CN103232979B - Cyanea capillata thioredoxin and coding gene and application thereof - Google Patents
Cyanea capillata thioredoxin and coding gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine and provides a cyanea capillata thioredoxin. Currently, researches and reports related to jellyfish thioredoxin are not seen yet. The cyanea capillata thioredoxin provided by the invention has protein formed by an amino acid sequence shown in SEQ ID NO: 2. The invention further provides a coding gene of the cyanea capillata thioredoxin, wherein the coding gene has a nucleotide sequence shown in SEQ ID NO: 1. Meanwhile, the invention further provides application of the cyanea capillata thioredoxin and the coding gene of the cyanea capillata thioredoxin in preparation of anti-oxidation drugs, anti-radiation drugs, skin protectants, food preservatives, anti-aging drugs and the like. The cyanea capillata thioredoxin and the coding gene of the cyanea capillata thioredoxin have greater clinical application values.
Description
Technical field
The invention belongs to biological medicine technology field, be specifically related to a kind of Cyanea capillata Trx and encoding gene 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 the new functional protein that some are active strongly, to have good DEVELOPMENT PROSPECT.Jellyfish class is swum in the dark seawater surface of 4-6 rice, lives in for a long time under strong optical radiation environment.Oxidative damage is that uv-radiation causes one of important mechanisms of body injury, there are some researches show, planktonic organism is avoid or reduce the infringement that uv-radiation causes, and selects through long adaptation, has produced anti-oxidation active substance of a great variety, that structure is special, activity is strong in its body.Flourishing antioxidant system is avoided playing an important role in light and photopigment equivalent damage at Cell protection and organism, can protect the radiation of high light ultraviolet to organism, removes the free radical producing in radioreaction.Studies have found that, Cyanea capillata tentacle extract has very strong antioxidation activity in vitro, strong more than conventional antioxidant vitamin C to the removing ability of active chalcogen.Also from jellyfish Rhopilema esculentum, Stomolophus meleagris, isolate respectively the multiple albumen with obvious removing free radical function by the method such as ammonium sulfate precipitation, gel permeation chromatography.These results all show, medusa contains active strong anti-oxidant activity component, is important sources anti-oxidant, anti-radiation active substances.However, the at present not yet systematic understanding such as the sequence information of the composition to antioxidant system in medusa, important antioxidant reductase (albumen), expression, anti-oxidant activity level.
Trx (Thioredoxin, Trx) is that a class is extensively present in the small molecules redox protein as hydrogen carrier in various organisms, and molecular weight is 10～12kDa, all contains the active structure site WCGPC of a high conservative.It is two sulfydryls that Trx can make the disulfide bond reduction of albumen, thereby multiple bioprocesses in body is regulated, comprise regulate transcribe factor D NA in conjunction with activity, cell anti-oxidation stress, apoptosis process, anti-coercion, antitumor, Promote immunity is replied etc.Trx and thioredoxin reductase, NADPH form thioredoxin system jointly; can the protein of oxidized damage in body be reduced and be repaired with again folding; plurality of enzymes vigor in control agent, is changed at protection body reply oxidative stress, is kept the aspects such as cell homeostasis to bring into play the effect of important reduction carrier by reversible cystine linkage and mercaptan.In addition,, the in the situation that of the excessive generation of some active oxygens, the expression amount of Trx also rolls up, and reduces or avoid the oxidative damage of body as active oxygen scavenger in organism.Cause because of aging that at body active oxygen increase causes in apoptotic process, Trx can also be in conjunction with regulation and control some cytokine, inhibited apoptosis and body aging.
At present, there is not yet the relevant research report to jellyfish Trx both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of Cyanea capillata Trx and encoding gene thereof, another object of the present invention is to provide this Cyanea capillata Trx in the application of preparing in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives, antiaging agent etc.
The present invention passes through to build Cyanea capillata tentacle tissue cDNA library, and analysis is measured and annotated to the sequence of this library recombinant clone, has obtained the encoding gene of Cyanea capillata Trx.This Cyanea capillata Trx is the jellyfish similar thioredoxin with obvious anti-oxidant activity of finding first, is the important activity component of antioxidant system in medusa, and 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, it checked order and screened, and obtains the encoding gene of Cyanea capillata Trx, and its anti-oxidant activity is studied.
A first aspect of the present invention, provides a kind of Cyanea capillata Trx, and described a kind of Cyanea capillata Trx has the protein of (I) or (II) as follows:
(I) has the protein of the aminoacid sequence composition shown in SEQ ID NO:2;
Amino acid order shown in (II) SEQ ID NO:2 be substituted, lack and/or add one or several amino acid and same function by (I) derivative protein.
Described a kind of Cyanea capillata Trx, has the aminoacid sequence as shown in SEQ ID NO:2, and this albumen, containing signal peptide, for non-secretory protein, is not positioned tenuigenin, and molecular weight is 11.5kDa, and iso-electric point is 5.1.
The present invention also provides a kind of encoding gene of Cyanea capillata Trx, the DNA molecular for following (I) or (II):
(I) has the nucleotide sequence shown in SEQ ID NO:1;
(II) and the nucleotide sequence of the nucleotide sequence homology shown in SEQ ID NO:1 more than 80%.
The encoding gene of described a kind of Cyanea capillata Trx, this nucleotide sequence total length 479bp, the open reading frame that comprises a 315bp, as shown in SEQ ID NO:1.
A second aspect of the present invention, provides a kind of preparation method of Cyanea capillata Trx, and the method comprises the steps:
(1) build Cyanea capillata tentacle tissue cDNA library;
(2) sequence of above-mentioned Cyanea capillata tentacle tissue cDNA library recombinant clone is measured and analyzed, obtain the nucleotide sequence of a coding Trx;
(3) expression plasmid of structure Cyanea capillata Trx, recombinant bacterial strain;
(4) expression of Cyanea capillata Trx;
(5) purifying of restructuring Cyanea capillata Trx.
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, synthetic Cyanea capillata tentacle tissue cDNA;
3) above-mentioned cDNA is inserted to 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 built into Cyanea capillata tentacle tissue cDNA library.
The expression plasmid of the structure Cyanea capillata Trx in described step (3), recombinant bacterial strain, concrete steps are:
1) according to the restriction enzyme site of Cyanea capillata thioredoxin gene sequence and prokaryotic expression carrier pET24a, design a pair of PCR primer with specificity restriction enzyme site Nde I and Xho I, as follows:
Upstream primer: 5 '-GCGGGAATTCCATATGGTTAGAGA-3 '
Downstream primer: 5 '-CCGCTCGAGTTTATGACTCTTAATC-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, 30 circulations; 68 DEG C of insulation 5min;
2) PCR product and the pET24a prokaryotic expression plasmid of enzyme after cutting back to close;
3) utilize two restriction enzyme sites that encoding sequence is connected on pET24a carrier, build recombinant expression plasmid, be then transformed into e. coli bl21 (DE3) and obtain recombinant bacterial strain.
The expression condition of the Cyanea capillata Trx in described step (4) is: under 25 DEG C, 1mM IPTG, 150rpm/min, induce 8 hours, under this inductive condition, recombinant protein is mainly expressed with soluble form, and expression amount accounts for the more than 50% of total protein concentration.
Purifying in described step (5) is for adopting nickel ion affinity chromatograph post single step purification and get final product, and elution requirement is to account for respectively the binding buffer liquid of cumulative volume 50% and 50% elution buffer; Wherein, binding buffer liquid is NaH
2pO
420mM; NaCl500mM; Imidazoles 30mM; PH7.4, elution buffer is NaH
2pO
420mM; NaCl500mM; Imidazoles 500mM; PH7.4.
A third aspect of the present invention, provides Cyanea capillata Trx and encoding gene thereof in the application of preparing in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives, antiaging agent.
The present invention, by building Cyanea capillata tentacle tissue cDNA library, adopts BLASTx analytical method, has obtained the sequence of a coding Trx, comprises the nucleotide sequence shown in sequence table SEQ ID NO.1.This nucleotide sequence total length 479bp, the open reading frame that comprises a 315bp, the protein containing 105 amino-acid residues of encoding, i.e. aminoacid sequence shown in sequence table SEQ ID NO.2.This albumen, containing signal peptide, for non-secretory protein, is not positioned tenuigenin, has typical Trx functional domain.
The present invention is by building the RT-PCR expression vector of thioredoxin gene, recombinant expression vector is transformed to e. coli host cell, screening obtains expressing the recombinant bacterium of Cyanea capillata Trx, after abduction delivering, utilize affinity chromatography purifying to obtain recombinant protein, this preparation method is simple, with low cost.
The restructuring Cyanea capillata Trx that the present invention obtains has significant anti-oxidant activity.In the experiment of Regular Insulin reduction method, in the situation that DTT exists, the disulfide linkage of INSULIN A, B interchain can be reduced and rupture by above-mentioned Trx, two chains dissociate, because the lower reaction solution that makes of solubleness of B chain becomes muddy, at 655nm place, light absorption value obviously raises, and the concentration of restructuring Cyanea capillata Trx is higher, light absorption value rises faster, illustrates that it has obvious disulfide bond reduction ability.In addition, above-mentioned Trx also has ABTS radical scavenging activity, and resistance of oxidation is 6.5 times of vitamins C Equivalent Trolox.Therefore the Cyanea capillata Trx, relating in the present invention will have very large using value aspect exploitation anti-oxidation medicine, antiaging agent, antiradiation drug, shielding medicine for skin, food preservatives.
Brief description of the drawings
Fig. 1 is the sequence multiple ratio pair of Cyanea capillata Trx and other species Trxs; Wherein,
Black represents the region of complete homology, and square frame has marked the active structure site WCGPC of its high conservative.
Fig. 2 is the PCR product nucleic acid electrophoresis result of Cyanea capillata Trx open reading frame encoding sequence of increasing in the present invention; Wherein, the nucleic acid molecular weight Marker of M:2kb; 1:PCR product.
Fig. 3 is the SDS-PAGE electrophoresis result of Cyanea capillata Trx abduction delivering of recombinating in the present invention;
Wherein, M: molecular weight of albumen Marker; 1: the not pET24a intestinal bacteria of induction; 2: through the pET24a intestinal bacteria of induction; 3: the Cyanea capillata Trx recombination bacillus coli before induction; 4: the Cyanea capillata Trx recombination bacillus coli after induction; 5: the Cyanea capillata Trx recombination bacillus coli ultrasonic degradation supernatant after induction; 6: the Cyanea capillata Trx recombination bacillus coli ultrasonic degradation precipitation after induction;
Fig. 4 is the SDS-PAGE electrophoresis result of Cyanea capillata Trx separation and purification of recombinating in the present invention;
Wherein, 1: the Cyanea capillata Trx recombination bacillus coli before induction; 2: the Cyanea capillata Trx recombination bacillus coli ultrasonic degradation supernatant after induction; 3: when purification of recombinant proteins, penetrate peak; 4: elution peak when purification of recombinant proteins;
Fig. 5 is for detecting purifying Cyanea capillata Trx resistance of oxidation (ABTS
+free radical scavenging method) time the typical curve (y=-0.0005x+0.2979, the R that make of vitamins C Equivalent Trolox reaction
2=0.9976).
Fig. 6 detects the graphic representation of purifying Cyanea capillata Trx to INSULIN A, B interchain disulfide bond reductive action ability.
Embodiment
Describe the present invention below in conjunction with embodiment and accompanying drawing.But the following example should not regarded limitation of the scope of the invention as.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
The Cyanea capillata Trx making with embodiment 1 carries out the experiment of embodiment 2-3.
The Cyanea capillata (Cyanea Capillata) that the present invention selects gathers from Zhejiang Province's nutrients, and through the professor of Aquatic Products Inst. Attached to Jimei Univ. qualification (L.Xiao et al, Toxicon, 2009,53:146 – 152).
Embodiment 1: the screening of Cyanea capillata Trx and preparation.
1. structure and the analysis in Cyanea capillata tentacle tissue cDNA library
1) extracting of Cyanea capillata tentacle total tissue RNA is carried out according to the Trizol reagent explanation of Invitrogen company, and chloroform is removed protein, obtains the total RNA of approximately 7 μ g;
2) separation of mRNA is carried out according to the Oligotex mRNA Spin-column Kit explanation of QIANGEN company, and the synthetic of cDNA carries out with reference to the SMART cDNA Library Construction Kit explanation of Clontech company;
3) cDNA is inserted to pUC19 plasmid vector (purchased from Takara company), then be converted in bacillus coli DH 5 alpha (purchased from Beijing Bo Maide company), coat 15CM culture dish and carry out blue hickie screening, be built into Cyanea capillata tentacle tissue cDNA library.
1923 of the total bacterium colonies in this library, wherein 35 of locus coeruleus, recombination fraction 98.18%, storage capacity 1.92*106,1035 of the ordered sequences of intubating length >=400bp, adopt 100bp, 90% principle to carry out unigene merger to these sequences, and obtaining unigene number is 528.Sequence is carried out to total length analysis simultaneously, have 12 sequences to have relevant homologous information in 20 sequences, result is 12 total lengths wherein, and integrity ratio: 12/12 × 100%=100%, shows that this cDNA library has preferable quality.This cDNA library is carried out to random sequencing, and institute's calling sequence carries out BLASTx analysis after unloading body
（http://blast.ncbi.nlm.nig.gov）。
2. the analysis of Cyanea capillata thioredoxin gene sequence
Cyanea capillata thioredoxin gene is from the clone who is numbered 2F09 in above-mentioned cDNA library.Sequence total length 479bp, the open reading frame that comprises a 315bp, coding is containing the protein of 105 amino-acid residues, molecular weight 11.5kDa, iso-electric point 5.1.Blastx Search Results shows the Trx height homology (as Fig. 1) of this albumen and multiple species, is the recruit of jellyfish source Trx family.
Its further bioinformatic analysis is shown, this albumen does not contain signal peptide, for non-secretory protein, is positioned tenuigenin.
3. the structure of Cyanea capillata Trx recombinant expression plasmid and engineering bacteria restructuring
1) according to Cyanea capillata thioredoxin gene sequence and prokaryotic expression carrier pET24a(purchased from
Novagen company) restriction enzyme site, 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), the sequence of two primers is specific as follows:
Upstream primer: 5 '-GCGGGAATTCCATATGGTTAGAGA-3 ' (SEQ ID NO:3)
Downstream primer: 5 '-CCGCTCGAGTTTATGACTCTTAATC-3 ' (SEQ ID NO:4)
After grads PCR experiment, selected 55.5 DEG C is optimum annealing temperature, goal gene is carried out to PCR and increase in a large number, and PCR reaction conditions is:
Obtain the PCR product that 5 ' end comprises respectively Nde I and Xho I restriction enzyme site, nucleic acid electrophoresis shows that PCR product band is in tram (as Fig. 2), and about 334bp, reclaims PCR product;
2) according to the Nde I of NEB company and Xho I endonuclease PCR product and the pET24a prokaryotic expression plasmid of enzyme after cutting back to close respectively for the description of product;
3) reclaim enzyme and cut after product the T4DNA ligase enzyme explanation according to NEB company again and carry out ligation, ligation product is converted into after e. coli bl21 (DE3) (purchased from Beijing Bo Maide company) and coats on the culture dish containing kantlex (100 μ g/ml) and cultivate 15 hours.Picking list bacterium colony shakes bacterium to the LB substratum containing 5ml with (100 μ g/ml) kantlex and cultivates 12 hours, after the method qualifications such as bacterium liquid PCR, plasmid enzyme restriction checking are recombinated successfully, taking T7Terminator as sequencing primer, recombinant plasmid is carried out to two-way order-checking, checking clone's gene is goal gene, illustrates that Cyanea capillata Trx recombinant expression plasmid correctly builds.
4. the expression of Cyanea capillata Trx
Correct recombination bacillus coli BL21 (DE3) the bacterium liquid of order-checking is added containing in the liquid LB substratum of (100 μ g/ml) kantlex, and 37 DEG C, 250rpm/min shaking table is cultured to OD
600during for 0.6-0.8, start to add inductor IPTG to induce.
The optimum condition of the expression of determining recombinant protein is: under 25 DEG C, 1mM IPTG, 150rpm/min, induce 8 hours.Centrifugal collection thalline after induction, through SDS-PAGE electrophoresis as seen under this inductive condition recombinant protein mainly express with soluble form, and expression amount accounts for the more than 50% of total protein, molecular weight and predictor (add His label after about 12.6kDa) conform to, as shown in Figure 3.
5. the purifying of restructuring Cyanea capillata Trx
Bacterium liquid centrifugal (10000 × g*10min) after induction is collected to thalline, then use binding buffer liquid (NaH
2pO
420mM; NaCl500mM; Imidazoles 30mM; PH=7.4) abundant resuspended thalline, carries out the ultrasonic bacterium of splitting, and 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 is carried out nickel ion affinity chromatograph, obtains purer Cyanea capillata Trx.
The visible object band of SDS-PAGE electrophoresis (as Fig. 4) conforms to predicted position, and purity is more than 95%, and the elution requirement of use is: 50% binding buffer liquid and 50% elution buffer (NaH
2pO
420mM; NaCl500mM; Imidazoles 500mM; PH7.4).
Embodiment 2: restructuring Cyanea capillata Trx resistance of oxidation detects (ABTS
+free radical scavenging method)
Obtain after comparatively single target protein by nickel ion affinity chromatograph method, the illustration method of the resistance of oxidation detection kit of producing according to green skies company, measures the Cyanea capillata Trx resistance of oxidation of purifying.First toward the ABTS that adds 200 μ l in all detections hole of 96 orifice plates
+solution, then add 10 μ l elutriants, typical curve group to add respectively the vitamins C Equivalent Trolox standardized solution of 10 μ l different concns, the Cyanea capillata Trx that sample sets adds the purifying of 10 μ l different concns toward blank group, mix, after 2～6 minutes, measure A750 in incubated at room.Obtain typical curve as shown in Figure 5.
Calculate 6.5 times that the resistance of oxidation of Cyanea capillata Trx is Trolox according to typical curve.
Embodiment 3: the detection of restructuring Cyanea capillata Trx disulfide bond reduction ability
The method that this experiment is introduced with reference to Holmgren (J.Boil.Chem, 1979,254:9627-9632.) is carried out.In 1ml reaction buffer, contain the Sigma I8405 that concentration is 1.25mg/ml, 0.1M PBS, 2mMEDTA, to adding in blank group 100 μ l elutriants, sample detection group to add 100 μ l concentration to be respectively the restructuring Cyanea capillata Trx of 4 μ M, 8 μ M, 12 μ M, then to add final concentration in each reaction solution be the DDT of 2mmol/l and mix to start reaction.Start immediately to measure an A655 every 1min, according to absorbance value over time, make response curve figure (as Fig. 6).
Result shows, the A655 curve of control group is very mild and it is worth close to zero, and each sample detection curve has the trend increasing in time and rise, and the larger rising of concentration is faster, illustrates that purifying protein has obvious disulfide bond reduction ability.
More than show and described 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; that in above-described embodiment and specification sheets, describes 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.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (8)
1. a Cyanea capillata Trx, is characterized in that, the aminoacid sequence of described Cyanea capillata Trx is as shown in SEQ ID NO:2.
2. an encoding gene for Cyanea capillata Trx as claimed in claim 1, is characterized in that, the nucleotide sequence of this encoding gene is as shown in SEQ ID NO:1.
3. a preparation method for Cyanea capillata Trx as claimed in claim 1, is characterized in that, the method comprises the steps:
(A) build Cyanea capillata tentacle tissue cDNA library, described Cyanea capillata collection is from Zhejiang Province's nutrients;
(B) sequence of above-mentioned Cyanea capillata tentacle tissue cDNA library recombinant clone is measured and analyzed, obtain the nucleotide sequence of a coding Trx;
(C) expression plasmid of structure Cyanea capillata Trx, recombinant bacterial strain;
(D) expression of Cyanea capillata Trx;
(E) purifying of restructuring Cyanea capillata Trx.
4. the preparation method of Cyanea capillata Trx according to claim 3, is characterized in that: the Cyanea capillata tentacle tissue cDNA library in described step (A) builds by the following method:
A) extracting Cyanea capillata tentacle total tissue RNA;
B) separating mRNA, synthetic Cyanea capillata tentacle tissue cDNA;
C) above-mentioned cDNA is inserted to 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 built into Cyanea capillata tentacle tissue cDNA library.
5. the preparation method of Cyanea capillata Trx according to claim 3, is characterized in that: the expression plasmid of the structure Cyanea capillata Trx in described step (C), and recombinant bacterial strain, concrete steps are:
A) design and synthesize PCR primer 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, 30 circulations; 68 DEG C of insulation 5min;
B) PCR product and the pET24a prokaryotic expression plasmid of enzyme after cutting back to close;
C) utilize two restriction enzyme sites that encoding sequence is connected on pET24a carrier, build recombinant expression plasmid, be then transformed into e. coli bl21 and obtain recombinant bacterial strain.
6. the preparation method of Cyanea capillata Trx according to claim 3, is characterized in that: the expression condition of the Cyanea capillata Trx in described step (D) is: under 25 DEG C, 1mM IPTG, 150rpm/min, induce 8 hours.
7. a Cyanea capillata Trx as claimed in claim 1 is in the application of preparing in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives or antiaging agent.
8. the encoding gene of a Cyanea capillata Trx as claimed in claim 2 is in the application of preparing in anti-oxidation medicine, antiradiation drug, shielding medicine for skin, food preservatives or antiaging agent.
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