention will have very large using value in exploitation anti-oxidation medicine, antiaging agent, antiradiation drug, shielding medicine for skin, food preservatives.
Accompanying drawing explanation
Fig. 1 is the multiple sequence comparison of Cyanea capillata superoxide-dismutase and other species superoxide-dismutases; Wherein, black represents the region of complete homology.
Fig. 2 is the PCR primer nucleic acid electrophoresis result of Cyanea capillata superoxide-dismutase open reading frame encoding sequence of increasing in the present invention; Wherein, the nucleic acid molecular weight Marker of M:2kb; 1-12:PCR product.
Fig. 3 is the SDS-PAGE electrophoresis result of Cyanea capillata superoxide-dismutase abduction delivering of recombinating in the present invention; Wherein, M: molecular weight of albumen Marker; 1: the recombination bacillus coli of not inducing; 2: ultrasonic degradation liquid after 1mM IPTG induces; 3: ultrasonic supernatant after 1mM IPTG induces; 4: ultrasound precipitation after 1mM IPTG induces; 5: the recombination bacillus coli of not inducing; 6: ultrasonic degradation liquid after 0.5mM IPTG induces; 7: ultrasonic supernatant after 0.5mM IPTG induces; 8: ultrasound precipitation after 0.5mM IPTG induces; The position of arrow instruction recombinant protein.
Fig. 4 is the SDS-PAGE electrophoresis result of Cyanea capillata superoxide-dismutase separation and purification of recombinating in the present invention; Wherein, 1: the Cyanea capillata superoxide-dismutase recombination bacillus coli before induction; 2: the Cyanea capillata superoxide-dismutase recombination bacillus coli ultrasonic degradation supernatant after induction; 3: during purification of recombinant proteins, penetrate peak; 4: elution peak during purification of recombinant proteins; The position of arrow instruction recombinant protein.
Fig. 5 is for detecting purifying Cyanea capillata superoxide-dismutase to the result of ultra-oxygen anion free radical rejection ability (WST-1 method); Can be observed the rising along with recombinant C uZnSOD protein concentration, in reaction system, the inhibiting rate of superoxide anion obviously increases, and when recombinant C uZnSOD reaches 500 μ g/mL level, superoxide anion is almost completely suppressed.
Embodiment
Describe the present invention below in conjunction with embodiment and accompanying drawing.But the following example should not regard limitation of the scope of the invention as.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
The Cyanea capillata (Cyanea capillata) that the present invention selects gathers from Zhejiang Province's nutrients, and through Aquatic Products Inst. Attached to Jimei Univ. professor qualification (L.Xiao et al, Toxicon, 2009,53:146 – 152).
Embodiment 1: the structure of Cyanea capillata tentacle tissue cDNA library and analysis
1) extracting of Cyanea capillata tentacle total tissue RNA illustrates according to the Trizol test kit of Invitrogen company and carries out, and chloroform removes protein, obtains about 7 μ g total serum IgE;
2) separation of mRNA illustrates according to the Oligotex mRNA Spin-column Kit of QIANGEN company and carries out, and the synthesis of cDNA is then carried out with reference to the SMART cDNA LibraryConstruction Kit explanation of Clontech company;
3) cDNA is inserted pUC19 plasmid vector (purchased from Takara company), be converted in bacillus coli DH 5 alpha (purchased from Beijing Bo Maide company) again, coat 15CM culture dish and carry out blue hickie screening, be namely built into Cyanea capillata tentacle tissue cDNA library.
This library has bacterium colony 1923, wherein locus coeruleus 35, recombination fraction 98.18%, storage capacity 1.92*106, the ordered sequence of intubating length >=400bp 1035, adopt 100bp, 90% principle unigene merger is carried out to these sequences, obtaining unigene number is 528.Carry out total length analysis to sequence simultaneously, have 12 sequences to have associated homologous information in 20 sequences, result is wherein 12 total lengths, and integrity ratio: 12/12 × 100%=100%, shows that this cDNA library has preferable quality.Carry out random sequencing to this cDNA library, gained sequence carries out BLASTx analysis (http://blast.ncbi.nlm.nig.gov) after unloading body.
4) Cyanea capillata superoxide dismutase gene is from the clone being numbered 13F9 in above-mentioned cDNA library.Sequence 745bp, comprises the open reading frame of a 465bp, and coding contains the protein of 154 amino-acid residues, molecular weight 16.0kDa, iso-electric point 6.2.Blastx Search Results shows the superoxide-dismutase very high homology (as Fig. 1) of this albumen and multiple species, is the recruit of jellyfish source superoxide-dismutase family.To its further bioinformatic analysis display, this albumen containing signal peptide, is not non-secreted protein, is positioned tenuigenin.
Embodiment 2: the structure of Cyanea capillata superoxide dismutase recombinant expression plasmid and engineering bacteria restructuring
1) according to the restriction enzyme site of Cyanea capillata superoxide dismutase gene sequence and prokaryotic expression carrier pET24a (purchased from Novagen company), design and synthesize pair of primers, wherein upstream primer comprises restriction enzyme site Nde I (CATATG), downstream primer comprises restriction enzyme site Xho I (CTCGAG), and the sequence of two primers is specific as follows:
Upstream primer: 5 '-GGGAATTCCATATGGCTACATTGAAAGC-3 ' (SEQ IDNO:3)
Downstream primer: 5 '-CGCTCGAGCGCATTGGCTATT-3 ' (SEQ ID NO:4)
After grads PCR experiment, selected 55.5 DEG C is optimum annealing temperature, and carry out PCR to goal gene and increase in a large number, PCR reaction conditions is: 95 DEG C of insulation 5min; 95 DEG C of insulation 30sec, 55.5 DEG C of insulation 30sec, 68 DEG C of insulation 1min, 35 circulations; 68 DEG C of insulation 5min.Obtain the PCR primer that 5 ' end comprises Nde I and Xho I restriction enzyme site respectively after PCR reaction, nucleic acid electrophoresis display PCR primer band is at tram (as Fig. 2), and about 481bp, reclaims PCR primer;
2) according to the Nde I of description of product NEB company and Xho I endonuclease respectively enzyme cut back to close after PCR primer and pET24a prokaryotic expression plasmid;
3) the T4DNA ligase enzyme reclaimed again according to NEB company after digestion products illustrated and carries out ligation, by cultivation on the culture dish coated after ligation product conversion to intestinal bacteria Rosetta (DE3) pLysS (purchased from Beijing Bo Maide company) containing kantlex (100 μ g/ml) and paraxin (34 μ g/ml) 15 hours.Picking list bacterium colony shakes bacterium to the LB substratum containing 5ml with kantlex (100 μ g/ml) and paraxin (34 μ g/ml) and cultivates 12 hours, recombinate successfully through method qualifications such as bacterium liquid PCR, plasmid enzyme restriction checkings, be that sequencing primer carries out two-way order-checking to recombinant plasmid with T7Terminator, gene for the purpose of the gene of checking clone, illustrates that Cyanea capillata superoxide dismutase recombinant expression plasmid correctly builds.
Embodiment 3: the expression of Cyanea capillata superoxide-dismutase
Added in the LB liquid medium containing kantlex (100 μ g/ml) and paraxin (34 μ g/ml) by correct recombination bacillus coli Rosetta (DE3) the pLysS bacterium liquid of order-checking, 37 DEG C, 250rpm shaking table is cultured to OD
600induce for starting to add inductor IPTG during 0.6-0.8.
Determine that the optimum condition of the expression of recombinant protein is: 12 DEG C, induction 10 hours under 0.5mM IPTG, 150rpm.Induction after collected by centrifugation thalline, through SDS-PAGE electrophoresis as seen under this inductive condition recombinant protein can express with soluble form, molecular weight conforms to predictor (after adding His label about 17.2kDa), as shown in Figure 3.
Embodiment 4: the purifying of restructuring Cyanea capillata superoxide-dismutase
Bacterium liquid centrifugal (10000 × g*10min) after induction is collected thalline, then uses binding buffer liquid (NaH
2pO
420mM; NaCl 500mM; Imidazoles 30mM; PH=7.4) abundant resuspended thalline, carries out ultrasonicly splitting bacterium, gets supernatant and carry out purifying after ultrasonic degradation liquid centrifugal (10000 × g*10min).Finally according to the HisTrap HP chromatography column of GE Healthcare company and
the operation instructions of separation and purification system carries out nickel ion affinity chromatograph, namely obtains purer Cyanea capillata superoxide-dismutase.
The visible object band of SDS-PAGE electrophoresis (as Fig. 4) conforms to predicted position, and purity is more than 90%, and the elution requirement of use is: the binding buffer liquid of 50% and the elution buffer (NaH of 50%
2pO
420mM; NaCl 500mM; Imidazoles 500mM; PH 7.4).
Embodiment 5: restructuring Cyanea capillata superoxide-dismutase removes the detection of superoxide anion ability
After obtaining comparatively single target protein by the nickel ion affinity chromatograph method of embodiment 4, superoxide-dismutase (SOD) detection kit (WST-1 method) using colleague's chemistry institute (Dojindo) to develop detects, and operates by shop instruction normal process.
The subsidiary high water soluble tetrazolium salts WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2 hydrogen-tetrazolium salts, disodium salt) of this test kit can with superoxide anion (O
2 -) reaction generates a kind of water miscible dyestuff, but superoxide anion is preferentially occurred by the reaction that SOD suppresses, and therefore can measure the rejection ability of recombinant protein rCcCuZnSOD to ultra-oxygen anion free radical by colorimetry.
Experimental result as shown in Figure 5, can be observed the rising along with recombinant C uZnSOD protein concentration, and in reaction system, the inhibiting rate of superoxide anion obviously increases, and when recombinant C uZnSOD reaches 500 μ g/mL level, superoxide anion is almost completely suppressed.
Illustrate that restructuring Cyanea capillata superoxide-dismutase of the present invention has significant anti-oxidant activity, can be used for exploitation anti-oxidation medicine, antiaging agent, antiradiation drug, shielding medicine for skin, food preservatives etc.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (9)
1. a Cyanea capillata superoxide-dismutase, is characterized in that, described Cyanea capillata superoxide-dismutase has the protein of (I) or (II) as follows:
The protein of the aminoacid sequence composition shown in (I) SEQ ID NO:2;
Aminoacid sequence shown in (II) SEQ ID NO:2 is substituted, lacks and/or adds one or several amino acid and the protein derivative by (I) of same function.
2. a kind of Cyanea capillata superoxide-dismutase according to claim 1, it is characterized in that: described Cyanea capillata superoxide-dismutase is the aminoacid sequence shown in SEQ ID NO:2, this albumen is not containing signal peptide, for non-secreted protein, be positioned tenuigenin, molecular weight is 16.0kDa, and iso-electric point is 6.2.
3. an encoding gene for Cyanea capillata superoxide-dismutase as claimed in claim 1, is characterized in that, this encoding gene is the DNA molecular of following (I) or (II):
Nucleotide sequence shown in (I) SEQ ID NO:1;
The nucleotide sequence of nucleotide sequence homology more than 80% shown in (II) Yu SEQ ID NO:1.
4. the encoding gene of a kind of Cyanea capillata superoxide-dismutase according to claim 3, it is characterized in that: the encoding gene of described Cyanea capillata superoxide-dismutase, this nucleotide sequence total length 745bp, comprises the open reading frame of a 465bp.
5. a preparation method for Cyanea capillata superoxide-dismutase as claimed in claim 2, it is characterized in that, the method comprises the steps:
(A) Cyanea capillata tentacle tissue cDNA library is built;
(B) sequence of above-mentioned Cyanea capillata tentacle tissue cDNA library recombinant clone is measured and analyzed, obtain the nucleotide sequence of coding Cyanea capillata superoxide-dismutase according to claim 3; Use PCR method to organize CDNA library from Cyanea capillata tentacle to increase to this sequence;
(C) expression plasmid of Cyanea capillata superoxide-dismutase is built, recombinant bacterial strain;
(D) expression of Cyanea capillata superoxide-dismutase;
(E) purifying of restructuring Cyanea capillata superoxide-dismutase.
6. the preparation method of Cyanea capillata superoxide-dismutase according to claim 5, is characterized in that: the expression plasmid of the structure Cyanea capillata superoxide-dismutase in described step (C), recombinant bacterial strain, and concrete steps are:
A) PCR primer is designed and synthesized as follows:
Upstream primer as shown in SEQ ID NO:3,
Downstream primer as shown in SEQ ID NO:4,
PCR reaction conditions is: 95 DEG C of insulation 5min; 95 DEG C of insulation 30sec, 55.5 DEG C of insulation 30sec, 68 DEG C of insulation 1min, 35 circulations; 68 DEG C of insulation 5min;
B) enzyme cut back to close after PCR primer and pET24a prokaryotic expression plasmid;
C) utilize two restriction enzyme sites to be connected to by encoding sequence on pET24a carrier, build recombinant expression plasmid, be then transformed into intestinal bacteria Rosetta and obtain recombinant bacterial strain.
7. the preparation method of Cyanea capillata superoxide-dismutase according to claim 5, is characterized in that: the expression condition of the Cyanea capillata superoxide-dismutase in described step (D) is: 12 DEG C, induction 10 hours in 0.5mMIPTG, 150rpm environment.
8. a Cyanea capillata superoxide-dismutase as claimed in claim 1 or 2 is preparing the application in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives or antiaging agent.
9. the encoding gene of a Cyanea capillata superoxide-dismutase as claimed in claim 3 is preparing the application in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives or antiaging agent.
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| CN107828749A (en) * | 2017-12-15 | 2018-03-23 | 中国科学院深海科学与工程研究所 | A kind of low temperature resistant superoxide dismutase MnSOD in deep-sea sea cucumber source and preparation method thereof |
| CN107955806A (en) * | 2017-12-15 | 2018-04-24 | 中国科学院深海科学与工程研究所 | A kind of preparation method and applications of the superoxide dismutase Cu, ZnSOD in abyss sea cucumber source |
| CN112480224A (en) * | 2020-11-19 | 2021-03-12 | 中国人民解放军海军特色医学中心 | Marine-derived antioxidant protein of fusion expression cell-penetrating peptide and preparation method and application thereof |
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| CN102807987A (en) * | 2012-08-27 | 2012-12-05 | 辽宁省海洋水产科学研究院 | Meretrix intracellular copper zinc superoxide dismutase gene and application thereof |
| CN103255113A (en) * | 2013-05-21 | 2013-08-21 | 中国人民解放军第二军医大学 | Cyanea capillata peroxide reductase as well as encoding gene and application thereof |
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| CN102807987A (en) * | 2012-08-27 | 2012-12-05 | 辽宁省海洋水产科学研究院 | Meretrix intracellular copper zinc superoxide dismutase gene and application thereof |
| CN103255113A (en) * | 2013-05-21 | 2013-08-21 | 中国人民解放军第二军医大学 | Cyanea capillata peroxide reductase as well as encoding gene and application thereof |
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| 陆佳等: "发形霞水母（Cyanea capillata）触手cDNA文库与蛋白组", 《中国优秀硕士学位论文全文数据库 农业科学辑（月刊）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107828749A (en) * | 2017-12-15 | 2018-03-23 | 中国科学院深海科学与工程研究所 | A kind of low temperature resistant superoxide dismutase MnSOD in deep-sea sea cucumber source and preparation method thereof |
| CN107955806A (en) * | 2017-12-15 | 2018-04-24 | 中国科学院深海科学与工程研究所 | A kind of preparation method and applications of the superoxide dismutase Cu, ZnSOD in abyss sea cucumber source |
| CN112480224A (en) * | 2020-11-19 | 2021-03-12 | 中国人民解放军海军特色医学中心 | Marine-derived antioxidant protein of fusion expression cell-penetrating peptide and preparation method and application thereof |
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