CN100489093C

CN100489093C - Protein and nuclear acid of antrodia camphoratu antioxidase and preparation method and use thereof

Info

Publication number: CN100489093C
Application number: CNB2005100898699A
Authority: CN
Inventors: 林棋财
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-08-09
Filing date: 2005-08-09
Publication date: 2009-05-20
Anticipated expiration: 2025-08-09
Also published as: CN1912113A

Abstract

The invention relates to antrodia camphorate antioxidase protein and nucleic acid contains Mn-SOD, 1-Cys Prx, 2-Cys Prx amino acid sequence with sequence identification number 1, 3, 5 and its nucleic acid. The invention aloes relates to its manufacturing method and use.

Description

The protein of camphor tree sesame antioxidase and nucleic acid and manufacture method and purposes

Technical field:

The present invention relates to a kind of protein, amino acid and nucleic acid thereof, Nucleotide of camphor tree sesame antioxidase, and can be for making this protein expression carrier, host cell and manufacture method, and contain this proteinic medical composition.

Background technology:

Antioxidase comprises sudismase, and (superoxide dismutase, SOD:EC1.15.1.1) and peroxidation reductase enzyme (peroxiredoxin, Prx, for example 1-Cys Prx and 2-Cys Prx etc.), they are to keeping healthy playing an important role.In 2003, Gems and McElweeShi point out that longevity gene is to comprise multiple antioxidase and heat shock protein(HSP), these antioxidases and albumen can recover the configuration and the activity of the protein (misfolded proteins) of undesired folding, and help ILS.The same year, people such as NeumannShi point out that Prx will cause serious hemolytic anemia and cancerization to cause the life-span to be reduced sharply as above-mentioned antioxidase is lost activity in mouse (mice), it serves to show that life-span, health and detoxifcation to organism play an important role, be worth the research division as follows:

Sudismase SOD: its function is to get rid of O ₂ ^-, have report to point out that SOD is applied in traumatic wounds energy healing acceleration and removes face's blackspot, freckle.Again, SOD has and regulates the function (Chang, et al., 2001) that neurocyte produces IL-1 β, NO and TNF-α, so SOD plays an important role in healthy keeping.SOD is present in aerobic or anaerobic eucaryon or the prokaryotic organism, superoxide radical can be catalyzed into oxygen molecule or hydrogen peroxide, and the organism opposing toxicity destruction that oxygen is brought and the function of injury are provided, and its reaction formula is as follows: 2O ₂ ^-+ 2H ⁺→ H ₂O ₂+ O ₂SOD is metallic ferment albumen, unusual copper zinc type, manganese type and three types of (Bannister of swage of being divided into according to its activity site institute bonded metal cofactor, et al., 1987), these three kinds of dissimilar SOD are distributed in different species and different positions.American scholar McCord in 1969 and Fridovich find that the protein of this kind cupric zinc has the function of removing superoxide radical, therefore with its called after SOD, find to have the existence of Cu/Zn-SOD subsequently successively in various organisms.In eukaryotic cell, with sweet potato and pawpaw Cu/Zn-SOD is example, secondly the cell cube molecular weight is 19kDa, the protein that purifying comes out is light blue, and stable between pH2.2～12, heat-resistant quality is very high, following its activity (Lin that still can keep of the environment of the imidazoles (imidazole) of 4% SDS or 2M, etal., 1995,1999).By the Cu/Zn-SOD protein molecular of X-optical lattice diffraction bovine (bovine), finding has three positively charged amino acid to form positively charged active channel, is respectively Arg ¹⁴¹, Lys ¹²⁰, Lys ¹³⁵, cupric ion in the position in active centre be by four His ( ^44,46,61,118) imidazoles cyclic group (imidazolering) form coordinate bond, zine ion then is with His ⁶¹Link as bridge and cupric ion, the amino-acid residue of other and zine ion bond still has His ⁶¹, His ⁶⁹, His ⁷⁸And Asp ⁸¹The endogenous cystine linkage of above-mentioned Cu/Zn-SOD protein molecular is positioned at Cys ⁶⁰And Cys ¹⁶¹(Steinman, et al., 1974; Schinina, etal., 1996).The speed of reaction of SOD and superoxide radical is exceedingly fast, almost approach the limit of diffusion, but the cupric ion in active centre is exposed to the area of solvent not as good as 0.1% of ferment surface-area, mostly Cu/Zn-SOD protein sub-cell surface is electronegative, so because repulsion of same sex electric charge, same electronegative superoxide radical is delivered to the active centre of positively charged, carry out the catalyzed reaction (Tainer of ferment, et al., 1983), thus electrostatic force be considered to promoter action is arranged to guiding electronegative superoxide radical to arrive the active centre.Cupric ion has been played the part of two kinds of roles of oxidation and reduction in disproportionation reaction, and zine ion does not participate in katalysis, and its function mainly is the structure at stable whole ferment.If when replacing cupric ion with other metal ion, ferment will lose activity, but if with Co ²⁺, Hg ²⁺Or Cd ²⁺Even room replacement zine ion, then the ferment molecule still has part activity (Fridovich, 1986).Mn-SOD separated in by intestinal bacteria in 1970 and is incarnadine, mainly was arranged in prokaryotic cell prokaryocyte and eukaryotic grain line body.Activity of proteins is along with the valence mumber of sub-cell body active centre mn ion changes and changes, the catalytic capability that when the positive divalent charge of active centre mn ion band, has ferment, when positive three valence charges of active centre mn ion band, then be resting state, do not have the catalytic capability of ferment.Mn-SOD mostly is the diploid of being made up of two protein sub-cell bodies, but also finds to have the existence of quaternary body even triplet.Mn-SOD with the T.thermophilus bacterium is an example, and the about 19～22kDa of its every cell cube molecular weight contains a mn ion in each protein cell cube.The Mn-SOD of this bacterium is by His ²⁶, His ⁸¹, Asp ¹⁷⁵, His ¹⁷⁹With mn ion bond (William, et al., 1984).Fe-SOD is distributed in the protokaryon bacterial cell matrix, in algae, some higher plant and the chloroplast(id) thereof, yet do not find all that in all animal tissuess Fe-SOD exists, but then finding in some higher plant tissues has Fe-SOD to exist, the floral organ of the rape of ginkgo (Ginkgo biloba), tomato, Cruciferae, paddy rice (Pan and Yan, 1991) for example.It is faint yellow that Fe-SOD is, similar (William, et al., 1984 to Mn-SOD on nucleic acid and aminoacid sequence; Wayne, et al., 1991), relatively poor to heat and pH value stability, not suppressed by prussiate (cyanide), but to hydrogen peroxide then than passivity power, and have significantly different with the aminoacid sequence of Cu/Zn-SOD.Fe-SOD normally is made up of two protein sub-cell bodies, about the about 18.5～22kDa of the molecular weight of each protein cell cube, but discovery is also arranged with quaternary body (Lim, et al., 1997) form exists, and contains 0.5～1 iron ion in each cell cube.With intestinal bacteria E.coli is example, and its Fe-SOD is with His ²⁷, His ⁸¹, Asp ¹⁶³, His ¹⁶⁷With iron ion bond (Lim, et al., 1997).The activity of ferment changes along with the valence mumber of cell cube active centre iron ion.When iron ion band positive divalence in active centre is electric, have the catalytic capability of ferment, ferment presents dormant state when being with positive trivalent electric.

Peroxidation reductase enzyme Prx: found to have seven kinds of gene products in the buds in Arabic mustard such as HorlingShi in 2003, adapt to multiple redox environment, divide another name 2-Cys PrxA, 2-Cys PrxB, PrxQ, PrxIIA, PrxIIB, PrxIIC, PrxIIE, especially PrxIIB is brought out by tB oxygenant (tertiarybutylhydroperoxide), and PrxQ is to the easiest being induced of processing of superoxide (peroxide) and diamine compound (diamide).People such as CastroShi report in other 2002, parasite (Leishmania infantum) can strengthen H because of the expression (expression) of 2-Cys Prx ₂O ₂Phylactic power defensive power with the tB oxygenant.The about 21kDa of its molecular weight.

Peroxidation reductase enzyme Prx major function is being got rid of biological intravital superoxide such as H ₂O ₂, 3-butylperoxide (t-butylperoxide), cumene peroxide (cumene peroxide) etc., these superoxide such as untimely removing, then can be to organism (as protein, lipid, and nucleic acid) cause many injuries, accumulating these materials that are hurt is the reasons that cause aging or neurodegenerative disease.By protein sequence relatively, according to the halfcystine position (cysteine residues) of conserved regions, peroxidation reductase enzyme Prx can be divided into two big groups: (1), 1-Cys Prx lack Cys ¹⁷⁰And only has Cys ⁴⁷Conserved regions.(2), 2-Cys Prx has Cys ⁴⁷And Cys ¹⁷⁰Two conserved regions.People such as KawazuShi are cloned into 2-Cys Prx and 1-Cys Prx respectively from malaria protozoon (P.falciparum).

Peroxidation reductase enzyme Prx is a kind of anti-oxidant albumen of novelty, belong to a member in the family of peroxidase peroxidase, the about 25KDa of molecular weight, its with the thioredoxin of thioredoxin system (Trx) system (Trx, Trx reductase and NADPH) as the electronics supplier.Peroxidation reductase enzyme Prx can protect some ferment system recovery activity that are subject to free radical injury (for example, via the metal-catalyzed oxidation systems of metal catalytic free-radical generating system: vitamin C oxidation system ascorbateoxidation system (Fe ³⁺, O ₂And ascorbate) and sulphur oxidation system thiol oxidation system (Fe ³⁺, O ₂And RSH)), in addition, peroxidation reductase enzyme Prx is the multiple function of tool still: translate regulation and control, and apoptosis, immunizing power and infectivity, the most apparent is its antioxidation property.Prx is according to aminoacid sequence, and tissue distribution and cell position can be divided into 5 big groups (Gourlay, et al.2003) at least.

The generation of active oxygen ROS (reactive oxygen speceies) mainly is to be to breathe or external sunlight, the incomplete reduction of the redox of radiation and medicine and the phagocytic cell of stimulation of host (phagocytes) oxygen that causes.The increase of ROS and the increase of oxidative pressure; the mechanism of biological all anti-oxidant pressure of tool such as antioxidase and anti-oxidant molecule such as SOD, catalase (catalase), peroxidase (peroxidase), thioredoxin (thioredoxin) and Selenoperoxidase (glutathione peroxidase) are protected, in order to avoid be subjected to the injury of ROS.

Peroxidation reductase enzyme Prx mainly is reduction H ₂O ₂And alkyl peroxide (alkyl hydroperoxide) becomes water and alcohols (Jeon and Ishikawa, 2003).Cell also can produce active sulphur (reactive sulfurspecies, RSS) as thionyl free radical (thinyl radicals, RS), two sulphur free radical anion (disulfideradical anions, RS SR) and peroxide sulfanilamide (SN) free radical (peroxysulfenyl radicals, RSOO.).Prx also can get rid of active sulphur, with the injury of avoiding active sulphur to cause.

Camphor tree sesame (Antrodia camphorata) has another name called Antrodia camphorata, Cinnamomum kanahirai hay mushroom (Zang Hesu, 1990), and report is pointed out the treatment of liver tumor and tumor of cervix very effective, because of can only growing in Taiwan Cinnamomum kanahirai hay tree, so among the people to look the camphor tree sesame be jewellery.Because of the camphor tree sesame is the mushroom class that a large amount of biocidal property safrole can be grown in the unique energy metabolism Cinnamomum kanahirai hay tree of mushroom class, be different from the physiological function that he plants the mushroom class because of having.The camphor tree sesame is the main foodborne illness that is applied on the folk tradition therapy, and diarrhoea, lower abdominal pain, skin are itched and symptom such as liver cancer.In numerous compositions with physiologically active, polyphenoils comes into one's own especially, therefore at its antioxidant component, the analysis of its antioxidant component, in deep layer cultivation (A.camphorata in submerged culture),, point out that in the extraction liquid of Antrodia Camphorata mycelium triterpenes has the function of inhibition for the peroxidation of lipid by detailed research, triterpenes and polyphenolic substance have important effect (Song and Yen, 2002) for removing oxyradical.Since contain the antioxidant component of a large amount of and complicated component in the camphor tree sesame, therefore a series of by improving the mechanism that antioxidant effect reaches protection normal cell or poisoning cancerization cell, also study successively.Secondly under low dosage, tumor cell of liver strain Hep G2 is had very strong cell toxic action with Antrodia Camphorata mycelium methanol extraction thing, is water extraction part, but ferment filtrate then hardly tool kill property.Test with C57BL/6 and two kinds of mouse of BALB/c, the result shows that TNF-α in the mouse blood, IFN-γ and IL-2 all obviously increase, IL-4 and IL-10 then with control group no significant difference.In addition, Antrodia camphorata mycelium fermented filtered liquid utilizes its polyphenoils and the ability of removing free radical, and the normal effect of its liver function of protection is arranged.See through the processing of Antrodia Camphorata mycelium extraction liquid, can avoid GSH and the consumption of ATP in the human normal red blood cell, and leukemia leukimia HL-60 cell is produced cytotoxicity, show that Antrodia Camphorata mycelium may have the effect (Hseu of polyphenoils and inhibition tumour, etal., 2002).The camphor tree sesame is very good for the curative effect of disease of liver function, and its polysaccharide body has the obvious suppression effect for hepatitis B virus.In the Mushroom polysaccharide body of having delivered, the camphor tree sesame is proved the direct inhibition effect of species (Lee, et al., 2002) its polysaccharide body has to(for) hepatitis B virus for first.But the research report about camphor tree sesame sporophore is less, and its curative effect should be higher than mycelium, so be material with fresh camphor tree sesame sporophore.

SOD is often different with activity because of different its proteinic stability that make of originating with anti-oxidant genes involved, but camphor tree sesame sporophore SOD and anti-oxidant genes involved product are known little, even other biology, especially above-mentioned anti-oxidant genes involved, also be report in recent years, therefore be worth camphor tree sesame sporophore SOD and anti-oxidant genes involved are done systematic research.

Fresh camphor tree sesame sporophore with the Nantou County Lu Gu township of picking up from the Taiwan is a material, extracts RNA, synthetic cDNA.By est sequence information design primer, cDNA is a template with camphor tree sesame sporophore, with PCR hyperplasia SOD and the Prx DNA row filter of going forward side by side, sieve the sheet segment DNA of camphor tree sesame sporophore SOD and Prx, carry out 5 ' RACE and 3 ' RACE again, obtain total length Mn-SOD and 1-Cys Prx at last, the cDNAs of 2-Cys Prx, that continues is connected with expression vector, implants E.coli and yeast and carries out mass production and purifying.SOD and Prx gene product are protein, molecular weight is another test and how to implant eukaryotic cell between 41～16kDa, therefore connects several amino acid (Tat) respectively at the N end, Tat-SOD then ,-Prx product will be implanted eukaryotic cell easily to bring into play its function.Can be applicable to the Whitening, spot makeup, or be added in the ointment for scald.

Summary of the invention:

The invention provides a kind of protein and nucleic acid of camphor tree sesame antioxidase, and make this proteinic carrier, host cell and method, and contain this proteinic composition.

The present invention expresses at least a antioxidase (Mn-SOD, 1-Cys Prx, 2-Cys Prx) by the clone and the selection of fresh camphor tree sesame sporophore.This zymoprotein can be removed superoxide and therefore can be applicable to makeup, ointment for scald and dermatoplasty etc.

Protein

The invention relates to a kind of antioxidase protein, its selection comprises sequence identification numbering: the aminoacid sequence of the sequence identification numbering 2,4 and 6 (SEQ ID NO:2, SEQ ID NO:4 and SEQIDNO:6) shown in sequence table.According to the present invention, all have the resemblance that go free radical and superoxide function identical with camphor tree sesame antioxidase protein of the present invention and gene thereof and also be contained in the scope of the present invention.As used herein, word ＂ goes the free radical and the functional similarity thing ＂ of superoxide one to have and the proteinic free radical protein identical with the superoxide function that goes of camphor tree sesame antioxidase of the present invention.Preferable, protein of the present invention comprises as the sequence identification numbers: the aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:4 and the SEQ ID NO:6.

According to preferred embodiment of the present invention, clone and have free radical and the active antioxidase protein of superoxide, it has sequence identification numbering: 2,4 and 6 aminoacid sequence, essence comprises 229,223 and 188 amino acid respectively.With the Mn-SOD of camphor tree sesame antioxidase aminoacid sequence of the present invention and other species, the Prx sequence is compared, and camphor tree sesame antioxidase of the present invention should belong to Mn-SOD, 1-Cys Prx, 2-Cys Prx.The present invention finds that also camphor tree sesame antioxidase protein has good specific activity, pH value stability, heat-resistant quality, proteolytic enzyme stability and chemical agent stability in addition.Camphor tree sesame antioxidase protein tool following properties for example of the present invention:

The Mn-SOD specific enzyme activity of camphor tree sesame antioxidase of the present invention is about 5320U/mg; After 7 minutes, still can keep the activity of quite high (about 50%) 80 ℃ of heating; PH value through 5.4～11.0 is handled, to its active nothing influence; Imidazoles (imidazole) through 2% SDS or 0.8M concentration is handled, and protein or activity all do not have obviously to change; Add enzyme liquid measure 1/20] Quimotrase (chymotrypsin) or trypsin trypsin), at 37 ℃ down behind the reaction 3h, carry out electrophoretic analysis, all there not be obviously change through the found that protein or the activity of protein staining and active coloring.

The 1-Cys Prx of camphor tree sesame antioxidase of the present invention still can keep 37% activity behind 60 ℃ of heating 16min; PH value through 5.4～11.0 is handled, to its active nothing influence; 1.6M the imidazoles of concentration is handled, and still can keep 14% activity; The Quimotrase or the trypsinase that add enzyme liquid measure 1/20 after 37 ℃ are reacted 20min down, still can be kept 15% activity.

The 2-Cys Prx of camphor tree sesame antioxidase of the present invention still can keep the activity of quite high (about 68%) behind 60 ℃ of heating 2min; PH value through 5.4～11.0 is handled, to its active nothing influence; Imidazoles through 2% SDS or 0.4M concentration is handled, and still can keep the activity of quite high (about 50%); The Quimotrase or the trypsinase that add enzyme liquid measure 1/20 behind reaction 3h under 37 ℃, still can be kept the activity of quite high (about 50%).

Have, according to the present invention, the proteinic preparation of camphor tree sesame antioxidase is under the condition of this protein expression of tolerable again, and cultivation comprises code book and invents the host cell of proteinic nucleic acid molecule and reclaim this protein.

Nucleic acid

The invention provides a kind of isolated nucleic acid molecule, comprise the protein amino acid sequence of coding tool sequence table coding SEQ ID NO:2, SEQID NO:4 and SEQ ID NO:6.The employed word ＂ of this paper nucleic acid molecule ＂ means DNA or RNA congener and derivative, fragment and the homologue that comprises that dna molecular (as cDNA or genomic dna), RNA molecule (as mRNA), use nucleic acid congener are produced.Nucleic acid molecule can be sub-thread or bifilar, but preferable with distrand DNA.The employed word ＂ of this paper isolating nucleic acid ＂ molecule means and separates other nucleic acid that is present in crude substance.

According to the present invention, nucleic acid can only comprise the bioactive part fragment of coding camphor tree sesame antioxidase protein.The employed words and phrases ＂ of this paper fragment ＂ means the still nucleotide sequence part of the bioactive camphor tree sesame of tool antioxidase protein fragments of encoding.

In a preferred embodiment of the present invention, isolated nucleic acid molecule of the present invention has nucleotide sequence or its degenerate sequence (wobble hypothesis, Wobble hypothesis) of sequence table coding SEQ ID NO:1, SEQID NO:3 and SEQ ID NO:5.In another preferred embodiment, isolated nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO:1 (Mn-SOD), and its total length is 891, and translating the district has 687bp, can translate out 229 amino acid.Nucleotide sequence shown in the SEQ ID NO:3 (1-Cys Prx), its total length is 837, translating the district has 669bp, can translate out 223 amino acid.Nucleotide sequence shown in the SEQ ID NO:5 (2-Cys Prx), its total length is 939, translating the district has 564bp, can translate out 188 amino acid.The sequence in nucleic acid molecule of the present invention and other source relatively has high similarity.The coded camphor tree sesame antioxidase protein of nucleic acid of the present invention can be with O ₂ ^-Be reduced into H ₂O ₂With with superoxide such as H ₂O ₂, t-butylperoxide, cumenene peroxide etc. is reduced into water and alcohols, the destruction and the injury that provide organism opposing toxicity oxygen to be brought.Those skilled in the art based on the degeneracy (wobble hypothesis) of gene-code, can make the proteinic nucleic acid of many code book invention camphor tree sesame antioxidases.Therefore, at a certified specific amino acids sequence, those skilled in the art can make various different nucleic acid by means of simple one or more password of modification under the situation that does not change the proteinic aminoacid sequence of camphor tree sesame antioxidase.

According to the present invention, coding camphor tree sesame antioxidase proteinic nucleic acid can the hybridization of use standard and clone technology separate.Especially, nucleic acid of the present invention can the use standard be cloned and triage techniques, separates from the cDNA storehouse of fresh camphor tree sesame sporophore.The amplification of nucleic acid of the present invention (amplification) can be according to Standard PC R amplifying technique, uses cDNA, mRNA or genomic dna to get as template and suitable Oligonucleolide primers.Nucleic acid through amplifying can be cloned into suitable carrier and borrow dna sequence analysis to identify.

Expression vector and host system

The present invention also provides an expression vector, comprises nucleic acid of the present invention.Expression vector of the present invention comprises a coding as SEQ ID NO:1, SEQ ID NO:3, the protein core acid sequence shown in the SEQ ID NO:5.

The employed word ＂ of this paper expression vector ＂ can directly express the gene nucleic acid molecule that is connected on it.Preferable carrier is for can duplicate and express connection nucleic acid thereon voluntarily.Generally speaking, the expression vector that can be used for recombinant DNA technology is generally ＂ carrier ＂ form, is generally the bifilar DNA of ring-type, does not merge to karyomit(e) when it is carrier format.

The encode nucleotide sequence of antioxidase protein or its functional similarity thing of the present invention can insert in the suitable expression to express the bioactive antioxidase protein of tool.This expression vector need contain the necessary assemblies such as transcribing and translate that inserts encoding sequence.According to the present invention, those skilled in the art can utilize the method structure of knowing to contain coding antioxidase protein and suitably transcribe and translate the expression vector of control unit.These methods comprise the extracorporeal recombinant DNA technology, synthetic technology and vivo gene recombinant technology etc.

Another purpose of the present invention provides the host cell that comprises expression vector, and this carrier comprises coding antioxidase nucleic acid sequences to proteins.The employed word ＂ of this paper host cell ＂ is can be through the host cell of carrier (as plastid) infection.According to the present invention, many host systems can be used for comprising and express coding antioxidase proteinic sequence.These host systems include but not limited to microorganism (as the bacterium that transforms with reorganization plastid or expression vector), yeast (as the yeast that transforms with yeast expressed carrier) or zooblast system.Host cell of the present invention is intestinal bacteria and yeast.The present invention at intestinal bacteria or yeast system expression, can give expression to the proteinic cDNA of antioxidase really and has active recombinant protein, after the affinity tubing string carries out fast purifying, obtains pure antioxidase protein.

According to a specific embodiment of the present invention: the Mn-SOD of camphor tree sesame antioxidase of the present invention is to be the structure of expression vector with pYEX-S1; Design N end contains restriction Eco RI fragment (5 ' GGAATTCGATG TCC ATG CTC AGT ACT GCT C3 ') and contains 6 His-tag fragments and EcoRI fragment (5 ' GGAATTCCTA GTG GTG GTG GTG GTG GTG TTT CTG TGC AGCTTC GAC GTA G 3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the proteinic cDNA of antioxidase.This DNA sent in the intestinal bacteria screen, the plastid DNA that extracts is to use restriction enzyme Eco RI to handle, and the gained fragment engages with the pYEX-S1 that the same restrictions enzyme was handled, and sends into yeast (yeast), and with medium YPD substratum (1% yeast extract yeast extract, 2% peptone peptone, 2% glucose glucose) cultivate 5D (170rpm) down at 30 ℃, break thalline, with 10, the centrifugal 5min of 000xg is after the collection supernatant liquor, after the affinity tubing string carries out fast purifying.Make protein staining behind the electrophoresis, through 12.5%SDS-PAGE, can see obvious colour band in the 25kDa place, this is antioxidase protein.For making things convenient for antioxidase protein to pass cytolemma, can connect at front end and contain nine amino acid whose Tat, and the antioxidase protein dna that will connect Tat is sent in the yeast and expressed.

The 1-Cys Prx of camphor tree sesame antioxidase of the present invention also is expression vector with pYEX-S1; Design N end contains restriction enzyme Eco RI fragment (5 ' GGAATTCG ATG CCT AGC CTC CGC CTT GGA 3 ') and contains 6 His-tag fragments and Eco RI fragment (5 ' GGAATTCCTCA GTG GTG GTGGTG GTG GTG TAC GTT GAG AGG GGT GGT TCG 3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the cDNA of 1-Cys Prx.All the other steps are identical with Mn-SOD.

The 2-Cys Prx of camphor tree sesame antioxidase of the present invention also is expression vector with pYEX-S1; Design N end contains restriction enzyme Eco RI fragment (5 ' GGAATTCG ATG GTC GCC ATC GTC CAG AA 3 ') and contains 6 His-tag fragments and Eco RI fragment (5 ' GGAATTCCTA GTG GTG GTG GTGGTG GTG CCA TGT TCG TCC TTT TAAACG G 3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the cDNA of 2-Cys Prx.All the other steps are identical with Mn-SOD.

Medical composition

The present invention also comprises a kind of medical composition, and it comprises protein of the present invention.Except that protein of the present invention, this medical composition can contain suitable pharmaceutically acceptable supporting agent.

Medical composition of the present invention can manner known in the art (for example utilizing common method) be made, and this common method comprises mixings, dissolving, granulation, makes ingot, levigate, emulsification, encapsulate, bag is sunken and/or step such as lyophilize.

Medical composition of the present invention can be borrowed any approach dispensing, and that these approach include, but is not limited to is oral, intravenously, intramuscular, intra-arterial, wear in skin, subcutaneous, the peritoneal cavity, in the nose or enterally administering.

Practicality

The present invention derives from the camphor tree sesame antioxidase protein of camphor tree sesame, and its activity is good than the antioxidase protein in other source all.Therefore, the camphor tree sesame antioxidase protein in camphor tree sesame of the present invention source has more utility value and contribution at aspects such as medical science, food, academic researches.

Camphor tree sesame antioxidase protein of the present invention can be with O ₂ ^-Be reduced into H ₂O ₂With superoxide such as H ₂O ₂, t-butylperoxide, cumenene peroxide etc. is reduced into water and alcohols, and the organism opposing destruction that peroxide brought and the ability of injury are provided.Antioxidase protein of the present invention is applied to makeup, can remove blackspot, freckle and prevent wrinkle; But make an addition to the ointment for scald accelerating wound healing; In orthopedic dermatoplasty and synthetic leather graft application, can prevent the tissue necrosis of cutify by antioxidase protein, and then quicken the stitching of cutify.

Sequence 1 is the nucleotide sequence of first kind of antioxidase protein of camphor tree sesame sporophore (Mn-SOD), and total length is 891bp; Transcriptional domain is 687bp; Coding region is 229 amino-acid residues.

Sequence 3 is the nucleotide sequence of second kind of antioxidase protein of camphor tree sesame sporophore (1-Cys Prx), and total length is 837bp; Transcriptional domain is 669bp; Coding region is 223 amino-acid residues.

Sequence 5 is the nucleotide sequence of the third antioxidase protein of camphor tree sesame sporophore (2-Cys Prx), and total length is 939bp; Transcriptional domain is 564bp; Coding region is 188 amino-acid residues.

Embodiment:

The following example further specifies the present invention, but non-ly desires to limit the scope of the invention, and substituting and change known to any those skilled in the art all is contained in the scope of the present invention, and do not depart from spirit of the present invention and purpose.

The clone of camphor tree sesame antioxidase (Mn-SOD, 1-Cys Prx, 2-Cys Prx) cDNA:

By the synthetic of the extraction of the total RNA of fresh camphor tree sesame sporophore, cDNA and set up the cDNA storehouse and est sequence information, by est sequence information design primer, with camphor tree sesame cDNA is template, with the anti-oxidant relevant DNA of the PCR hyperplasia row filter of going forward side by side, carry out RACE again, obtain total length antioxidase cDNA at last.

The extraction of the total RNA of camphor tree sesame sporophore:

Get 4g camphor tree sesame sporophore with the liquid nitrogen IQF, take out after in mortar, claying into power, in the 30mL centrifuge tube, mix with the dissolving damping fluid (lysis buffer) of 20mL (Novalgen ' s Straight ' s mRNA Isolation System) at once, place behind 4 ℃ of following 5min with 9,000xg is at 4 ℃ of centrifugal 15min, get top aqueous layer after centrifugal, nearly 20mL.The aqueous solution is drawn to adding 3mg magnetized particles (magnetight particles) mixing among the 50mL Falcon, place under 4 ℃ and hold magnetized particles (magnetightparticles) with magnetic support, with 1.5mL wash buffer washing three times, wash from being RNA (10ug) with 0.5mL at 60 ℃, add 0.1 vol.3M sodium-acetate (sodium acetate) and 0.8 vol. Virahol (isopropanol) places-75 ℃ of 20min, subsequently with 12,000rpm is centrifugal 15min under 4 ℃, clean altogether three times with 70% ethanol (ethanol), be stored in after the drying-70 ℃ standby.

Camphor tree sesame 5 '-RACE-Ready cDNA and 3 '-RACE-Ready cDNA's is synthetic: Based on BDBiosciences Clontech ' s SMART RACE cDNA Amplification Kit.

5’RACE-Ready?cDNA：

Following all reaction solns are incorporated in the microtest tube of 1.5mL and and finish on ice:

mRNA(ug/uL)(1ug) 1uL

5’-CDS?primer 1uL

Smart?II?A?Oligo 1uL

H ₂O 2uL

Amount to 5uL

3’-RACE-Ready?cDNA：

mRNA(ug/uL)(1ug) 1uL

3’-CDS?primerA 1U1

H ₂O 3uL

Amount to 5uL

Careful uniform mixing reaction soln, and centrifugally a little be placed on 2min in 72 ℃ of constant temperature water baths.After the effect centrifuge tube placed 2min on ice.Add 2uL 5X first-strand buffer, 1uL 20mM DTT, 1uL 10mM dNTP mixes, 1uL powerscript reverse transcriptase 1.5h in 42 ℃ of constant temperature water baths.The back add 200uL tricine-EDTA buffer in 72 ℃ of constant temperature water baths, be stored in behind the 7min-70 ℃ standby.

Camphor tree sesame antioxidase Mn-SOD, 1-Cys Prx, the clone of 2-Cys Prx gene:

By camphor tree sesame est sequence information design primer, with camphor tree sesame 5 '-RACE-Ready cDNA and 3 '-RACE-Ready cDNA is template, with the anti-oxidant genes involved DNA of the PCR hyperplasia row filter of going forward side by side, get the sheet segment DNA of the anti-oxidant gene of camphor tree sesame, carry out 5 ' RACE and 3 '-RACE again, obtain the cDNA of total length antioxidase at last.

Mn-SOD, 1-Cys Prx, the expression of 2-Cys Prx cDNA:

Via PCR, colloidal preparation and electrophoresis, subclone (subcloning), the structure of yeast expressed type carrier (pYEX-S1), and proteinic bringing out with the electrophoretic analysis supervisor of sample reach.

The PCR program:

Following reaction test portion is added in the 0.5mL microtest tube, adds following reagent in regular turn:

Template DNA 0.2ug

5 '-sense primer 10pmol

3 '-antisense primer 10pmol

10X Taq dna polymerase buffer liquid 5uL

15mM?MgCl ₂ 6uL

Taq archaeal dna polymerase 2.5units

10mM?dNTP 1.5uL

Add sterilized water to amounting to 50uL

Add 50～100uL mineral oil, carry out 25 cycles of PCR (94 ℃ of for 30min, 50 ℃ of for 30min, 72 ℃ of for 1.5min) after the PCR reaction finishes, can get the DNA product, after reclaiming, can be used to carry out next step experiment, for example carry out the DNA reorganization, analyze its sequence.

Colloidal preparation and electrophoresis:

Used colloid is 1.0% agarose gel, and pouring 1X TAE damping fluid into is principle can cover glue fully.Get 15uL PCR product, add tracking stain (0.25% tetrabromophenol sulfonphthalein of its 1/10 volume, 0.25% xylene cyanol blue FF, 30% glycerine is in water), in the filling orifice, the 1kb dna marker that also injects 6uL in addition is used for understanding the size of DNA sample, and the voltage of following with 100 volts carries out electrophoresis, treat that the DNA dyestuff moves to 2/3 place of glue, can end electrophoresis.To finish electrophoretic glue with bromine second pyridine (EtBr) dyeing 15min after, put into water and move back and dye, can place afterwards the ultraviolet source case under and observe, in addition also to clap upright photograph.

Subclone:

1.PCR product engages with carrier with host cell and transforms

Get 1uL PCR product, add the 1uL salts solution, after the sterilized water of 1uL and the pCR4 of 0.5uL, reaction 5min is to connect under room temperature.Get the aforementioned recombinant DNA that has engaged, add in the 18uL TOPO10 active cells, place 30min on ice, be placed on that 30sec carries out placing 2min on ice immediately after the heat-shocked in 42 ℃ the water-bath, add 150uL SOC substratum, at 37 ℃ of following shaking culture 60min, it is uniformly coated on the culture dish of the LB agarose that contains 50ug/mL microbiotic penbritin (ampicillin), after 37 ℃ are cultivated 12-16h, select 50-80 single bacterial strain and place on the LB agarose of new ampicillin, treat further experiment.

2. the preparation of the transfer printing of thalline and radioactive probe

Transfer paper is overlying on the culture dish surface, and with after the syringe needle Zha Dong work location, takes out transfer paper gently, the bacterium colony (colony) on the culture dish is transferred on the transfer paper at this moment, and the clamper finishes.Prepare a tray, on cover 3mm filter paper, pour denaturing soln (0.5M NaoH into, 1.5M NaCl), pouring volume faces up transfer paper abundant moistening 3mm filter paper being principle with the absorption bacterium, be placed on 7min on the moistening 3mm filter paper, then get another tray with neutralization solution (1MTris-HCl, 1M NaCl, pH7.5) 7min on the abundant moistening 3mm filter paper.Repeat after the neutralizer step transfer paper to be placed baking oven,, take 40min with 60-70 ℃ of oven dry.Afterwards, transfer paper is put in the UV-crosslinked instrument machine (Amersham), carry out netted binding 15sec after, treat further to screen with radioactive probe.

The preparation of radioactive probe:

Oligonucleotide (3pmole/uL) 0.5uL

10X T ₄Polynucleotide base enzyme buffer liquid 3.0uL

Polynucleotide base enzyme (12unit) 1.5uL

[r-32p]ATP 3.5uL

H ₂O 21.5uL

Amount to 30.0uL

Mix, place 37 ℃ down behind the reaction 30min.Promptly finish the preparation of radioactive probe, radioactive probe is added in the hybridization solution, be used for screening desired target gene.

3. target gene DNAThe clone

Take out aforementioned through netted banded transfer paper, the lotion that adding is mixed with 1.5X SSC and 0.1% SDS, under 44 ℃, jolt 30min, outwell lotion, add hybridization solution (the 5X SSC that does not contain radioactive probe, 5X Denhardt solution, 0.5% SDS, the 100ug/mL salmon sperm dna), purpose is for removing some nonspecific combinations, to reduce background value, this process is called prehybridization.Pour out hybridization solution, add the hybridization solution that contains radioactive probe, place 44 ℃ of water-baths to jolt more than the 16h, pour out the hybridization solution that contains radioactive probe, use aforementioned lotion, jolt 15min, repeat this step 2-4 time at 44 ℃.Press dry transfer paper, carry out automatic radiography, after the punching, choose the positive reaction strain.

The purifying of bacterium plastid with inspect

Choose the positive reaction strain and be incubated at the LB that contains ampicillin (50ug/mL), cultivate 6-8h down in 37 ℃.With 10, the centrifugal 3min of 000xg obtains the bacterial sediment thing, adds 200uL resuspending solution (50mM Tris, pH7.5,10mM EDTA) vibration, makes the thalline suspended state.Add the molten liquid (0.2M NaOH, 1% SDS) that splits of 200uL, mixing gently can be dissolved away thalline fully, and plastid can be molten from coming out.Add 200uL neutralizer (1.32M Potassium ethanoate), careful mixing, then with 12, the centrifugal 10min of 000xg, get supernatant liquor, annotate in 0.5mL centrifuge tube (containing the DNA adsorption film), with 12, the centrifugal 1min of 000xg, make DNA can be adsorbed on the film, the washing soln that adds 700mL afterwards is with 12, behind the centrifugal 2min of 000xg.Again with 12, the centrifugal 2min of 000xg to be to remove residual alcohol, and after the hot water (60 ℃) that adds 50uL at last left standstill 30sec, with 12, the centrifugal 2min of 000xg collected restriction enzyme reaction and electrophoretic analysis that the centrifugal plastid DNA that gets off is made plastid.Promptly get DNA and an amount of restriction enzyme, add 10 x reactin damping fluids, adding sterilized water at last, to make volume be 10uL, reacts 2h under suitable temperature, with 1% agarose gel electrophoresis analysis, to determine the size of the intercalation of DNA.

5. the interpretation of sequence

Utilize ABI PRISM377-96 DNA sequencing instrument to carry out automatic sequencing.

Structure at yeast expressed type carrier:

Expression vector is pYEX-S1, and Mn-SOD is to be the structure of expression vector with pYEX-S1; Design N end contains restriction enzyme Eco RI fragment (5 ' GGAATTCG ATG TCC ATG CTC AGT ACT GCTC3 ') and contains 6 His-tag fragments and Eco RI fragment (5 ' GGAATTCCTA GTG GTGGTG GTG GTG GTG TTT CTG TGC AGC TTC GAC GTA G 3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the proteinic cDNA of antioxidase.This DNA sent in the intestinal bacteria screen, the plastid DNA that extracts is to use restriction enzyme Eco RI to handle, and the gained fragment engages with the pYEX-S1 that the same restrictions enzyme was handled, and sends into yeast, and with YPD substratum (1% yeast extract, 2%peptone, 2% glucose) cultivate 5D (170rpm) down at 30 ℃, break thalline, with 10, the centrifugal 5min of 000xg is after the collection supernatant liquor, after the affinity tubing string carries out fast purifying.Make protein staining behind the electrophoresis, through 12.5% SDS-PAGE, can see obvious colour band in the 25kDa place, this is antioxidase protein.Better, pass cytolemma, can connect at front end and contain nine amino acid whose Tat, and the antioxidase protein dna that will connect Tat is sent in the yeast and expressed for making things convenient for antioxidase protein.

1-Cys Prx also is expression vector with pYEX-S1; Design N end contains Eco RI fragment (5 ' GGAATTCG ATG CCT AGC CTC CGC CTT GGA 3 ') and contains 6His-tag fragment and Eco RI fragment (5 ' GGAATTCCTCA GTG GTG GTG GTGGTG GTG TAC GTT GAG AGG GGT GGT TCG3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the cDNA of 1-CysPrx.All the other steps are identical with Mn-SOD.

2-Cys Prx is to be expression vector with pYEX-S1; Design N end contains limiting enzyme EcoRI fragment (5 ' GGAATTCG ATG GTC GCC ATC GTC CAG AA 3 ') and contains 6His-tag fragment and EcoRI fragment (5 ' GGAATTCCTA GTG GTG GTG GTG GTG GTG CCA TGTTCG TCC TTT TAAACG G 3 ') with C end primer, is that template and primer carry out the DNA that PCR obtains with the cDNA of 2-CysPrx.All the other steps are identical with Mn-SOD.

Its commentaries on classics is grown into yeast [trp ^--Ada ^---Ura ^---] as expressive host.PYEX-S1 (all handle with Eco RI earlier and connect) gets the aforementioned recombinant DNA that has engaged, adds in the yeast active cells, and screening is chosen the positive reaction strain to carry out protein expression.

Transform at yeast:

1. preparation transformant

(1) method 1: choose single bacterium colony and ruling completely on the YPD culture dish, cultivate 8～96h at 28-32 ℃, scrape from the bacteria culture medium top layer and get bacterium, add Yeastern commodity SCOS mixed solution to 110 μ L and make that bacterial concentration reaches 5 x 10 in the test tube ⁷Individual/test tube.

(2) method 2: choose single fresh bacterium colony and add in 1～10mL YPD nutrient solution, rock in 28～32 ℃ and cultivate 20～30h.To leave standstill 8～24h in nutrient solution adding 10～100mL YPD nutrient solution, with the centrifugal 10min of 2000 xg, remove supernatant,, the throw out mixing is stirred into 110-115 μ L SCOS mixed solution reaches 5 x 10 until bacterial concentration with the YPD nutrient solution of twice removal remnants of sterile water wash ⁷Individual/test tube.

2. preparation SCOS mixes liquid

In the 1.5mL test tube, add 100 μ L SCOS and cushion night, add 5 μ L 1M DTT (being stored in-20 ℃), add 5 μ L ss DNA (being stored in-20 ℃), add plastid DNA (being no more than 5 μ L).

3. prepare dry type selectivity culture dish

Therefore the culture dish of complete drying can provide good conversion reaction in Yeastern ' s SCOS conversion system, suggestion is first culture dish is repeatedly put before places the uncovered culture dish in the aseptic technique platform about 1h, treats its complete drying.

4.SCOS transform

(1) the SCOS mixed solution is mixed stirring with the yeast suspension cell and make SCOS conversion mixed solution.

(2) test tube is added a cover, place 45.5 ℃ of 10～60min.

(3) will transform mixed solution and be applied directly to dry culture dish [YNBD (1.7g yeast nitrogen base, the 5g sulfate of ammoniac, 20g glucose, the 15g agarose is dissolved in 1L water) contain 20ug Trp/mL], finish in this program 20sec, place 28-32 ℃ to descend 2～4 days in culture dish.

Protein expression:

(1～2mm) in 10mL YWAAND (1.7g yeast nitrogen base to get single bacterium colony, the acid of 5g l-asparagine, 20g glucose in one liter) contains 20ug Trp/mL nutrient solution, this container uses the 125mL flask, and in 30 ℃ of incubators, shake overnight incubation with 250rpm, add the 5mL nutrient solution again next day and make OD ₆₀₀After reaching at 0.1 o'clock and continuing to cultivate 72h, add 10mLYPD again and continue and cultivate 48h with 30 ℃, 250rpm concussion with 30 ℃, 250rpm concussion.

All bacterium liquid with 5000xg, 4 ℃ of centrifugal 10min, are collected bacterium and added 0.5g glass sphere and 2mLPBS (pH8.0), break thalline, with 10, the centrifugal 5min of 000xg after the collection supernatant liquor, adds the 2mL damping fluid again, repeat this extraction step, extract once with the 2mL damping fluid at last again, collect the 6mL supernatant liquor altogether, this is the crude protein sample, treat further electrophoresis, electrophoretic colloid size is 10cm * 8cm * 0.75mm.

1. electrophoresis colloidal preparation

With aluminium sheet and glass plate wipe away clean after, and parting bead (spacer) combines, and puts into the glue model, again according to the listed consumption preparation colloidal solution of following table: separating gel, in mixing and the injection model, it is flat to add suitable hydraulic pressure again, passes through 1h approximately, after colloid forms, remove water layer, the spacer gel that reinjects, and plug the groove comb, treat into glue and get final product.

2. electrophoresis

Get and inject the colloid hole after an amount of sample and sample are written into the damping fluid mixing, compress into capable electrophoresis with 100 voltaisms, native-PAGE needs 65min approximately, and SDS-PAGE needs to end electrophoresis behind the 130min approximately, carefully takes off colloid, carry out Coomassie blue (Coomassie blue) dyeing: colloid is dipped in the stain, evenly shake 30min, remove stain, with sterile water wash once after, add and to move back stain, move back and dye 12h and get final product.

3. colloidal drying

To desire the exsiccant colloid and be dipped in 30min in 10% glycerine solution, get a glass plate and two glassine papers that cooperate glass plate size, the abundant moistening glassine paper of water, after will be wherein a Zhang Ping be laid on the glass plate, put the exsiccant colloid of wanting again, spread another glassine paper at last, fix, place under the room temperature and dry in the shade with plastic clip.

4.Mn-SOD, 1-Cys Prx, the purifying of 2-Cys Prx

Because of the C end has 6 His, with Ni ²⁺-nitrilotriacetic acid Sepharose superflow carries out affinity tubing string purifying, method is as follows: the resin tubing string of 3mL, with the PBS balance of 5vol., afterwards the crude protein sample of 6mL is injected and collect the about 6mL of liquid (pass through) that flows down.Contain 20mM imidazole with 3vol.PBS again and wash tubing string, contain 100mM imidazole towards carrying, collect 6 parts altogether, wherein have 2 parts to be total to 3mL and collect protein, because contain imidazole, so need remove through dialysis succeeded by 1.5mL PBS.

Dialyzate: the about 3mL of sample is packed in the dialysis membrane, clip with dialysis clamp, put into and contain the beaker that 200mL 1/3PBS contains 1mM DTT and 5% glycerol dialyzate,, repeat this dialysis step and can packing treat further analysis in 4 ℃ down more than the dialysis 4h.

5.Mn-SOD, 1-CysPrx, the protein content of 2-Cys Prx

Make protein typical curve (BSA standard curve) earlier: promptly get protein standard substance (BSA respectively, 0.5mg/mL) 1,2,3,4,5,6uL is in its pairing aqua sterilisa, make last volume reach 800uL, add respectively afterwards under the photoghraphic coupler 200uL room temperature and react 5min, survey OD _595nmAnd obtain the protein typical curve according to light absorption value.The sample of gathering is recorded light absorption value according to same procedure can try to achieve protein content by typical curve.

6. determination of activity

Mn-SOD:(1) at first will finish electrophoretic colloid, and be soaked in the aqueous solution of 21.6mL 2.5mM NBT (nitroblue tetrazolium), lucifuge is shaken 15min, removes NBT solution, behind the washed with de-ionized water colloid, is soaked in 21mL again and contains 8 * 10 ^-5In the aqueous solution of M riboflavin (riboflavin) and 30mM TEMED, irradiation shakes, and the zone that presents transparent bright band is for having the active part of SOD.This method is for utilizing under riboflavin and the illumination condition and the TEMED reaction, and as the system that produces super oxygen, NBT is as photoghraphic coupler (chromogenic reagent).The colloid zone that has SOD to exist, super oxygen is decomposed, and reduction reaction does not take place in NBT, and presents transparent bright band.The zone that no SOD exists, reduction reaction takes place in super oxygen and NBT, and generates hepatic formaza (Beauchamp and Fridovich, 1971).

(2) (Ardmore UK), gets different extent of dilution S1～S6 according to RNASOD kit; S1:0.05mLMn-SOD standard substance (5.9U/mL), the mixed matrix that adds 1.7mL (contains 0.05mM xanthine, 0.025mM INT[2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride], 0.94mM EDTA, 50mM CAPS buffer, pH10.2).After mixing evenly, add 0.25mLxanthine oxidase (0.02U), mixing is in 37 ℃ of reactions, when 30sec and 3min, measure its light absorption value A1 and A2 respectively, after the following formula of foundation calculates and suppresses percentage at wavelength 595, take the logarithm as transverse axis with the activity of standard substance, suppress percentage as the longitudinal axis,, obtain typical curve through after the linear regression.

(A2—A1)/2.5＝ΔA _{standardorsample/min}

100—(ΔA _{standard?orsample/min}×100)/ΔA _S1/min

＝％inhibition

The enzyme of getting an amount of purifying is identical with the measuring method of standard substance, obtain reaction times 30sec and 3min variation at wavelength 595nm light absorption value, and being converted into the inhibition percentage, the reference standard curve can be obtained the SOD activity (U/mg) of protein example (sample).

1-Cys Prx, 2-Cys Prx active testing:

Make H earlier ₂O ₂, t-butylperoxide and cumenene peroxide typical curve: promptly get H respectively ₂O ₂T-butylperoxide and cumenene peroxide standard substance (1mM) each 1,2,4,8,16uL is in its corresponding 1/3 PBS containing (5% glycerol and 1mM DTT), make last volume reach 50uL, add 20uL 26.3% TCA afterwards, then add 20uL 10mM Fe ²⁺, adding 10uL 2.5MKSCN at last, cumulative volume 100uL measures OD _475nmLight absorption value is also obtained H according to light absorption value ₂O ₂, t-butylperoxide and cumenene peroxide typical curve.The sample of gathering is recorded light absorption value according to same procedure can try to achieve protein is removed superoxide (peroxide) in Eppendorf tube ability: promptly add 1/3 PBS in the Eppendorf tube and contain (5% glycerine and 1mM DTT), an amount of 1mM H by typical curve ₂O ₂Reach 50uL with an amount of enzyme (enzyme) cumulative volume, react 10min under the room temperature, promptly add 20uL 26.3% TCA, then add 20uL 10mM Fe ²⁺, adding 10uL 2.5M KSCN at last, cumulative volume 100uL measures OD _475nmLight absorption value.

7.Mn-SOD, 1-Cys Prx, 2-Cys Prx character

Thermostability

Get an amount of enzyme liquid respectively in the 1.5mL test tube, each 5 pipe, respectively at 80 ℃ (for Mn-SOD), 60 ℃ (for 1-Cys Prx), behind 60 ℃ (for 2-Cys Prx) heating 0,2,4,8, the 16min, place on ice, carry out 15% Native-gel electrophoresis, make protein staining (1.5ug/Mn-SOD respectively, 3.6ug/1-Cys Prx, 2ug/2-Cys Prx) and active testing (1.5ug/Mn-SOD, 1.8ug/1-Cys Prx, 1ug/2-Cys Prx).The result shows that this enzyme is very stable, and Mn-SOD is 7min at its active half required time of minimizing of 80 ℃ of heating, and its degradation constant is 1.02 x 10 ^-1Min ^-1

The influence of pH

Get an amount of enzyme liquid respectively, in the 1.5mL test tube, totally 6 manage, add different pH buffer: 0.2M sodium citrate buffer solution (pH2.3, or5.4), 0.2M Tris-Hcl damping fluid (pH7.8, or9.0), 0.2M glycine NaOH damping fluid (pH 10.4 or 11.2), behind reaction 1h under 37 ℃, place on ice, carry out respectively making protein staining (1.5ug/Mn-SOD, 3.6ug/1-Cys Prx, 2ug/2-Cys Prx) and active testing (1.5ug/Mn-SOD behind the 15% Native-gel electrophoresis, 1.8ug/1-Cys Prx, 1ug/2-Cys Prx).The result shows that this enzyme is very stable, shows very stablely with acid-alkali treatment, especially handles 1h to not influence of enzymic activity to pH8～pH11.

The influence of SDS

Get an amount of enzyme liquid respectively, in the 1.5mL test tube, totally 5 pipes add different 20% SDS that measure respectively, and making the SDS ultimate density is 0,1,2,3,4%, behind reaction 1h under 37 ℃, carry out respectively making protein staining (1.5ug/Mn-SOD, 3.6ug/1-Cys Prx, 2ug/2-Cys Prx) and active testing (1.5ug/Mn-SOD behind the 15% Native-gel electrophoresis, 1.8ug/1-Cys Prx, 1ug/2-Cys Prx).The result shows that Mn-SOD is also very stable with the processing of SDS, only suppresses 23% during concentration height to 4%.

The influence of imidazoles

Get an amount of enzyme liquid respectively, in the 1.5mL test tube, totally 5 pipes add the imidazoles of different amounts respectively, make that the imidazoles ultimate density is 0,0.2,0.4,0.8,1.6M, behind reaction 1h under 37 ℃, carry out respectively making protein staining (1.5ug/Mn-SOD, 3.6ug/1-Cys Prx, 2ug/2-Cys Prx) and active testing (1.5ug/Mn-SOD behind the 15%Native-gel electrophoresis, 1.8ug/1-Cys Prx, 1ug/2-Cys Prx).The result shows that Mn-SOD is also very stable with the processing of imidazoles, must be high activity be just had inhibition (70%) during to 1.6M.

The influence of proteolytic enzyme

Get an amount of enzyme liquid respectively, in the 1.5mL test tube, totally 4 pipes, (pH8.5 contains 20mM CaCl in the Tri-HCl damping fluid to add the trypsinase of suitable enzyme liquid measure 1/20 or Quimotrase respectively ₂) respectively at 37 ℃ of following reactions 1,2,3h, carry out respectively making protein staining (1.5ug/Mn-SOD, 3.6ug/1-CysPrx, 2ug/2-Cys Prx) and active testing (1.5ug/Mn-SOD behind the 15% Native-gel electrophoresis, 1.8ug/1-Cys Prx, 1ug/2-Cys Prx).The result shows that Mn-SOD is very stable with tryptic processing, and behind the 3h, activity only descends 23%.Quimotrase is handled also very stable, and behind the 3h, activity only descends 33%.

Though the present invention has utilized aforementioned preferred embodiment to melt in detail; right its is not in order to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; can do various changes and modification, so protection scope of the present invention is when being as the criterion with claims restricted portion.

Sequence table

＜110〉woods chess wealth

＜120〉protein of camphor tree sesame antioxidase and nucleic acid and manufacture method thereof and purposes

<160>6

<170>PatentIn?version?3.3

<210>1

<211>891

<212>DNA

＜213〉camphor tree sesame (Antrodia camphorata)

<220>

<221>CDS

<222>(36)..(725)

<400>1

<210>2

<211>229

<212>PRT

＜213〉camphor tree sesame (Antrodia camphorata)

<400>2

<210>3

<211>837

<212>DNA

＜213〉camphor tree sesame (Antrodia camphorata)

<220>

<221>CDS

<222>(53)..(724)

<400>3

<210>4

<211>223

<212>PRT

＜213〉camphor tree sesame (Antrodia camphorata)

<400>4

<210>5

<211>939

<212>DNA

＜213〉camphor tree sesame (Antrodia camphorata)

<220>

<221>CDS

<222>(13)..(579)

<400>5

<210>6

<211>188

<212>PRT

＜213〉camphor tree sesame (Antrodia camphorata)

<400>6

Claims

1, a kind of protein of camphor tree sesame antioxidase, it belongs to the protein of camphor tree sesame sporophore antioxidase, and it is selected from the sudismase Mn-SOD of camphor tree sesame, and the aminoacid sequence of the sudismase Mn-SOD of this camphor tree sesame is shown in SEQ ID NO:2.

2, the encode proteinic aminoacid sequence of camphor tree sesame antioxidase as claimed in claim 1 of a kind of isolating nucleic acid of camphor tree sesame antioxidase, this separated nucleic acid sequence.

3, the isolating nucleic acid of camphor tree sesame antioxidase according to claim 2, wherein this isolating nucleic acid is expressed in yeast.

4, a kind of expression vector of camphor tree sesame antioxidase, it comprises the nucleotide sequence of the isolating nucleic acid of camphor tree sesame antioxidase as claimed in claim 2.

5, a kind of host cell of camphor tree sesame antioxidase, it comprises the expression vector of camphor tree sesame antioxidase as claimed in claim 4.

6, a kind of proteinic manufacture method of camphor tree sesame antioxidase, it is in order to make the protein of camphor tree sesame antioxidase as claimed in claim 1, and this manufacture method comprises the following step:

Obtain the nucleotide sequence of a camphor tree sesame antioxidase, the proteinic aminoacid sequence of its this camphor tree sesame antioxidase of encoding;

The nucleotide sequence of this camphor tree sesame antioxidase is written in the expression vector;

The expression vector of this camphor tree sesame antioxidase is written in the host cell;

The host cell of this camphor tree sesame antioxidase is cultivated in a nutrient solution, and this nutrient solution is allowed the nucleic acid molecule encoded protein matter expression of this camphor tree sesame antioxidase, with the protein of synthetic camphor tree sesame antioxidase; And

In the nutrient solution of this host cell, reclaim the protein of this camphor tree sesame antioxidase.

7, a kind of medical composition of camphor tree sesame antioxidase, it comprises the protein of camphor tree sesame antioxidase as claimed in claim 1.

8, the medical composition of camphor tree sesame antioxidase according to claim 7, wherein the medical composition of this camphor tree sesame antioxidase is makeup or ointment for scald.

9, the proteinic application of the described camphor tree sesame of claim 1 antioxidase, it is in order to make the makeup of removing blackspot, freckle and preventing wrinkle.

10, the proteinic application of the described camphor tree sesame of claim 1 antioxidase, it is in order to make the ointment for scald of accelerating wound healing.