CN104342408A

CN104342408A - Preparation method of recombinant cordyceps sinensis superoxide dismutase (rcSOD)

Info

Publication number: CN104342408A
Application number: CN201410535820.0A
Authority: CN
Inventors: 刘爽; 邓小霞; 孙晓悦; 庞玉; 王子华
Original assignee: JILIN JINZIYUAN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Current assignee: JILIN JINZIYUAN BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Priority date: 2014-06-18
Filing date: 2014-10-12
Publication date: 2015-02-11
Anticipated expiration: 2034-10-12
Also published as: CN104342408B

Abstract

The invention provides a nucleotide sequence of a signal peptide suitable for escherichia coli to carry out secretory expression on recombinant cordyceps sinensis superoxide dismutase (rcSOD), an SOD nucleotide sequence for coding cordyceps sinensis SOD, a carrier containing the nucleotide sequences, an engineering cell containing the carrier and a method for purifying rcSOD from the engineering cell. An optimized SOD gene is very suitable for being expressed by escherichia coli and has the characteristics of high expression, high stability and high secretion. The rcSOD pure product can be efficiently, simply and conveniently obtained at a low cost. The invention also provides application of rcSOD prepared by the method to preparation of medicines, food, health care products and cosmetics for treating human SOD related diseases or pathologic symptoms.

Description

The preparation method of restructuring Cordyceps sinensis superoxide-dismutase

Technical field

The present invention relates to E. coli secretion and express the nucleotide sequence of the nucleotide sequence of the coded signal peptide of restructuring Cordyceps sinensis superoxide-dismutase and coding Cordyceps sinensis SOD, the carrier containing above-mentioned nucleotide sequence, the engineering cell containing aforementioned bearer and from method of this project cell purification rcSOD and uses thereof, belong to genetically engineered and art of protein biochemistry.

Background technology

Superoxide-dismutase (superoxide dismutase, SOD) a kind ofly can be converted into the enzyme of oxygen and hydrogen peroxide by disproportionation reaction by catalyzes superoxide.It is extensively present in all kinds of animal, plant, microorganism, is a kind of important antioxidant, protects the cell be exposed in oxygen.Can be divided into three kinds by its containing metal prothetic group difference, the first is the title (Cu.Zn-SOD) of cupric (Cu) zinc (Zn) metal prothetic group, a kind of enzyme the most common, in green, is mainly present in body cell slurry; The second is the title (Mn-SOD) containing manganese (Mn) metal prothetic group, in purple, is present in eukaryotic plastosome and prokaryotic cell prokaryocyte; The third is the title (Fe-SOD) of iron content (Fe) metal prothetic group, in tawny, is present in prokaryotic cell prokaryocyte.

Superoxide-dismutase can remove super-oxide, and Cell protection is from oxidative damage.The reaction of super-oxide and non-free radical is spin forbidden.In biology system, this just means that it mainly reacts with self (disproportionation) or another biology free radical (as nitrogen protoxide).Superoxide anion radical (O ₂ ^-) can (when pH=7, speed of response is 10 quickly ⁵m ^-1s ^-1) disproportionation is O ₂with hydrogen peroxide (H ₂o ₂).But super-oxide can react at faster speed with specific molecule (as NO free radical), generate peroxynitrite (O=N-O-O ^-), therefore the katalysis of superoxide-dismutase just seems particularly important.And, the disproportionation reaction of super-oxide and its starting point concentration square relevant, although therefore the super-oxide transformation period of high density very short (being 0.05 second under such as 0.1mM concentration), the transformation period of the super-oxide of lower concentration quite grows (can reach 14 hours under 0.1nM concentration).Comparatively speaking, the disproportionation reaction of superoxide-dismutase catalysis is first order reaction for super-oxide starting point concentration, and in all known enzymes, have the fastest turnover number (with substrate reactions speed) (7x10 ⁹m ^-1s ^-1) (Loeffler Petrides Heinrich.Biochemie & Pathobiochemie.8th Edition.2007.123.), therefore the restriction of speed of reaction is just enzyme and the intermolecular collision frequency of super-oxide, and namely speed of reaction is " diffusion limited ".

Super-oxide is one of active oxygen main in cell, and therefore superoxide-dismutase plays the effect of crucial antioxidant.Superoxide-dismutase has important physiological role, and the mouse of this enzyme of shortage after genetic engineering modified can suffer from serious disease.Such as, a couple of days dies from serious oxidation emergency (Li to the mouse of shortage SOD2 after birth, et al., Y.Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.Nat.Genet.1995,11:376 – 381.doi:10.1038/ng1295-376.), the mouse lacking SOD1 has pathological characters widely, comprise hepatocellular carcinoma (hepatocellular carcinoma) (Elchuri, et al., S.CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life..Oncogene.2005, 24:367 – 380.doi:10.1038/sj.onc.1208207.), the quality of the muscle relevant to the age is accelerated to reduce (Muller, et al., F.L.Absence of CuZn superoxide dismutase leads to elevated oxidative stress, mutagenesis and acceleration of age-dependent skeletal muscle atrophy.Free Radic.Biol.Med.2006, 40:1993 – 2004.doi:10.1016/j.freeradbiomed.2006.01.036.), cataract occurs ahead of time and the life-span reduces, the mouse lacking SOD3 does not show any obvious defect and has the normal life-span, but they are easier to high oxygen injury occurs.The mouse knocking out any one superoxide-dismutase is easier to the medicament dying from super-oxide production, as Paraquat (paraquat) and diquat (diquat) (Sentman, et al., M.L.Phenotypes of mice lacking extracellular superoxide dismutase and copper-and zinc-containing superoxide dismutase.J.Biol.Chem.2006,281:6904 – 6909.doi:10.1074/jbc.M510764200.).

The fruit bat lacking SOD1 has the life-span significantly shortened, and the fruit bat lacking SOD2 is then in utero just dying.In nematode nematode, knock out SOD does not cause serious physiology disorderly.SOD1 knock out or deletion mutantion very harmful in oxygen-free environment growth for yeast saccharomyces cerevisiae, and cause the remarkable shortening of rear diauxic growth phase (post-diauxic lifespan); SOD2 knock out or deletion mutantion then can cause growth-inhibiting and same reduce after the diauxic growth phase.

Cordyceps sinensis (cordyceps sinensis) calls Chinese caterpillar fungus, Cordyceps, cordyceps sinensis, is the Hepialus larva parasitized by the Cordyceps fungus of the snakelike Cordyceps of Hypocreales snakelike Chinese caterpillar fungus section in alpine meadow soil, and larva body is ossify.And under optimum conditions, summer goes out long bar-shaped stroma by bombys batryticatus head end pumping and is formed, i.e. the complex body that forms of the sporophore of Cordyceps fungus and bombys batryticatus sclerotium (larva corpse).Main active ingredient is cordycepin, and it has immunity moderation system function, antitumor, antifatigue, invigorates the lung and the kidney, hemostasis and phlegm, the multiple efficacies such as secret lean gas.Eating method have beat powder, steep in wine, soaked etc.Cordyceps sinensis mainly originates in Qinghai, China's Mainland, Tibet, Sichuan, Yunnan, Gansu and Guizhou etc. to be economized and municipal severe cold areas and snow-capped mountains and marshlands.Cordyceps sinensis is also containing SOD, but content is extremely low.In order to obtain Cordyceps sinensis SOD, we utilize modern genetic engineering technology, expression Cordyceps sinensis SOD protein engineering bacterium and carrier thereof are attempted, present system optimization, change the method for the engineered E. coli cell obtaining Cordyceps sinensis SOD, the transport piece utilizing bacillus coli gene to express and SOD amalgamation and expression obtain more satisfied result, and by the improvement of zymotechnique, easy purifying process simultaneously, obtains high expression level, high reactivity and high yield pulp1, has a kind of copper zinc type rcSOD production method of industrialization value.

Summary of the invention

Based on the shortcoming of prior art, the object of this invention is to provide the nucleotide sequence of above-mentioned signal peptide of encoding, carrier, the engineering cell containing aforementioned bearer and the method from this project cell purification rcSOD containing this signal peptide nucleotide sequence and Cordyceps sinensis SOD nucleotide sequence.

The present inventor is surprised to find, before expressing the Cordyceps sinensis SOD nucleotide sequence as shown in SEQ ID NO:3, add the signal peptide nucleotide sequence as shown in SEQ ID NO:1, unexpectedly can obtain the carrier of the Cordyceps sinensis SOD of high amalgamation and expression, and use the SOD of this vector expression to be easy to purifying, purity is high, receipts amount large, active high.

Of the present invention in another, provide a kind of add before the nucleotide sequence of coding Cordyceps sinensis superoxide-dismutase one can make Cordyceps sinensis Expression of Superoxide Dismutase after be secreted into the transport piece nucleotide sequence in cell walls and plasmalemma gap, described nucleotide sequence SEQ ID NO:1 can encode the aminoacid sequence shown in SEQ ID NO:2, and described nucleotide sequence SEQ ID NO:1 and Cordyceps sinensis superoxide-dismutase coding region sequence SEQ ID NO:3 merges and forms nucleotide sequence SEQ ID NO:5.

In another aspect of the invention, provide a kind of expression vector, it contains the above-mentioned nucleotide sequence of the present invention.Preferably, described expression vector is pET22b.

In another aspect of the invention, provide a kind of engineering cell, it is obtained by the Sequence Transformed host cell of described expression vector, and is integrated with the nucleotide sequence of coding recombinant superoxide dismutase in expression vector.

Preferably, described engineering cell is coli strain is BL21 (DE3).

Of the present invention further in, provide a kind of expression method of producing Cordyceps sinensis superoxide-dismutase, comprise the following steps: under applicable expression condition, cultivate the host cell that the present invention is above-mentioned, thus secreting, expressing Cordyceps sinensis superoxide-dismutase; Preferably, described host cell is coli strain is BL21 (DE3).Described cultivation comprises: cultivate and be divided into cultivation stage and induction period, and cultivation stage is cultivated bacterial concentration and reached OD600, and time inductive phase is 18-21hr, and fermentation and inducing temperature remain on 16-25 DEG C, and IPTG consumption is 0.05-1mM/L, and the pH value of inductive phase is 6-8.Described e. coli host cell comprises can use IPTG Induced synthesis Cordyceps sinensis superoxide-dismutase, and can be secreted into the Bacillus coli cells in cell walls and plasmalemma gap.

In another aspect of the present invention, carry out slightly carrying and being further purified to restructuring Cordyceps sinensis SOD after expression.It comprises the steps: (1) collects thalline to fermented sample by centrifugal and/or filter type, with N,O-Diacetylmuramidase process thalline, clear liquid is obtained again by centrifugal method, (2) by saltout and/or after ultrafiltration carries out preliminary purification, or directly by anionresin, hydrophobic chromatography method, obtain the sterling that purity reaches more than 95% (as 95-99.9%), the restructuring Cordyceps sinensis SOD prepared by the present invention is active in 5500U or more than 6000U.

In another aspect of the present invention, the invention provides and use the purposes of the restructuring Cordyceps sinensis SOD for preparing of aforesaid method in the medicine of preparation treatment and human superoxide dismutase relative disease or pathological symptom, food, healthcare products and makeup thereof.

Major advantage of the present invention is: the link secretion signal peptide gene after (1) optimization is very applicable to escherichia coli expression, has the feature of high expression level, high stable, hypersecretion; (2) host strain of the superoxide dismutase gene containing secreting signal peptide has the characteristic of high-density growth, is very suitable for the fermentative production of genetically engineered drug large scale and high density.The hgSOD of escherichia coli expression is secreted into cell walls and plasmalemma gap with solubility, tool activity form; Product must renaturation, directly can carry out purifying; Simultaneously because expression level is high, purifying process complexity simplifies greatly, and yield is greatly improved; (3), compared with expressing with eukaryotic system, cost reduces greatly, and industrialization value is improved significantly.

Accompanying drawing explanation

Fig. 1 display is used for the nucleotide sequence of code book invention signal peptide.

The aminoacid sequence of Fig. 2 signal peptide of the present invention.

Fig. 3 display is used for the nucleotide sequence of code book invention natural cordyceps SOD.

The aminoacid sequence of Fig. 4 natural cordyceps SOD of the present invention.

Fig. 5 display is used for the nucleotide sequence of code book invention restructuring Cordyceps sinensis SOD.

Fig. 6 the present invention recombinates the aminoacid sequence of Cordyceps sinensis SOD.

Fig. 7 is used for the nucleotide sequence of code book invention signal peptide and the corresponding diagram of the aminoacid sequence of the signal peptide of the present invention estimated by it.

Fig. 8 is used for code book and invents the nucleotide sequence of the restructuring Cordyceps sinensis SOD of warm expression and the corresponding diagram of the aminoacid sequence of the restructuring Cordyceps sinensis SOD of the warm expression of the present invention estimated by it.

Fig. 9 is the structure schematic diagram of a kind of expression plasmid pET-rcSOD of the present invention.

Figure 10 is that under shaking flask 37 DEG C of conditions, rcSOD expresses electrophorogram.Each swimming lane is as follows: before swimming lane 1, induction; After swimming lane 2, induction, induce sampling in 4 hours, SDS-PAGE electrophoresis detection expression.

Figure 11 is that (IPTG concentration is 1mM/L) rcSOD expresses electrophorogram under condition of different temperatures.Induce after 20 hours, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, gets supernatant, and SDS-PAGE detects expression.

Figure 12 is the impact of IPTG concentration on rcSOD expression level.Under 20 DEG C of conditions, use the IPTG abduction delivering of 0.6mM, 0.4mM, 0.2mM, 0.1mM concentration respectively.Induce after 20 hours, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, gets supernatant, and SDS-PAGE detects expression.

Figure 13 is the impact of induction time on rcSOD expression level.Under 25 DEG C of conditions, after inducing 8,10,16,18,20,30 hours respectively, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, gets supernatant, and SDS-PAGE detects expression.

Figure 14 is under 25 DEG C of conditions, the expression electrophorogram of rcSOD in 6.5L fermentor tank.Each swimming lane is as follows: 1, sampling before induction; 2, sampling in 20 hours is induced.

Figure 15 is the electrophorogram of the rcSOD after purifying.In 6.5L fermentor tank, express rcSOD, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, gets supernatant, with the protein SDS-PAGE electrophorogram that anion-exchange chromatography, hydrophobic chromatography are later.

Figure 16 is the chromatography collection of illustrative plates of purifying postlyophilization powder product molecular sieve Superdex75 (Phamarcia) method testing product purity.

Embodiment

In order to provide, substance of the present invention be understood, describe some aspect of the present invention, pattern, embodiment, modification and feature with different the level of details hereinafter.

In the practice of the invention, employ molecular biology, genetic engineering, genetics, Cell. Mol, biological chemistry, protein biochemistry, protein biochemistry engineering, pharmaceutical technology, medical science, physiology, pathology, experimentation on animals, (the Molecular Cloning:A Laboratory Manual such as Sambrook, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) etc. a lot of conventional art and basic theory.These technology are known.

In an embodiment of the invention, provide a kind of restructuring Cordyceps sinensis superoxide-dismutase,

Wherein, described Cordyceps sinensis superoxide-dismutase comprises arbitrary following aminoacid sequence:

A) aminoacid sequence shown in SEQ ID NO.2; And/or

B) aminoacid sequence shown in SEQ ID NO.4; Or

C) aminoacid sequence shown in SEQ ID NO.6.

In another embodiment of the present invention, provided the nucleotide sequence of the described signal peptide of coding and Cordyceps sinensis superoxide-dismutase by molecular cloning.

Term of the present invention " nucleotide sequence " comprises DNA and RNA of genomic dna, cDNA, synthesis.Preferably, it refers to DNA, is more preferably the cDNA of encoding sequence.The present invention is also contained coding and is had the enzyme of special properties as herein defined or the nucleotide sequence of albumen.Term used herein " nucleotide sequence " refers to oligonucleotide sequence or polynucleotide sequence and its variant, homologue, fragment and derivative (as its part).Described nucleotide sequence can be derived from genome or synthetics or thing of recombinating, and it can be (no matter it represents positive-sense strand or antisense strand) nucleic acid molecule of double-strand or strand.

In a specific embodiment of the present invention, provide a kind of nucleic acid molecule of separation, wherein, the nucleic acid molecule of described separation comprises arbitrary following nucleotide sequence:

A) nucleotide sequence shown in SEQ ID NO.1 of the aminoacid sequence of coding SEQ ID NO:2; And/or

B) nucleotide sequence shown in SEQ ID NO.3 of the aminoacid sequence of coding SEQ ID NO:4; Or

C) nucleotide sequence shown in SEQ ID NO.5 of the aminoacid sequence of coding SEQ ID NO:6.

The nucleotide sequence that coding has enzyme as herein defined or albumen can obtain from producing any cell of described enzyme or albumen or biology to be separated.Various methods for separating of nucleotide sequence are all well known in the art.Be only citing character herein, such as utilize strong denaturant to extract total serum IgE, utilize and transcribe and reverse transcription method, the chromosomal DNA or the messenger RNA(mRNA) (mRNA) that produce the biology of described enzyme or albumen build genomic dna and/or cDNA library.

Further, also can be prepared the nucleotide sequence of the described enzyme of coding or albumen by the standard method of maturation by synthesis, for another example, utilize synthetic oligonucleotide on automatic dna synthesizer, be then purified, anneal, connect and be cloned in suitable carrier.

Therefore, described nucleotide sequence can be derived from the genome of mixing and synthesis source, the synthesis of mixing and the genome of cDNA source or mixing and cDNA source, its according to standard technique by connect be derived from synthesis, fragment that is genomic or cDNA obtains as required.The fragment of each connection corresponds to the different piece of whole nucleotide sequence.Described DNA sequence dna also can use specific primer to pass through polymerase chain reaction (PCR) to prepare.

Term " PCR " is referred to as polymerase chain reaction (polymerase chain reaction herein, PCR) refer under the catalysis of archaeal dna polymerase, by sex change, withdraw from a secret society or underworld gang, polymerization amplification waits iterative cycles to reach the region of DNA section of geometry order of magnitude amplification between two sections of known arrays.

Polynucleotide of the present invention (nucleotide sequence) can be used to prepare primer, as PCR primer, for the primer of optional amplified reaction; Maybe polynucleotide can be cloned in carrier.The length of described primer and other fragment can be at least 12, as at least 15, be preferably at least 20, as at least 25,30,35 or 40 Nucleotide, and it is also encompassed in as the term is employed herein within polynucleotide.

Usually, recombinant DNA technology (DNA namely recombinated) preparation coding is used to have the enzyme of special properties as herein defined or the nucleotide sequence of albumen.Therefore, in an Alternate embodiments of the present invention, chemical process known in the art can be used to synthesize all or part of nucleotide sequence.

Usually, prepare primer by synthetic method, the method comprises progressively prepares required nucleotide sequence in the mode of next Nucleotide every.Be easy in this area obtain and use automatic technology to realize the technology of aforesaid method.Design of primers can need to consider that the nucleotide sequence held at target protein N end or C adds the nucleotide sequence of code tag albumen (Tagged-protein expression system) according to improving purity, common label protein includes but not limited to His-tag albumen, Flag-tag albumen, GST-tag albumen etc.

Can recombinate, synthesize or be prepared according to polynucleotide of the present invention (as DNA polynucleotide) by the available any method of those skilled in the art.They also can be cloned by standard technique.Usual use recombination method, prepares longer polynucleotide as used PCR (polymerase chain reaction) clone technology.This comprises the pair of primers (according to appointment 12 to 40 Nucleotide) that preparation is positioned at the lipid targeted sequence area flank of required clone, primer is made to contact mRNA or cDNA obtained from Cordyceps sinensis, polymerase chain reaction is carried out under the condition that desired zone can be made to increase, be separated the fragment (as by purification reaction mixture on sepharose) of amplification, and reclaim the DNA of amplification.Can design to introduce makes it comprise applicable Restriction Enzyme recognition site, so that can by the DNA clone of amplification in the cloning vector be applicable to.

As mentioned above, can recombinate, synthesize or be prepared according to polynucleotide of the present invention (as DNA polynucleotide) by the available any method of those skilled in the art.They also can be cloned by standard technique.Usual use recombination method, prepares longer polynucleotide as used PCR (polymerase chain reaction) clone technology.This comprises the pair of primers (according to appointment 15 to 30 Nucleotide) that preparation is positioned at the lipid targeted sequence area flank of required clone, primer is made to contact mRNA or cDNA obtained from Cordyceps sinensis, polymerase chain reaction is carried out under the condition that desired zone can be made to increase, be separated the fragment (as by purification reaction mixture on sepharose) of amplification, and reclaim the DNA of amplification.Can design to introduce makes it comprise applicable Restriction Enzyme recognition site, so that can by the DNA clone of amplification in the cloning vector be applicable to.The design of restriction enzyme site can set according to the nucleotide sequence of vector cloning sites and the encode enzyme or albumen with special properties defined herein; often add several protection bases above, in detail with reference to the protection base setting reference manual that PROMEGA company provides.

In a specific embodiment of the present invention, described Cordyceps sinensis superoxide-dismutase obtains by expressing arbitrary nucleotide sequence shown below:

Term " construct ", namely refers to the carrier built, and it comprises the nucleotide sequence of carrier vector itself and has the enzyme of special properties defined herein or the nucleotide sequence of albumen according to the coding that the present invention uses, and it is connected with promotor directly or indirectly.In some embodiments, preferred construct at least comprises nucleotide sequence of the present invention, or coding has the enzyme of special properties as defined herein or albumen and the nucleotide sequence be operationally connected with promotor.

Term " expression vector " refers to can in vivo or the construct of vivoexpression.It comprises the nucleotide sequence of carrier itself and has the enzyme of special properties defined herein or the nucleotide sequence of albumen according to the coding that the present invention uses, and it is connected with strong promoter directly or indirectly.Preferably, expression vector of the present invention comprises the nucleotide sequence of carrier itself and the nucleotide sequence of described signal peptide-Cordyceps sinensis SOD combination, and expression vector be directed in the genome of biology (as escherichia coli host is biological).Stable being incorporated in genome preferably contained in term " introducing ".

In a specific embodiment of the present invention, provide a kind of expression vector, wherein said expression vector contains arbitrary following nucleotide sequence:

A) nucleotide sequence shown in SEQ ID NO.1; And/or

B) nucleotide sequence shown in SEQ ID NO.3; Or

C) nucleotide sequence shown in SEQ ID NO.5.

Preferably, described expression vector is pET22b.

Nucleotide sequence and the signal peptide sequence of coding Cordyceps sinensis SOD as herein defined may reside in carrier, described in this carrier, nucleotide sequence is operably connected to regulating and controlling sequence, make described regulating and controlling sequence can by be applicable to host cell (as intestinal bacteria) express as described in nucleotide sequence, namely described carrier is expression vector.Carrier of the present invention can be transformed in above-mentioned applicable host cell, to express the enzyme or albumen with coding Cordyceps sinensis SOD activity as herein defined.Usually according to it, host cell be introduced into is selected carrier (as plasmid, clay, virus or phage vector, genomic inserts).The present invention can contain performance identical functions and the expression vector of other known in the art or known form.Once be transformed in selected host cell, carrier can not rely on the genome of host cell and copies and play function, or can be integrated into genome voluntarily.

Described carrier can comprise one or more selected marker, as provided the gene of antibiotics resistance, as provided the gene of amicillin resistance, kalamycin resistance, chlorampenicol resistant or tetracyclin resistance.Carrier can use in vitro, such as, for the preparation of RNA or for transfection or transformed host cell.

Expression vector can comprise the nucleotide sequence that this carrier can be made to copy in described host cell further.Described carrier can be pET1-46 serial carrier (NOVAGEN company), pGEX serial carrier, pUC serial carrier, pACYC serial carrier, pUB serial carrier, pE serial carrier, pAMB serial carrier and pIJ serial carrier.Be preferably pET serial carrier, most preferably be pET22b.

The detection of above-mentioned nucleotide sequence has been come by nucleotide sequence sequenator, and whether analyze obtained gene by various molecular biology software is goal gene simultaneously.Conventional analysis software has GENTYX, Vector NTI, GenDOC, DNAman, also can carry out off-line and/or on-line search and retrieval analysis in order to comparison nucleotide sequence and protein sequence by means of BLAST and FASTA.

Term as used herein " enzyme " and term " aminoacid sequence " and/or term " enzyme or albumen " and/or term " albumen " synonym.In some instances, term " aminoacid sequence " and term " peptide " and/or " enzyme " synonym, can alternative each otherly use.

Term as used herein " restructuring Cordyceps sinensis SOD " and term " the restructuring Cordyceps sinensis SOD of warm expression " synonym.

Term " signal peptide " is according to sequence used and/or carrier herein, and the Cordyceps sinensis SOD produced by the nucleotide sequence of encoding Cordyceps sinensis SOD by host cell expression can be secreted or be included in cell.Signal sequence may be used for instructing encoding sequence to secrete by specific cytolemma.Described signal sequence can be natural or external source concerning Cordyceps sinensis SOD encoding sequence.Any signal coding sequence that expressed Cordyceps sinensis SOD can be instructed to enter selected host cell (being preferably e. coli bl21 host cell) Secretory Pathway can be used.

The Cordyceps sinensis SOD that can encode natural for the nucleotide sequence of the coding Cordyceps sinensis SOD in either method of the present invention and/or application or Cordyceps sinensis SOD variant.Such as, for the nucleotide sequence of coding Cordyceps sinensis SOD of the present invention can be described in one.These documents of NCBI are incorporated to herein by reference.

Term used herein " Cordyceps sinensis SOD " preferably refers to that having catalysis superoxide radical is converted into hydrogen peroxide and the enzyme of the function that produces oxygen.

Preferably, the superoxide radical in various organism can be converted into hydrogen peroxide for the Cordyceps sinensis SOD in either method of the present invention and/or application and produce oxygen function.Described organism includes but not limited to animal, plant and microorganism.First-selection is animal, is preferably the mankind.

In certain embodiments of the present invention, the nucleotide sequence of coded signal peptide can be operably connected to the nucleotide sequence of the selected Cordyceps sinensis SOD of coding.Selected Cordyceps sinensis SOD can as expressing fusion protein in host cell defined herein.

The aminoacid sequence coded by the nucleotide sequence for the coding Cordyceps sinensis superoxide-dismutase in either method of the present invention and/or application is also contained in the present invention.

The invention provides a kind of transport piece encoding sequence of the Cordyceps sinensis superoxide-dismutase being particularly suitable for expressing in Bacillus coli cells.This sequence filters out according to principles such as e. coli codon preferences.The special encoding sequence ordinary method for rcSOD secreting, expressing designed carries out full genome synthesis, or realized by PCR method.

Term " cell " herein comprises any cell of product that nucleotide sequence of the present invention or coding have the enzyme of special properties as defined herein or the nucleotide sequence of albumen and/or obtained by it, itself and term " biology " and/or term " bacterium " and/or term " microorganism " and/or term " bacterial micro-organism " synonym, replace use each other.As being also contained in this paper term " cell " at the natural cordyceps vegetable cell of the native nucleotide sequence containing coding natural cordyceps SOD enzyme described by background technology.

The term " engineering cell " relevant with the present invention or " host cell of conversion " comprise and anyly comprise the encode enzyme of special properties that has as defined herein or the nucleotide sequence of albumen and/or the cell of product that obtained by it, and/or wherein promotor can allow the to encode enzyme of the special properties had as defined herein or the nucleotides sequence of albumen is listed in described biology and expresses.Preferred nucleotide sequence is introduced in biological genome.

Term " genetically modified organism " is not encompassed in and is in self natural surroundings and is subject to the natural nucleus glycoside coding sequences that it is in the natural promoter control in self natural surroundings together simultaneously.Therefore, genetically modified organism of the present invention comprises the biology comprising following any one or combination: coding has enzyme or the nucleotide sequence of albumen, construct defined herein, carrier defined herein, plasmid defined herein, fixed cell or its product of special properties as defined herein herein.Such as, described genetically modified organism can also comprise coding to be had the enzyme of special properties as defined herein or albumen and is subject to the nucleotide sequence that promotor controls, and wherein said promotor is not connected with restructuring Cordyceps sinensis superoxide-dismutase encoding gene originally.

In yet further embodiment of the invention, provide a kind of engineering cell, wherein said engineering cell is the bacterial cell by obtaining after transforming above-mentioned expression vector and entering host cell.

Described host microorganism can be protokaryon or eukaryote.The nucleotide sequence of Cordyceps sinensis SOD of the present invention or coding Cordyceps sinensis SOD can available from, include but not limited to the bacterial micro-organism with subordinate: Escherichia (Escherichia), Mycobacterium (Mycobacterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus), Desulfitobacterium (Desulfitobacterium), bacillus (Bacillus), campylobacter (Campylobacter), Vibrio (Vibrionaceae), XyZella (Xylella), sulfolobus solfataricus belongs to (Sulfolobus), Aeromonas (Aeromonas), streptomyces (Streptomyces), Saccharomycodes (Saccharomyces), lactococcus (Lactococcus), , Aspergillus (Aspergillus), Schizosaccharomyces (Schizosaccharomyces), listeria (Listeria), Neisseria (Neisseria), Autoinducer belongs to (Mesorhizobium), Lei Er Bordetella (Ralstonia), xanthomonas (Xanthomonas) and mycocandida (Candida), be preferably Escherichia (Escherichia). be preferably Escherichia (Escherichia).

Aptly, the nucleotide sequence of Cordyceps sinensis SOD of the present invention or coding Cordyceps sinensis SOD can available from, preferably available from following bacterial micro-organism: intestinal bacteria (E.coli), genus bacillus (Bacillus sp), campylobacter jejuni (Campylobacter jejuni), vibrios (Vibrionaceae), xyllela fastidiosa (Xylella fastidiosa), solfatara sulfolobus (Sulfolobus solfataricus), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Aeromonas hydrophila (Aeromonas hydrophila), aeromonas salmonicida (Aeromonas salmonicida), streptomyces coelicolor (Streptomyces coelicolor), streptomyces rimosus (Streptomyces rimosus), mycobacterium (Mycobacterium), micrococcus scarlatinae (Streptococcus pyogenes), Lactococcus lactis (Lactococcus lactis), Ralstonia solanacearum (Ralstonia solanacearum), xanthomonas campestris (Xanthomonas campestris), Xanthomonas axonopodis (Xanthomonas axonopodis), Candida parapsilosis (Candida parapsilosis), thermophilus streptococcus (Streptococcus thermophilus), lactobacterium helveticus (Lactobacillus helveticus), dehalogenation desulfiting bacterium (Desulfitobacterium dehalogenans), terreus (Aspergillus terreus), schizosaccharomyces pombe (Schizosaccharomyces pombe), listera innocua (Listeria innocua), listerisa monocytogenes in mjme (Listeria monocytogenes), Neisseria meningitidis (Neisseria meningitidis), Root or stem of Littleleaf Indianmulberry Autoinducer (Mesorhizobium loti).Be preferably Bacillus coli cells, be more preferably BL21 (ED3) Bacillus coli cells.

In some embodiments of the invention, prepared some competent cells to be beneficial to external carrier and to be transferred in host cell.Conventional prepare competent cell and comprise subzero treatment, such as 16 DEG C.

Moreover being suitable for host living beings of the present invention also can be plant.Therefore, the invention still further relates to the method producing and there are the transgenic plant of enhancement of SOD expression level, it comprises the following steps: utilize Cordyceps sinensis superoxide dismutase defined herein (especially utilizing the expression vector or the construct that comprise Cordyceps sinensis superoxide dismutase defined herein) transformed plant cells, and go out plant by the culture plant cell transformed.In one embodiment, Cordyceps sinensis SOD according to the present invention can be can available from, preferably available from the Cordyceps sinensis SOD (Classification And Nomenclature: colon bacillus in e. coli bl21 JZY001, EscherichiaColi), the microbial preservation budapest treaty that described coli strain JZY001 is used for patented procedure by Jin Zi source, Jilin Province Bioisystech Co., Ltd according to international recognition is deposited in China General Microbiological Culture Collection Center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center, postcode: 100101) preservation day is on 09 29th, 2014, preserving number is CGMCC 9744.

In certain embodiments of the present invention, carried out experimental research to being applicable to the bacterial classification of bacterial micro-organism of the present invention, culture condition, inductive condition and expression fermentation condition etc., described condition also comprises the outside various conditions such as dissolved oxygen, rotating speed and temperature.

After acquisition engineering cell, just can culturing engineering cell under the suitable conditions.Allly be suitable for Escherichia coli Growth, the substratum of expression all can be used for the present invention.Such as, suitable substratum comprises (but being not limited to) following substratum: LB substratum, potato sugar nutrient agar, bean sprouts medium, pea nutrient agar, potato-sucrose-nutrient agar, potato-sucrose-nutrient agar etc., is preferably LB substratum.

Dissolved oxygen condition can control, and the dissolved oxygen be applicable to controls at 20-90%.The maintenance of dissolved oxygen can solve with oxygen, passing into of air gas mixture.

In another preference, the present invention also by fermentation technology optimization, optimizes the expression condition of rcSOD further.Because expression level is high, reach more than 100mg/L, and be solubility expression, great convenience is brought to purifying process, by two-step chromatography technique, increase substantially product yield, obtain the rcSOD product that expression amount is high, specific activity is high, purification is convenient, yield is high and proterties is stable, greatly reduce production cost, be very applicable to scale operation.

Hold at the 5 ' end and 3 ' of optimized gene and introduce specific cleavage site respectively, by the ordinary method of molecular cloning, optimized gene is cloned into expression vector (as pET28, pET22 etc.).Then, be transformed into BL21 (DE3) host cell, with IPTG induction, shaking flask is expressed and is selected high expression level engineering cell.

After acquisition engineering cell, just can culturing engineering cell under the suitable conditions.Allly be suitable for Escherichia coli Growth, the substratum of expression all can be used for the present invention.Such as, suitable substratum includes, but are not limited to following substratum:

A kind of preferred shake flask culture conditions is: the mono-clonal on picking LB flat board, is shake flask culture with LB, is cultured to OD600, use IPTG abduction delivering, and induction 18-21 hour, rcSOD output can reach 50-200mg/L.

In order to scale operation rcSOD, need culturing engineering cell on fermentor tank, express rcSOD, be therefore optimized the pilot scale expression condition of e. coli bl21 (DE3) engineering cell, fermentation-scale is from 1L to 300L, and fermentation capacity is linearly amplified.After optimizing, expression amount is greater than 400mg/L fermented product.

Repeatedly test through of the present invention, expression condition is optimized as follows:

1. for the selection of substratum, during ferment tank, can minimal medium be selected, also necessarily can change on minimal medium basis, but contained ion composition should be similar to minimal medium.

2., with regard to the control of temperature, fermentation and the inducing temperature of rcSOD remain on 16-25 DEG C.

3., with regard to the pH value of inductive phase, inductive phase, pH controlled at 6-8;

4., with regard to the control of dissolved oxygen (DO), DO controls at 20-90%.The maintenance of dissolved oxygen can solve with passing into of oxygen/air mixed gas.

5. with regard to the stream of feed supplement adds, feed supplement kind should comprise the carbon source such as starch, glucose, can feed supplement or mixing feed supplement separately.

6., with regard to inductive phase IPTG concentration, conventional induced concentration all can be used for the present invention, and usual IPTG concentration controls at 0.1-1mM/L.

7. with regard to the usage quantity of Ampicillin Trihydrate, require 100mg-400mg, effectively can suppress varied bacteria growing.

8. with regard to induction time, being not particularly limited, being generally 18-21 hour, is preferably 20 hours.

In certain embodiments of the present invention, extracting Cordyceps sinensis SOD albumen from the bacterium of fermentation.This extractive process is exactly described slightly carrying, and specifically comprises collection thalline, centrifugal acquisition biomass, broken wall stripping albumen, saltouts, the step such as filtration.

The rcSOD expressed is present between cell walls and plasmalemma, and expression level is greater than 200mg/L fermented product, and purifying complex degree is declined greatly.Usually, fermented sample, first with mode collecting cells such as centrifugal or filtrations, is then used N,O-Diacetylmuramidase (4-10mg/g thalline) dissolved cell wall polysaccharide, is made target protein enter in supernatant fluid, and supernatant liquor is containing target protein matter rcSOD.Supernatant liquor by saltouing, the method such as ultrafiltration carries out chromatography purification after carrying out preliminary purification again, also directly chromatography purification can be carried out, describedly saltout as with the thick leach protein of 0-60% neutral sulphates ammonium classification, ultrafiltration ultra-filtration membrane filters, such as, filter with the ultra-filtration membrane that molecular weight cut-off is 10KD.

In certain embodiments of the present invention, the albumen after slightly carrying is further purified.Chromatographic technique comprises the technology such as cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affinity chromatography.Compare through different chromatographic technique, chromatography process, the chromatography method of optimization comprises: 1. anion-exchange chromatography: anion-exchange chromatography medium includes but not limited to Q-Sepharose, DEAE-Sepharose.If the salt concn of fermented sample is higher, impact and the combination of Ion Exchange Medium, then need to reduce salt concn before carrying out ion exchange chromatography.Sample can carry out the replacing of level pad by means such as dilution, ultrafiltration, dialysis, gel permeation chromatographies, until with corresponding ion exchange column balance liquid system similarity, then loading, carry out the gradient elution of salt concn or pH; 2. hydrophobic chromatography: hydrophobic chromatoghaphy medium includes but not limited to Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose.Sample is by adding NaCl, (NH4) ₂sO ₄salt concn is improved, then loading, by reducing salt concn method wash-out etc. mode.The foreign protein of larger difference is had by hydrophobic chromatography removing hydrophobicity.3. gel permeation chromatography hydrophobic chromatoghaphy medium includes but not limited to Sephacryl, Superdex, Sephadex class.By gel permeation chromatography exchange buffering system, or consummate further.

Can obtain rcSOD sterling through above-mentioned purifying, purification yield is generally more than 40%, purity more than 95%, and sterling yield is about 200mg/L culture.RcSOD using conventional procedures after purifying makes various formulation, as lyophilized injectable powder.Select certain stablizer, also can add the protective material such as tensio-active agent, antioxidant simultaneously, and suitably damping fluid carries out lyophilize.The goods obtained have that loss of activity is little, moisture content is low, good stability, be easy to the features such as long-term preservation.RcSOD also can make aqueous injection, sprays, microsphere sustained-release agent, and makes prolonged action preparation etc. through PEG modification.

In an example of the present invention, the BL21 (ED3) constructing rcSOD expresses engineering cell, and IPTG induces, and high-level secretory expression rcSOD, does not need renaturation.

In another example of the present invention, parameter optimization by fermentation, improves the expression level of rcSOD.

Because expressing protein belongs to exocytosis type, do not need brokenly bacterium process.In another example of the present invention, through purifying, obtain rcSOD sterling, purification yield more than 50%, purity more than 95%, sterling yield is about 200mg/L culture.

In another embodiment of the present invention, after purifying, stoste adds suitable auxiliary material, makes the powder ampoule agent for injection of rcSOD.Stability test shows, this powder injection is stablized.

Preferably, the combination of described incubation temperature and incubation time effectively can ensure that the SOD of at least 5% is active, preferably the SOD of at least 10% is active, the preferably enzymic activity of at least 15%, 20%, 25%, 26%, 28%, 30%, 40%, 50%, 60% or 75%.

In yet another embodiment, sample obtains after the displacement of 10KD film bag ultrafiltration and concentration damping fluid, Q Sepharose F.F. and phenyl sepharose F.F. chromatography that purity is greater than 95%, the sterling of yield more than 50%.

In sum, in a complete embodiment, the invention provides a kind of method preparing restructuring Cordyceps sinensis superoxide-dismutase, wherein, described method comprises the steps:

A) following arbitrary nucleotide sequence is cloned into the step of expression vector pET22b, described nucleotides sequence is classified as: the nucleotide sequence shown in SEQ ID NO.1, and/or the nucleotide sequence shown in SEQ ID NO.3, or the nucleotide sequence shown in SEQ ID NO.5;

B) described expression vector pET22b is transferred to the step of host cell E. coli BL21 (ED3) cell;

C) host cell E. coli BL21 (ED3) cell is carried out culturing step, culture condition comprises: cultivate and be divided into cultivation stage and induction period, cultivation stage is cultivated bacterial concentration and is reached OD600, induction time is 18-21hr, fermentation and inducing temperature remain on 16-25 DEG C, and inductive phase, IPTG concentration was at 0.1-0.2mM/L;

D) separation and purification goes out to melt the step of the restructuring Cordyceps sinensis superoxide-dismutase of nuclear expression, thalline is collected, with N,O-Diacetylmuramidase 1mg/g thalline, under 37 DEG C of conditions by centrifugal and/or filter type to fermented sample, process 2 hours, then by centrifugal acquisition clear liquid;

E) by saltout and/or any preliminary purification step is carried out in ultrafiltration, described in saltout as with the thick leach protein of 0-60% neutral sulphates ammonium classification, ultrafiltration ultra-filtration membrane filters, the ultra-filtration membrane of described ultra-filtration membrane to be molecular weight cut-off be 10KD;

F) by anionresin, hydrophobic chromatography method, thus the step that purity reaches the restructuring Cordyceps sinensis superoxide-dismutase of 95-99.9% is obtained.

In yet another embodiment of the present invention, there is provided and use the purposes of the Cordyceps sinensis SOD for preparing of aforesaid method in the medicine of preparation treatment and human superoxide dismutase relative disease or pathological symptom, food, healthcare products and makeup thereof, described restructuring Cordyceps sinensis superoxide-dismutase contains arbitrary following aminoacid sequence:

A) aminoacid sequence shown in SEQ ID NO.2; And/or

B) aminoacid sequence shown in SEQ ID NO.4; Or

C) aminoacid sequence shown in SEQ ID NO.6.

Above-mentioned relative disease or pathological symptom include but not limited to: cataract, degenerative ocular fundus pathology, newborn infant's retinopathy, atopic dermatitis, pigmentation blackspot, senile plaque, allergic, hypertension, arteriosclerosis, myocardial infarction, amyocardia, Arrhythmias, nose is irritated, asthma, pulmonary emphysema, pulmonary fibrosis, adult respiratory distress, senile dementia, Parkinson's disease, disturbance of cerebral circulation, sickle anaemia, broad bean disease, lead poisoning, nephropathy, proteinuria, urocystitis, behign prostate hypertrophy, the inflammation of kidney pompon, constipation, halitosis, diabetes and common complication, rheumatic arthritis, cancer, chemotherapy side effect is performed the operation, organ transplantation, hepatitis, climacterium, ageing diseases (aging, aging) etc.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, such as Sambrook etc., Molecular Cloning: A Laboratory guide (New York:ColdSpring Harbor Laboratory Press, 1989); Condition described in Protein Purification (Jan-Christer Janson etc., John Wiley & Sons, 1998), or according to the condition that manufacturer advises.

Embodiment

The structure of embodiment 1.rcSOD coli expression carrier:

The design of membranin signal peptide shown in transport piece gene reference e. coli k12 strain secretory protein and SEQ ID NO:2 (Fig. 2), and full genome synthesis, the DNA fragmentation (Fig. 1) of its sequence as shown in SEQ ID NO:1.Aminoacid sequence wherein for the nucleotide sequence of code book invention signal peptide and the signal peptide of the present invention of its deduction is shown in Fig. 7.Introduce NdeI restriction enzyme site at its 5 ' end simultaneously.As shown in SEQ ID NO:4, shown in the aminoacid sequence (Fig. 4) of natural cordyceps superoxide-dismutase and SEQ ID NO:3, nucleotide sequence (Fig. 3) is known, the Genbank number of logging in as NCBI is respectively AY195841.1 and AAO40743.1, the acquisition of goal gene is the aminoacid sequence according to natural cordyceps superoxide-dismutase, use conventional gene synthesis method, transport piece gene is introduced at 5 ' end of the aminoacid sequence of Cordyceps sinensis superoxide-dismutase, and introduce XhoI restriction enzyme site at 3 ' end of the aminoacid sequence of Cordyceps sinensis superoxide-dismutase, this genes of SEQ ID NO:5 encodes secretion peptide-Cordyceps sinensis superoxide-dismutase aminoacid sequence as shown in SEQ ID NO:6 (Fig. 5 and Fig. 6).The aminoacid sequence corresponding relation inventing the restructuring Cordyceps sinensis SOD of the nucleotide sequence of the restructuring Cordyceps sinensis SOD of warm expression and the warm expression of the present invention of deduction thereof for code book is shown in Fig. 8.

The structure of expression plasmid pET-rcSOD and the acquisition building process of high expression level engineering cell are as shown in Figure 9.Secretion peptide-superoxide-dismutase full genome NdeI+XhoI restriction endonuclease (the deriving from NEB Biolab) enzyme of synthesis is cut, carrier pET22b is carried out enzyme with identical NdeI+XhoI to cut simultaneously, then NEB TAKARADNA ligase enzyme (solution1 is used, TAKARA, DaLian, China) connect in pET22b carrier, obtain pET-rcSOD, order-checking confirms that DNA sequence dna is correct.Then, by pET-rcSOD plasmid, be converted in e. coli bl21 (DE3), under 37 DEG C of conditions, determine after inducing 4 hours with IPTG to express Cordyceps sinensis superoxide-dismutase (Figure 10).

Embodiment 2 temperature is on the impact of rcSOD expression level

Get mono-clonal, be inoculated in LB primary seed solution, cultivate 17-20hr; Get 3 50mL culture tubes, ratio in 1: 50 is inoculated in the 50mL culture tube of three 5mL substratum respectively, cultivate about 4hr, 1mM/LIPTG induction, inducing temperature is respectively 16 DEG C, 25 DEG C, 37 DEG C, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, get supernatant, SDS-PAGE detects expression.Experimental result shows, expresses rcSOD at 16 DEG C, 25 DEG C all almost almost in shaking flask, but 37 DEG C of expression-secretion albumen relatively less (Figure 11).

Embodiment 3IPTG concentration is on the impact of rcSOD expression level

Get mono-clonal, be inoculated in LB primary seed solution, cultivate 17-20hr; Ratio in 1: 50 is inoculated in 5 respectively and is equipped with in the 50mL culture tube of 5mL substratum, cultivate about 4hr for 37 DEG C, be cooled to 20 DEG C, add IPTG respectively, make concentration in substratum be 0.6mM, 0.4mM, 0.2mM, 0.1mM induction, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, get supernatant, SDS-PAGE detects expression.Experimental result shows, several gradient concentration IPTG induces rcSOD expression amount similar, with the IPTG concentration induction ratio less expensive of 0.1mM, cost-saving (Figure 12).

Embodiment 4 induction time is on the impact of rcSOD expression level.Get mono-clonal, be inoculated in LB primary seed solution, cultivate 5hr; Ratio in 1: 50 is inoculated in 5 respectively and is equipped with in the 50mL culture tube of 5mL substratum, cultivate about 4hr for 37 DEG C, be cooled to 25 DEG C, add the IPTG of 0.1mM concentration respectively, induce 8,10,16,18,20,30 hours respectively, collect thalline, with N,O-Diacetylmuramidase under 37 DEG C of conditions, process 2 hours, centrifugal 10 minutes of 15000rpm, get supernatant, SDS-PAGE detects expression.Experimental result shows, induces 20 hourly outputs the highest (Figure 13).

Embodiment 5 pilot scale fermentation condition is selected and is optimized NBS BioFlo 3000 fermentor tank (6.5L) after above-mentioned optimization, and rcSOD expression amount is about 300mg/L fermented liquid, and expression amount obviously increases (Figure 14).

In embodiment 6 rcSOD purifying embodiment 4 fermented product 4 DEG C at centrifugal 10 minutes of 5000rpm, collect thalline, dissolve thalline with N,O-Diacetylmuramidase, lytic enzyme usage quantity is 1mg/g thalline, 37 DEG C of temperature baths 2 hours.From 15000rpm centrifugal segregation thalline, collect clear liquor.Use Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes phegma during ultrafiltration.Supernatant ultrafiltration is left about 500ml to volume, adds with 10mM phosphate buffered saline buffer (PB, pH7.4), continues ultrafiltration; This program repeatedly, until the conductance of the conductivity water of sample gentle 10mM PB (pH7.4) damping fluid is on close level.

Anion-exchange chromatography: chromatography media: Q Sepharose F.F. (Phamarcia) damping fluid: solution A: 10mM PBS (pH7.4), solution B: 10mM PB+1M NaCl (pH7.4) loading: by rcSOD solution (about the 1L of ultrafiltration and concentration, conductance is about 4000us/cm) loading cleaning: by the solution A cleaning chromatography column gradient of 6CV (column volume, lower same) after loading: directly collect with 30% solution B wash-out target protein after cleaning: the protein chromatographic under collection 30%B wash-out.

Hydrophobic chromatography: phenyl-sepharose (Phamarcia) damping fluid: solution A: 10mM PBS (pH7.4), solution C: 10mM PB+3M NaCl (pH7.4) loading: the sample that chromatography 1 obtains is added solid NaCl to 3.0M, loading gradient: 100%A directly cleans collection: the target protein sample under collection 100%A washes is through this 2 step chromatography, namely after anion-exchange chromatography, hydrophobic chromatography, purity is increased to 95% (Figure 15 and Figure 16), the yield rcSOD sterling more than 50%, sterling yield 200mg/L culture.

The vigour-testing method of SOD

The assay NBT photoreduction of improvement is simple, comparatively practical.Chemoluminescence method and Nitrite formation method are not suitable for mensuration Mn-SOD, but extremely sensitive for Cu/Zn-SOD reaction.Cyte reduction method is used for Mn-SOD vitality test result and stablizes, reproducible.But specificity and insufficient sensitivity ideal, and need specific apparatus, practical application is restricted.Nitrite forming method combines with CN-inhibitor or SDS process, be applied to Mn-SOD to measure, remolding sensitivity Cyte reduction method improves several times, and specificity is strong, reproducible, easy to operate, do not need specific apparatus and equipment, being easy to practical application and popularization, is current one of measuring method preferably.Under normal circumstances, SOD enzyme activity measures and can only apply indirect activity assay method.According to SOD national standard, our company adopts improvement Marklund method, i.e. mouse thymus cells method.

1 definition: suppress pyrogallol required SOD amount when oxygen words speed 50% to be a unit of activity when 25 DEG C.

2 principles: in the basic conditions, can there is autoxidation in pyrogallol, mouse thymus cells ability can be suppressed to measure SOD vigor according to SOD.

3 reagent: A liquid: pH8.200.1mol/L tri-rifle aminomethane (Tris)-hydrochloride buffer (including 1mmol/L EDTA.2Na).Take 1.214g Tris and 37.2mg EDTA.2Na is dissolved in 62.4mL0.1mol/L hydrochloric acid soln, be settled to 100ml with distilled water.B liquid: 4.5mmol/L pyrogallol hydrochloric acid soln.Take pyrogallol (A.R) 56.7mg to be dissolved in a small amount of 10mmol/L hydrochloric acid and to be dissolved in, and be settled to 100mL.

4 instruments: ultraviolet-visible spectrophotometer, secret acidometer, tolerance range 0.01pH, whizzer, 10mL cuvette, 10mL centrifuge tube, glass mortar.

The preparation of 5 samples:

6 analytical procedures: mouse thymus cells rate determination, at about 25 DEG C, once add A liquid 2.35mL in 10mL cuvette, distilled water 2mL, B liquid 0.15mL.Add B liquid to mix immediately and impouring cuvette, light absorption value after initial and 1min under being determined at 325 wavelength condition respectively, the difference of the two and mouse thymus cells speed △ A ₃₂₅(min ^-1). △ A is determined in this test ₃₂₅(min ^-1) be 0.060.SOD determination of activity application of sample programsheet is as follows:

Test solution	Blank	Sample liquid	SOD liquid
				A liquid/ml	2.35	2.35	2.35
Distilled water/ml	2	1.8	1.8
				Sample liquid or SOD liquid/ul	0	20	20
B liquid/ml	0.15	0.15	0.15

7 results calculate: liquid sample is calculated as follows:

In formula:

U/mL:SOD enzyme activity unit

△ A325: mouse thymus cells speed

△ A ' 325: sample liquid or SOD enzyme liquid suppress mouse thymus cells speed

V: the enzyme-added liquid of institute or sample liquid volume, unit is milliliter (mL)

D: the extension rate of enzyme liquid or sample liquid

4.5: reaction solution cumulative volume, unit is milliliter (mL)

Calculation result retains 3 position effective digitals.

Solid sample calculation formula is as follows:

In formula:

U/g:SOD enzyme activity unit

△ A325: mouse thymus cells speed

V1: sample liquid cumulative volume, unit is milliliter (mL)

D: the extension rate of enzyme liquid or sample liquid

M: sample quality, unit is gram (g)

4.5: reaction solution cumulative volume, unit is milliliter (mL)

Calculation result retains three position effective digitals.

The all documents mentioned are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

The present invention does not limit to the particular implementation described in this application, as the unitary declaration of independent aspect of the present invention.Various amendment and change is carried out when it will be understood by those skilled in the art that the spirit and scope that can not depart from the application.According to above description, except enumerating herein, the functionally equivalent purposes in the scope of the present disclosure is obvious for those skilled in the art of this area.Such change and amendment are intended to fall within the scope of the appended claims.The disclosure only by claims and with such claim the four corner that is equal to of scope limit.Should be appreciated that the disclosure is not limited to specific method, reagent, composition and biosystem, certainly, described method, reagent, composition and biosystem can change.It can also be appreciated that term used herein only for describing specific embodiment, it is restrictive for not being used for.

All patents that are referred herein or that quote, patent application, earlier application and publication are incorporated to herein by reference and in full, comprise all accompanying drawings and form, they are not contradicted with the clearly instruction of this specification sheets.Other embodiment is proposed in right.

Claims

1. a restructuring Cordyceps sinensis superoxide-dismutase, it is characterized in that, described Cordyceps sinensis superoxide-dismutase comprises arbitrary following aminoacid sequence:

A) aminoacid sequence shown in SEQ ID NO.2; And/or

B) aminoacid sequence shown in SEQ ID NO.4; Or

C) aminoacid sequence shown in SEQ ID NO.6.

2. the nucleic acid molecule be separated, it is characterized in that, the nucleic acid molecule of described separation comprises arbitrary following nucleotide sequence:

3. a restructuring Cordyceps sinensis superoxide-dismutase, is characterized in that, described Cordyceps sinensis superoxide-dismutase obtains by expressing arbitrary nucleotide sequence shown below:

4. an expression vector, is characterized in that, described expression vector contains arbitrary following nucleotide sequence:

A) nucleotide sequence shown in SEQ ID NO.1; And/or

B) nucleotide sequence shown in SEQ ID NO.3; Or

C) nucleotide sequence shown in SEQ ID NO.5.

5. expression vector as claimed in claim 4, it is characterized in that, described expression vector is pET22b.

6. an engineering cell, is characterized in that, described engineering cell is the bacterial cell by obtaining after transforming expression vector according to claim 4 and entering host cell.

7. engineering cell as claimed in claim 5, it is characterized in that, described engineering cell is e. coli bl21 (ED3) cell.

8. prepare a method for restructuring Cordyceps sinensis superoxide-dismutase, it is characterized in that, described method comprises:

A) arbitrary nucleotide sequence according to claim 3 is cloned into the step of expression vector pET22b;

9. restructuring Cordyceps sinensis superoxide-dismutase is preparing the purposes in the medicine for the treatment of and human superoxide dismutase relative disease or pathological symptom, food, healthcare products and makeup thereof.

10. method as claimed in claim 8 or purposes according to claim 10, it is characterized in that, described restructuring Cordyceps sinensis superoxide-dismutase contains arbitrary following aminoacid sequence:

A) aminoacid sequence shown in SEQ ID NO.2; And/or

B) aminoacid sequence shown in SEQ ID NO.4; Or

C) aminoacid sequence shown in SEQ ID NO.6.