CN104342408B - Recombinate the preparation method of Cordyceps militaris superoxide dismutase - Google Patents
Recombinate the preparation method of Cordyceps militaris superoxide dismutase Download PDFInfo
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- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The present invention provides a kind of signal peptide nucleotide sequence of suitable E. coli secretion expression restructuring Cordyceps militaris superoxide dismutase (recombinant cordyceps militaris superoxide dismutase, rcSOD) and the SOD nucleotide sequences of coding Cordyceps militaris SOD, the carrier containing above-mentioned nucleotide sequence, the engineering cell containing aforementioned bearer and the method from engineering cell purifying rcSOD.Superoxide dismutase gene after optimization is very suitable for Bacillus coli expression, has the characteristics that high expression, high stable, hypersecretion.The present invention can efficiently, it is easy, obtain rcSOD sterlings at low cost.Present invention additionally comprises preparing treatment and the application in the drug of human superoxide dismutase relevant disease or pathological symptom, food, health products and its cosmetics by restructuring Cordyceps militaris superoxide dismutase prepared by the above method.
Description
Technical field
The nucleosides of the encoded signal peptide of restructuring Cordyceps militaris superoxide dismutase is expressed the present invention relates to E. coli secretion
The nucleotide sequence of acid sequence and coding Cordyceps militaris SOD, the carrier containing above-mentioned nucleotide sequence, the engineering containing aforementioned bearer
Cell and the method and application thereof from engineering cell purifying rcSOD, belong to genetic engineering and art of protein biochemistry.
Background technology
Superoxide dismutase (superoxide dismutase, SOD) is that one kind can be catalyzed superoxides and pass through discrimination
Change the enzyme that reaction is converted into oxygen and hydrogen peroxide.It is widely present in all kinds of animals, plant, microorganism, is a kind of important
Antioxidant, cell of the protection in the oxygen.Three kinds can be divided by its contained metal prothetic group difference, the first is cupric
(Cu) title (Cu.Zn-SOD) of zinc (Zn) metal prothetic group, a kind of most commonly seen enzyme in green, are primarily present in body cell
In slurry；It is for second the title (Mn-SOD) containing manganese (Mn) metal prothetic group, in purple, is present in the mitochondria and original of eukaryocyte
In nucleus；The third is the title (Fe-SOD) of iron content (Fe) metal prothetic group, in yellowish-brown, is present in prokaryotic cell.
Superoxide dismutase can remove superoxides, protect cells from oxidative damage.Superoxides with it is non-free
The reaction of base is spin forbidden.In biology system, this means that it is mainly and itself (disproportionation) or another life
Object free radical (such as nitric oxide) reacts.Superoxide anion radical (O2 -) (in pH=7, can react quickly
Speed is 105M-1s-1) it is disproportionated into O2With hydrogen peroxide (H2O2).But superoxides can be with specific molecule (such as NO free radicals)
It reacts at faster speed, generation peroxynitrite (O=N-O-O-), therefore the catalytic action of superoxide dismutase
Just it is particularly important.Moreover, the disproportionated reaction of superoxides and its initial concentration is square related, thus while high concentration
Superoxides half-life period is very short (for example being 0.05 second under 0.1mM concentration), but the half-life period of the superoxides of low concentration is considerably long
When small (under 0.1nM concentration up to 14).In comparison, the disproportionated reaction of superoxide dismutase catalysis is at the beginning of superoxides
Beginning concentration is first order reaction, and has most fast conversion number (with substrate reactions rate) (7x 10 in all known enzymes9M-1s-1)(Loeffler Petrides Heinrich.Biochemie&Pathobiochemie.8th
Edition.2007.123.), therefore the limitation of reaction rate is simply enzyme and the intermolecular collision frequency of superoxides, i.e.,
Reaction rate is " diffusion limited ".
Superoxides is one of main active oxygen in cell, therefore superoxide dismutase has played the anti-oxidant of key
The effect of agent.Superoxide dismutase has important physiological role, the improved mouse meeting for lacking the enzyme of genetic engineering
Suffer from serious disease.For example, lack SOD2 mouse after birth a couple of days die of serious oxidation emergency (Li, 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 for lacking SOD1 has extensive pathological characters, including 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.), accelerate with the quality of age relevant muscle 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 service life is reduced；The mouse for lacking SOD3 does not show
Go out any apparent defect and with the normal service life, however they are easier to that high oxygen injury occurs.Knock out any super oxygen
The mouse of compound mutase is easier to die of the medicament of superoxides production, such as paraquat (paraquat) and diquat dibromide
(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.M5 10764200.)。
The drosophila for lacking SOD1 has the service life significantly shortened, and the drosophila for lacking SOD2 then just dies before birth.
SOD is knocked out in nematode nematode does not cause serious physiology disorderly.The knockout of SOD1 or deletion mutation are for saccharomyces cerevisiae in nothing
Oxygen environment growth is very harmful, and causes the notable shortening of rear diauxic growth phase (post-diauxic lifespan)；And SOD2
Knockout or deletion mutation can then cause growth inhibition and equally reduce after the diauxic growth phase.
Cordyceps militaris also contains SOD, but content is extremely low.In order to obtain Cordyceps militaris SOD, we utilize modern genetic engineering skill
Art, has attempted expression Cordyceps militaris SOD protein engineering bacterium and its carrier, and present system optimization changes acquisition Cordyceps militaris SOD's
The method of engineered E. coli cell is obtained more using the secretory piece of bacillus coli gene expression with SOD amalgamation and expressions
It is satisfied as a result, and pass through the improvement of zymotechnique, while easy purifying process, obtain high expression, high activity and high yield pulp1, pole
Have a kind of copper zinc type rcSOD production methods of industrialization value.
The content of the invention
Shortcoming based on the prior art, the object of the present invention is to provide encode the nucleotide sequence of above-mentioned signal peptide, contain
The carrier of the signal peptide nucleotide sequence and Cordyceps militaris SOD nucleotide sequences, the engineering cell containing aforementioned bearer and from this
The method that engineering cell purifies rcSOD.
Inventors of the present invention have surprisingly found that in expression such as SEQ ID NO：Cordyceps militaris SOD nucleotide sequences shown in 3
Before, add in such as SEQ ID NO：Signal peptide nucleotide sequence shown in 1 can unexpectedly obtain the Cordyceps militaris of high amalgamation and expression
The carrier of SOD, and be easy to purify using the SOD that the carrier is expressed, purity is high, receipts amount is big, and activity is high.
The nucleotide sequence of Cordyceps militaris superoxide dismutase is being encoded it is still another aspect of the present invention to provide a kind of
It is preceding to add in a secretory piece that make to be secreted into cell membrane and plasmalemma gap after Cordyceps militaris Expression of Superoxide Dismutase
Nucleotide sequence, the nucleotide sequence SEQ ID NO：1 can encode SEQ ID NO：Amino acid sequence shown in 2, and
The nucleotide sequence SEQ ID NO：1 and Cordyceps militaris superoxide dismutase coding region sequence SEQ ID NO：3 fusions are formed
Nucleotide sequence SEQ ID NO：5.
In another aspect of the invention, a kind of expression vector is provided, it contains the above-mentioned nucleotide sequence of the present invention.
Preferably, the expression vector is pET22b.
In another aspect of the invention, a kind of engineering cell is provided, it is Sequence Transformed by the expression vector
Host cell and obtain, and be integrated in expression vector coding recombinant superoxide dismutase nucleotide sequence.
Preferably, the engineering cell is that coli strain is BL21 (DE3).
In further aspect of the invention, a kind of expression for producing Cordyceps militaris superoxide dismutase is provided, is wrapped
Include following steps：In the case where being suitble to expression condition, the above-mentioned host cell of the culture present invention, so as to secreting, expressing Cordyceps militaris super oxygen
Object mutase；Preferably, the host cell is that coli strain is BL21 (DE3).The culture includes：Culture is divided into training
The stage of supporting and induction period, cultivation stage culture bacterial concentration reach OD600, and time induction period is 18-21hr, fermentation and induction
Temperature is maintained at 16-25 DEG C, and IPTG dosages are 0.05-1mM/L, and the pH value of induction period is 6-8.The e. coli host cell
Including synthesis Cordyceps militaris superoxide dismutase can be induced with IPTG, and cell membrane and plasmalemma gap can be secreted into
Bacillus coli cells.
In another aspect of the present invention, to recombinating Cordyceps militaris SOD after expression slightly carry and be further purified.It is wrapped
Include following steps：(1) thalline is collected by centrifugation and/or filter type to fermented sample, with bacteriolyze enzymatic treatment thalline, then passes through
Centrifugal method obtains clear liquid, and (2) are by saltouing and/or ultrafiltration is carried out after preliminary purification or directly by anion exchange, hydrophobic
Chromatography method obtains the sterling that purity reaches more than 95% (such as 95-99.9%), the restructuring Cordyceps militaris SOD prepared by the present invention
Activity is in 5500U or more than 6000U.
In another aspect of the present invention, the present invention is provided the restructuring Cordyceps militaris SOD prepared using the above method and prepared
Treatment and the purposes in the drug of human superoxide dismutase relevant disease or pathological symptom, food, health products and its cosmetics.
Main advantages of the present invention are：(1) the link secretion signal peptide gene after optimizing is very suitable for Escherichia coli table
It reaches, there is high expression, high stable, hypersecretion；(2) host of the superoxide dismutase gene containing secreting signal peptide
Bacterial strain has the characteristic of high-density growth, is very suitable for the fermenting and producing of genetically engineered drug large scale and high density.Escherichia coli
The hgSOD of expression is secreted into cell membrane and plasmalemma gap in the form of soluble, active；Product is not necessary to renaturation, Ke Yizhi
Tap into capable purifying；Simultaneously because expression is high, purifying process complexity greatly simplifies, and yield is greatly improved；(3)
Compared with eukaryotic system is expressed, cost substantially reduces, and industrialization value is improved significantly.
Description of the drawings
Fig. 1 shows to encode the nucleotide sequence of signal peptide of the present invention.
The amino acid sequence of Fig. 2 signal peptides of the present invention.
Fig. 3 shows to encode the nucleotide sequence of Natural C.militaris SOD of the present invention.
The amino acid sequence of Fig. 4 Natural C.militaris SOD of the present invention.
Fig. 5 shows to encode the nucleotide sequence of present invention restructuring Cordyceps militaris SOD.
The amino acid sequence of Fig. 6 present invention restructuring Cordyceps militaris SOD.
Fig. 7 is used to encode the amino acid sequence of the nucleotide sequence and the signal peptide of the present invention by its presumption of signal peptide of the present invention
The corresponding diagram of row.
The nucleotide sequence and this hair by its presumption that Fig. 8 is used to encode the restructuring Cordyceps militaris SOD of the warm expression of the present invention
The corresponding diagram of the amino acid sequence of the restructuring Cordyceps militaris SOD of bright warm expression.
Fig. 9 is the structure schematic diagram of expression plasmid pET-rcSOD of the present invention a kind of.
Figure 10 is rcSOD expression electrophoretograms under the conditions of 37 DEG C of shaking flask.Each swimming lane is as follows：Before swimming lane 1, induction；Swimming lane 2 lures
After leading, induction 4 samples when small, SDS-PAGE electrophoresis detection expressions.
Figure 11 is that (IPTG concentration is 1mM/L) rcSOD expresses electrophoretogram under condition of different temperatures.Induce 20 it is small when after,
Thalline is collected, with lysozyme under the conditions of 37 DEG C, when processing 2 is small, 15000rpm is centrifuged 10 minutes, takes supernatant, SDS-PAGE inspections
Survey expression.
Figure 12 is influence of the IPTG concentration to rcSOD expressions.Under the conditions of 20 DEG C, respectively with 0.6mM, 0.4mM,
The IPTG induced expressions of 0.2mM, 0.1mM concentration.Induce 20 it is small when after, collect thalline, with lysozyme under the conditions of 37 DEG C, processing
2 it is small when, 15000rpm is centrifuged 10 minutes, takes supernatant, SDS-PAGE detection expressions.
Figure 13 is influence of the induction time to rcSOD expressions.Under the conditions of 25 DEG C, respectively induce 8,10,16,18,
20th, 30 it is small when after, collect thalline, with lysozyme under the conditions of 37 DEG C, processing 2 it is small when, 15000rpm centrifuge 10 minutes, take
Clearly, SDS-PAGE detects expression.
Figure 14 is the expression electrophoretogram of rcSOD in 6.5L fermentation tanks under the conditions of 25 DEG C.Each swimming lane is as follows：1st, lure
Leading samples；2nd, sampled when induction 20 is small.
Figure 15 is the electrophoretogram of rcSOD after purification.RcSOD is expressed in 6.5L fermentation tanks, thalline is collected, uses lysozyme
Under the conditions of 37 DEG C, processing 2 it is small when, 15000rpm centrifuge 10 minutes, take supernatant, with anion-exchange chromatography, hydrophobic chromatography with
Protein SDS-PAGE electrophoretogram afterwards.
Figure 16 is that freeze dried powder product molecular sieve Superdex75 (Phamarcia) method detection product is pure after purification
The chromatography collection of illustrative plates of degree.
Specific embodiment
The substantive of the present invention is understood in order to provide, the present invention certain is hereinafter described with different the level of detail
A little aspects, pattern, embodiment, modification and feature.
In implementing the present invention, it may, molecular biology, genetic engineering, science of heredity, cellular elements biology are used
, biochemistry, protein biochemistry, protein biochemistry engineering, pharmaceutical technology, medicine, physiology, pathology,
(the Molecular Cloning such as zoopery, Sambrook:A Laboratory Manual,2nd edition,1989,
Cold Spring Harbor Laboratory Press) etc. many traditional technologies and basic theory.These technologies are known
's.
In an embodiment of the invention, a kind of restructuring Cordyceps militaris superoxide dismutase is provided, wherein, it is described
Cordyceps militaris superoxide dismutase includes any following amino acid sequence：
A) amino acid sequence shown in SEQ ID NO.2；And/or
B) amino acid sequence shown in SEQ ID NO.4；Or
C) amino acid sequence shown in SEQ ID NO.6.
In the another embodiment of the present invention, provided by molecular cloning and encode the signal peptide and Cordyceps militaris super oxygen
The nucleotide sequence of compound mutase.
The term " nucleotide sequence " of the present invention includes genomic DNA, cDNA, the DNA and RNA of synthesis.Preferably, refer to
The cDNA of DNA, more preferably coded sequence.Present invention also contemplates that enzyme or egg of the coding with special properties as herein defined
White nucleotide sequence.The term as used herein " nucleotide sequence " refers to oligonucleotide sequence or polynucleotide sequence and its change
Body, homologue, segment and derivative (such as its part).The nucleotide sequence can be derived from genome or synthetic or restructuring
Object can be double-strand or single-stranded (no matter it represents positive-sense strand or antisense strand) nucleic acid molecules.
In the specific embodiment of the present invention, a kind of separated nucleic acid molecules are provided, wherein, it is described separated
Nucleic acid molecules include any following nucleotide sequence：
A) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.1 of 2 amino acid sequence；And/or
B) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.3 of 4 amino acid sequence；Or
C) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.5 of 6 amino acid sequence.
Nucleotide sequence of the coding with enzyme as herein defined or albumen can be from times for generating the enzyme or albumen
It is isolated in what cell or biology.Various methods for isolated nucleic acid sequence are all well known in the art.It is only herein
Examples Propertiess, such as total serum IgE is extracted using strong denaturant, using transcription and reverse transcription method, generate the life of the enzyme or albumen
The chromosomal DNA or mRNA (mRNA) of object builds genomic DNA and/or cDNA library.
Also it is possible to the nucleotides sequence for encoding the enzyme or albumen is prepared by synthesizing by ripe standard method
Row, for another example, using synthetic oligonucleotide on automatic dna synthesizer, be then purified, anneal, connect and be cloned into it is appropriate
In carrier.
Therefore, the nucleotide sequence can be derived from the genome and synthesis source of mixing, the synthesis of mixing and cDNA
Source or the genome of mixing and cDNA sources, according to standard technique by connecting from synthesis, genome or cDNA
Segment as needed and be made.The segment each connected corresponds to the different piece of entire nucleotide sequence.The DNA sequence dna
It can also be prepared using specific primer by PCR (PCR).
The terms " PCR " are referred to as PCR (polymerase chain reaction, PCR)
Refer under the catalysis of archaeal dna polymerase, by being denatured, withdrawing from a secret society or underworld gang, the iterative cycles such as polymerization amplification reach geometry order of magnitude amplification position
DNA section between two sections of known arrays.
The polynucleotides (nucleotide sequence) of the present invention can be used to prepare primer, such as PCR primer, it is anti-for optional amplification
The primer answered；Or polynucleotides can be cloned into carrier.The length of the primer and other segments can be at least 12
It is a, such as at least 15, be preferably at least 20, at least such as 25,30,35 or 40 nucleotide, and it is also covered by such as
Within the term as used herein polynucleotides.
In general, preparing coding using recombinant DNA technology (DNA recombinated) has special properties as herein defined
The nucleotide sequence of enzyme or albumen.Therefore, in the optional embodiment of the present invention, known in the artization can be used
Method synthesizes all or part of nucleotide sequence.
In general, primer can be prepared by synthetic method, this method includes progressively making in a manner of per next nucleotide
Standby required nucleotide sequence.It is easily obtained in this field using automatic technology to realize the technology of the above method.Primer is set
Meter can need to consider to add in code tag albumen in destination protein N-terminal or the nucleotide sequence of C-terminal according to purity is improved
The nucleotide sequence of (Tagged-protein expression system), common label protein include but not limited to His-
Tag albumen, Flag-tag albumen, GST-tag albumen etc..
It can recombinate, synthesize or according to the present invention more to prepare by the available any method of those skilled in the art
Nucleotide (such as DNA polynucleotide).They can also be cloned by standard technique.Usually using recombination method, such as use
PCR (PCR) clone technologies prepare longer polynucleotides.This includes preparing the fat positioned at required clone
Matter targets the pair of primers (such as from about 12 to 40 nucleotide) of sequence area flank, makes what primer contact was obtained from Cordyceps militaris
MRNA or cDNA carries out PCR under conditions of it can expand desired zone, separates the segment of amplification (as led to
Cross the purification reaction mixture on Ago-Gel), and recycle the DNA of amplification.Introducing, which can be designed, makes that it includes suitable limits
Property enzyme recognition site processed, so as to the DNA clone that will expand into suitable cloning vector.
As set forth above, it is possible to it recombinates, synthesize or basis is prepared by the available any method of those skilled in the art
The polynucleotides (such as DNA polynucleotide) of the present invention.They can also be cloned by standard technique.Usually using restructuring side
Method such as prepares longer polynucleotides using PCR (PCR) clone technologies.This includes preparing positioned at required
The pair of primers (such as from about 15 to 30 nucleotide) of the lipid targeted sequence area flank of clone makes primer contact from Cordyceps militaris
The mRNA or cDNA of acquisition carry out PCR under conditions of it can expand desired zone, separate the piece of amplification
Section (such as passing through the purification reaction mixture on Ago-Gel), and recycle the DNA of amplification.Introducing, which can be designed, makes that it includes suitable
The Restriction Enzyme recognition site of conjunction, so as to the DNA clone that will expand into suitable cloning vector.The design of restriction enzyme site
It can be set according to the nucleotide sequence of the enzyme of vector cloning sites and coding with special properties as defined herein or albumen
It is fixed, several protection bases are often added in front, and hand is referred to referring in detail to the protection base setting that PROMEGA companies provide
Volume.
In the specific embodiment of the present invention, the Cordyceps militaris superoxide dismutase is any following by expressing
Shown nucleotide sequence and obtain：
A) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.1 of 2 amino acid sequence；And/or
B) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.3 of 4 amino acid sequence；Or
C) SEQ ID NO are encoded:Nucleotide sequence shown in the SEQ ID NO.5 of 6 amino acid sequence.
Term " construct " refers to the carrier of structure, it includes carrier vector nucleotide sequence in itself and according to this hair
It is bright use coding with special properties as defined herein enzyme or albumen nucleotide sequence, and its directly or indirectly with
Promoter connects.In some embodiments, preferably construct includes at least the nucleotide sequence of the present invention or coding tool
Enzyme or albumen and the nucleotide sequence that is operationally connected with promoter just like special properties defined herein.
Term " expression vector " is the construct for referring to express in vivo or in vitro.It includes the nucleotide of carrier in itself
The nucleotide sequence of sequence and the enzyme or albumen of the coding with special properties as defined herein that are used according to the present invention, and its
Directly or indirectly it is connected with strong promoter.Preferably, expression vector of the invention include the nucleotide sequence of carrier in itself and
The nucleotide sequence that the signal peptide-Cordyceps militaris SOD is combined, expression vector be directed into biological (such as escherichia coli host
Biology) genome in.Term " introducing " preferably covers stable be introduced into genome.
In the specific embodiment of the present invention, a kind of expression vector is provided, wherein the expression vector contains
Any 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, the expression vector is pET22b.
The nucleotide sequence of coding Cordyceps militaris SOD and signal peptide sequence can reside in carrier as herein defined,
Nucleotide sequence is operably connected to regulating and controlling sequence described in the carrier so that the regulating and controlling sequence can by be suitble to
Host cell (such as Escherichia coli) expresses the nucleotide sequence, i.e., described carrier is expression vector.The carrier of the present invention can be with
Be transformed into above-mentioned suitable host cell, with express with as herein defined coding Cordyceps militaris SOD activity enzyme or
Albumen.Host cell being introduced into is selected into carrier (such as plasmid, clay, virus or phage vector, gene generally according to it
Group insert).The present invention can cover the expression vector for playing identical functions and known in the art or knowable other forms.One
Denier is transformed into selected host cell, carrier can not depend on the genome of host cell and replicate and function or
Genome can be voluntarily integrated into.
The carrier can include one or more selected markers, such as provide the gene of antibiotic resistance, such as carry
For the gene of amicillin resistance, kalamycin resistance, chlorampenicol resistant or tetracyclin resistance.Carrier can make in vitro
With for example, being used to prepare RNA or for transfecting or converting host cell.
Expression vector can further include the nucleotide sequence that the carrier can be made to be replicated in the host cell.It is described
Carrier can be pET 1-46 serial carriers (NOVAGEN companies), pGEX serial carriers, pUC serial carriers, pACYC series loads
Body, pUB serial carriers, pE serial carriers, pAMB serial carriers and pIJ serial carriers.Preferably pET serial carriers, most preferably
For pET22b.
The detection of above-mentioned nucleotide sequence is completed by nucleotide sequence sequenator, while passes through various molecular biosciences
Software is learned to analyze whether obtained gene is target gene.Common analysis software has GENTYX, Vector NTI,
GenDOC, DNAman can also carry out offline and/or on-line search and retrieval analysis to compare by means of BLAST and FASTA
Nucleotide sequence and protein sequence.
Term as used herein " enzyme " and term " amino acid sequence " and/or term " enzyme or albumen " and/or term " egg
It is in vain " synonymous.In some instances, term " amino acid sequence " is synonymous with term " peptide " and/or " enzyme ", alternative each other can make
With.
Term as used herein " restructuring Cordyceps militaris SOD " is synonymous with term " the restructuring Cordyceps militaris SOD of warm expression ".
This paper terms " signal peptide " are according to sequence and/or carrier used, by encoding pupa worm by host cell expression
Cordyceps militaris SOD can be secreted or comprising in the cell caused by the nucleotide sequence of careless SOD.Signal sequence can be used for
Coded sequence secretion is instructed to pass through specific cell membrane.The signal sequence can be day for Cordyceps militaris SOD coded sequences
It is right or external source.Can use can instruct expressed Cordyceps militaris SOD into selected host cell (preferably Escherichia coli
BL21 host cells) secretory pathway any signal coding sequence.
Nucleotide sequence for the coding Cordyceps militaris SOD in either method of the present invention and/or application can encode naturally
Cordyceps militaris SOD or Cordyceps militaris SOD variants.For example, during the nucleotide sequence for the coding Cordyceps militaris SOD of the present invention can be
Described one kind.These documents of NCBI are incorporated herein by reference.
The term as used herein " Cordyceps militaris SOD " preferably refer to have catalysis superoxide radical be converted into hydrogen peroxide and
Generate the enzyme of oxygen functionality.
Preferably, being for the Cordyceps militaris SOD in either method of the present invention and/or application can will be various organic in vivo
Superoxide radical is converted into hydrogen peroxide and generates oxygen functionality.The organism includes but not limited to animal, plant and micro- life
Object.First choice is animal, is preferably the mankind.
In certain embodiments of the present invention, the nucleotide sequence of encoded signal peptide can be operably connected to volume
The nucleotide sequence of Cordyceps militaris SOD selected by code.Selected Cordyceps militaris SOD can be in host cell as defined herein as fusion
Protein expression.
Present invention also contemplates that by the coding Cordyceps militaris superoxide dismutase being used in either method of the present invention and/or application
The encoded amino acid sequence of nucleotide sequence.
The present invention provides a kind of Cordyceps militaris superoxide dismutases for being particularly suitable for expressing in Bacillus coli cells
Secretory piece coded sequence.The sequence is filtered out according to principles such as e. coli codon preferences.That designs is special
Door carries out full genome synthesis or by PCR method realization for the coded sequence of rcSOD secreting, expressings with conventional method.
Nucleotide sequence or coding that term " cell " herein includes the present invention have specificity as defined herein
The enzyme of matter or the nucleotide sequence of albumen and/or any cell by its product obtained, with term " biology " and/or term
" bacterium " and/or term " microorganism " and/or term " bacterial micro-organism " are synonymous, are replaced each other.As in background technology institute
The Natural C.militaris plant cell of the native nucleotide sequence containing coding Natural C.militaris SOD enzymes of description is also contained in herein
In term " cell ".
Term " engineering cell " related to the present invention or " host cell of conversion ", which include any coding that includes, to be had such as
The enzyme of special properties defined herein or the nucleotide sequence of albumen and/or by its acquisition product cell and/or wherein open
Mover can allow for coding to be listed in table in the biology with the enzyme of special properties as defined herein or the nucleotides sequence of albumen
It reaches.Preferred nucleotide sequence is introduced in the genome of biology.
Term " genetically modified organism " is not covered in itself natural surroundings and that it is subject to be in itself together simultaneously is natural
The natural nucleus glycoside coding sequences of natural promoter control in environment.Therefore, genetically modified organism of the invention include comprising with
Lower any or combination biology：The nucleotide sequence of enzyme or albumen of the coding with special properties as defined herein, sheet
The construct of text definition, carrier defined herein, herein plasmid defined herein, fixed cell or its product.For example, described turn
Gene biological can also include enzyme or albumen of the coding with special properties as defined herein and the core for being activated sub- control
Nucleotide sequence, wherein the promoter is not connected originally with restructuring Cordyceps militaris superoxide dismutase encoding gene.
In yet further embodiment of the invention, a kind of engineering cell is provided, wherein the engineering cell is by turning
Change above-mentioned expression vector and enter the bacterial cell obtained after host cell.
The host microorganism can be protokaryon or eucaryote.The Cordyceps militaris SOD of the present invention or coding Cordyceps militaris SOD
Nucleotide sequence available from including but not limited to the bacterial micro-organism of subordinate：Escherichia (Escherichia),
It is Mycobacterium (Mycobacterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus), de-
Sulfurous acid Pseudomonas (Desulfitobacterium), bacillus (Bacillus), campylobacter
(Campylobacter), vibrio (Vibrionaceae), XyZella (Xylella), solfataricus genus
(Sulfolobus), Aeromonas (Aeromonas), streptomyces (Streptomyces), Blastocystis
(Saccharomyces), lactococcus (Lactococcus), aspergillus (Aspergillus), Schizosaccharomyces
(Schizosaccharomyces), listeria (Listeria), eisseria (Neisseria), in take root slowly knurl
Pseudomonas (Mesorhizobium), Lei Er Bordetellas (Ralstonia), xanthomonas (Xanthomonas) and candida
(Candida)；Preferably Escherichia (Escherichia) is preferably Escherichia (Escherichia).
Expediently, the nucleotide sequence of the Cordyceps militaris SOD or coding Cordyceps militaris SOD of the present invention can be obtained from preferably
Obtained from following bacterial micro-organism：Escherichia coli (E.coli), bacillus (Bacillus sp), campylobacter jejuni
(Campylobacter jejuni), vibrios (Vibrionaceae), xyllela fastidiosa (Xylella fastidiosa), sulphur
Ore deposit sulfolobus (Sulfolobus solfataricus), saccharomyces cerevisiae (Saccharomyces cerevisiae), thermophilic aqueous vapor list
Born of the same parents bacterium (Aeromonas hydrophila), aeromonas salmonicida (Aeromonas salmonicida), streptomyces coelicolor
(Streptomyces coelicolor), streptomyces rimosus (Streptomyces rimosus), mycobacteria
(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), streptococcus thermophilus (Streptococcus thermophilus), Lactobacillus helveticus (Lactobacillus
Helveticus), dehalogenation desulfiting bacterium (Desulfitobacterium dehalogenans), Aspergillus terreus
(Aspergillus terreus), schizosaccharomyces pombe (Schizosaccharomyces pombe), listera innocua
(Listeria innocua), listerisa monocytogenes in mjme (Listeria monocytogenes), meningitis Neisser
Family name coccus (Neisseria meningitidis), crowtoe Autoinducer (Mesorhizobium loti).Preferably
Bacillus coli cells, more preferably BL21 (ED3) Bacillus coli cells.
In some embodiments of the invention, it is thin so that external carrier is transferred to host that some competent cells are prepared for
In born of the same parents.The common competent cell for preparing includes low-temperature treatment, such as 16 DEG C.
Furthermore it is suitable for host organism or the plant of the present invention.Therefore, the invention further relates to generate to have to increase
The method of the genetically modified plants of strong SOD expressions, comprises the following steps：Utilize Cordyceps militaris superoxide dismutase defined herein
Change enzyme (especially with the expression vector or construct for including Cordyceps militaris superoxide dismutase defined herein) and convert plant
Cell and the culture plant cell by converting go out plant.
In one embodiment, Cordyceps militaris SOD according to the present invention can be available from being preferably obtained from large intestine bar
Cordyceps militaris SOD (the Classification And Nomenclatures of bacterium BL21JZY001：Escherichia coli, Escherichia Coli), the Escherichia coli
Bacterial strain JZY001 is used for the microbial preservation of proprietary program by Jin Zi sources Bioisystech Co., Ltd of Jilin Province according to international recognition
Budapest treaty is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center China General
Microbiological Culture Collection Center (addresses：In Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology of the academy of sciences of state, postcode：100101) preservation day is on 09 29th, 2014, preserving number CGMCC
9744。
In certain embodiments of the present invention, to the strain, condition of culture, induction of suitable bacterial micro-organism of the invention
Condition and expression fermentation condition etc. have carried out experimental research, and it is external various that the condition further includes dissolved oxygen, rotating speed and temperature etc.
Condition.
It, can culturing engineering cell under the suitable conditions after engineering cell is obtained.It is all be suitable for Escherichia coli Growth,
The culture medium of expression can be used in the present invention.For example, suitable culture medium includes (but being not limited to) following culture medium：LB is cultivated
Base, potato sugar agar medium, bean sprouts medium, pea agar medium, potato-sucrose-agar medium, Ma Ling
Potato-sucrose-agar medium etc. is preferably LB culture mediums.
Dissolved oxygen condition can control, and suitable dissolved oxygen is controlled in 20-90%.The maintenance of dissolved oxygen can use oxygen, air
Mixed gas is passed through to solve.
In another preference, the present invention further optimizes the expression condition of rcSOD also by fermentation technology optimization.
Since expression is high, reach more than 100mg/L, and be solubility expression, bring great convenience to purifying process, pass through two steps
Chromatography technique, increases substantially product yield, has obtained high expression quantity, specific activity height, purification is convenient, yield is high and character is stablized
RcSOD products, substantially reduce production cost, be very suitable for mass producing.
Specific cleavage site is introduced respectively with 3 ' ends at 5 ' ends of optimization gene, it, will with the conventional method of molecular cloning
Optimization gene is cloned into expression vector (such as pET28, pET22).Then, BL21 (DE3) host cell is transformed into, is lured with IPTG
It leads, high expression engineering cell is selected in shaking flask expression.
It, can culturing engineering cell under the suitable conditions after engineering cell is obtained.It is all be suitable for Escherichia coli Growth,
The culture medium of expression can be used in the present invention.For example, suitable culture medium includes but is not limited to following culture medium：
A kind of preferred shake flask culture conditions are：Monoclonal on picking LB tablets, using LB as shake flask culture, culture is extremely
OD600, with IPTG induced expressions, when induction 18-21 is small, rcSOD yield is up to 50-200mg/L.
In order to mass produce rcSOD, it is necessary on fermentation tank culturing engineering cell, rcSOD is expressed, therefore to large intestine bar
The pilot scale expression condition of bacterium BL21 (DE3) engineering cell optimizes, and fermentation-scale from 1L to 300L, linearly put by fermentability
Greatly.Expression quantity is more than 400mg/L fermentates after optimization.
By the experiment repeatedly of the present invention, expression condition optimizes as follows：
1. for the selection of culture medium, minimal medium may be selected in when ferment tank, can also be trained in inorganic salts
It is centainly changed on the basis of foster base, but contained ion composition is preferably similar to minimal medium.
2. for the control of temperature, the fermentation of rcSOD and inducing temperature are maintained at 16-25 DEG C.
3. for the pH value of induction period, induction period pH is controlled in 6-8；
4. for the control of dissolved oxygen (DO), DO is controlled in 20-90%.The maintenance of dissolved oxygen can be mixed with oxygen/air
Gas is passed through to solve.
5. for the stream of feed supplement adds, feed supplement species preferably includes the carbon sources such as starch, glucose, can individually feed supplement or mixing mend
Material.
6. for induction period IPTG concentration, conventional induced concentration can be used in the present invention, and usual IPTG concentration control exists
0.1-1mM/L。
7. for the usage amount of ampicillin, it is desirable that 100mg-400mg can effectively inhibit varied bacteria growing.
8. for induction time, be not particularly limited, be usually 18-21 it is small when, preferably 20 it is small when.
In certain embodiments of the present invention, Cordyceps militaris SOD albumen is extracted from the bacterium of fermentation.The extractive process is just
Be it is described it is thick carry, specifically include collection thalline, centrifugation obtains biomass, broken wall dissolution albumen, saltouts, filters.
The rcSOD of expression is present between cell membrane and plasmalemma, and expression is more than 200mg/L fermentates, makes pure
Change complexity to be greatly reduced.In general, fermented sample first collects cell in a manner of centrifuging or filter etc., then with lysozyme (4-
10mg/g thalline) dissolving cell wall polysaccharides, target protein is made to enter in supernatant fluid, supernatant rcSOD containing target protein.On
Clear liquid can by saltouing, ultrafiltration the methods of carry out preliminary purification after carry out chromatographic purifying again, also can directly carry out chromatographic purifying, institute
It states and saltouts to be classified thick leach protein with 0-60% neutral sulphates ammonium, ultrafiltration is filtered with ultrafiltration membrane, such as uses molecular cut off
It is filtered for the ultrafiltration membrane of 10KD.
In certain embodiments of the present invention, the albumen after slightly carrying is further purified.Chromatographic technique includes sun
The technologies such as ion-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affinity chromatography.Through different chromatography skills
Art, chromatography process compare, and the chromatography method of optimization includes：1. anion-exchange chromatography：Anion-exchange chromatography medium include but
It is not limited to Q-Sepharose, DEAE-Sepharose.If the salinity of fermented sample is higher, influence and Ion Exchange Medium
Combination, then needed before ion-exchange chromatography is carried out reduce salinity.Sample can use dilution, ultrafiltration, dialysis, Gel filtration
The means such as analysis are balanced the replacement of buffer solution, until similar with corresponding ion exchange column equilibrium liquid system, then loading, into
The gradient elution of row salinity or pH；2. hydrophobic chromatography：Hydrophobic chromatoghaphy medium include but not limited to Phenyl-Sepharose,
Butyl-Sepharose、Octyle-Sepharose.Sample is by adding NaCl, (NH4)2SO4Etc. modes improve salinity, so
Loading afterwards is eluted by reducing salinity method.Removing hydrophobicity by hydrophobic chromatography has the foreign protein of larger difference.3. gel
Filtration chromatography hydrophobic chromatoghaphy medium includes but not limited to Sephacryl, Superdex, Sephadex class.Pass through Gel filtration
Buffer system or further consummate is replaced in analysis.
RcSOD sterlings are can obtain by above-mentioned purifying, purification yield is usually more than 40%, purity more than 95%, sterling
Yield about 200mg/L cultures.Various dosage forms, such as freeze drying powder injection can be made in rcSOD after purification of conventional method.Selection one
Fixed stabilizer, while can also add the protective agents such as surfactant, antioxidant and appropriate buffer solution is freeze-dried.
The active loss of obtained product is small, moisture is low, stability is good, the features such as being easy to preserve for a long time.RcSOD can also make
Liquid drugs injection, spray, microsphere sustained-release agent and through PEG modification durative action preparation etc. is made.
In an example of the present invention, BL21 (ED3) the expression engineering cells of rcSOD, IPTG inductions, Gao Shui are constructed
Flat secreting, expressing rcSOD, is not required to renaturation.
In another example of the present invention, optimize by fermentation parameter, improve the expression of rcSOD.
Because expressing protein belongs to exocytosis type, bacterium processing is not required to brokenly.In another example of the present invention, process is pure
Change, obtain rcSOD sterlings, purification yield more than 50%, purity more than 95%, sterling yield about 200mg/L cultures.
In another embodiment of the present invention, stoste adds in appropriate auxiliary material after purification, and the injection powder pin of rcSOD is made
Agent.Stability test shows that the powder-injection is stablized.
Preferably, at least 5% SOD activity can be effectively ensured in the combination of the incubation temperature and incubation time, preferably
At least 10% SOD activity, preferably at least 15%, 20%, 25%, 26%, 28%, 30%, 40%, 50%, 60% or 75%
Enzymatic activity.
In yet another embodiment, displacement buffer solution, Q Sepharose F.F. is concentrated by ultrafiltration through 10KD films bag in sample
And purity is obtained after phenyl sepharose F.F. chromatographies more than 95%, sterling of the yield more than 50%.
In conclusion in a complete specific embodiment, the present invention provides a kind of Prepare restructuring Cordyceps militaris to surpass
The method of superoxide dismutase, wherein, described method includes following steps:
A) the step of following any nucleotide sequence being cloned into expression vector pET22b, the nucleotide sequence
For：The nucleotide sequence or SEQ ID NO.5 shown in nucleotide sequence and/or SEQ ID NO.3 shown in SEQ ID NO.1
Shown nucleotide sequence；
B) the step of expression vector pET22b being transferred to host cell E. coli BL21 (ED3) cell；
C) host cell E. coli BL21 (ED3) cell is subjected to incubation step, condition of culture includes：Culture is divided into training
The stage of supporting and induction period, cultivation stage culture bacterial concentration reach OD600, induction time 18-21hr, fermentation and induction temperature
Degree is maintained at 16-25 DEG C, and induction period IPTG, concentration was in 0.1-0.2mM/L；
D) the step of isolating and purifying out the restructuring Cordyceps militaris superoxide dismutase for melting nuclear expression, to fermented sample by from
The heart and/or filter type collect thalline, with lysozyme 1mg/g thalline, under the conditions of 37 DEG C, when processing 2 is small, then are obtained by centrifugation
Obtain clear liquid；
E) by saltout and/or ultrafiltration carry out any preliminary purification step, it is described saltout for 0-60% neutral sulphates ammonium be classified
Thick leach protein, ultrafiltration are filtered with ultrafiltration membrane, and the ultrafiltration membrane is the ultrafiltration membrane that molecular cut off is 10KD；
F) by anion exchange, hydrophobic chromatography method, the restructuring Cordyceps militaris that 95-99.9% is reached so as to obtain purity surpasses
The step of superoxide dismutase.
In yet another embodiment of the present invention, provide and preparing treatment using Cordyceps militaris SOD prepared by the above method
It is described with the purposes in the drug of human superoxide dismutase relevant disease or pathological symptom, food, health products and its cosmetics
Restructuring Cordyceps militaris superoxide dismutase contains any following amino acid sequence：
A) amino acid sequence shown in SEQ ID NO.2；And/or
B) amino acid sequence shown in SEQ ID NO.4；Or
C) amino acid sequence shown in SEQ ID NO.6.
Above-mentioned relevant disease or pathological symptom include but not limited to：Cataract, degenerative eyeground pathological changes, newborn's retina
Lesion, Atopic dermatitis, pigmentation blackspot, age spot, skin allergy, hypertension, artery sclerosis, myocardial infarction, cardiac muscle
Powerless, cardiac arrhythmia, nose allergy, asthma, pulmonary emphysema, pulmonary fibrosis, adult respiratory distress, senile dementia, op parkinson's
Disease, disturbance of cerebral circulation, sickle anaemia, broad bean disease, lead poisoning, kidney trouble, albuminuria, cystitis, behign prostate hypertrophy, kidney pompon
Inflammation, constipation, halitosis, diabetes and common complication, rheumatic arthritis, cancer, side effects of chemotherapy operation, organ transplant, liver
Inflammation, climacteric, ageing diseases (aging, aging) etc..
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to conventional strip
Part, such as Sambrook etc., Molecular Cloning:A Laboratory guide (New York：ColdSpring Harbor Laboratory
Press, 1989)；Protein Purification (Jan-Christer Janson etc., John Wiley＆Sons, 1998)
Described in condition or according to the condition proposed by manufacturer.
Embodiment
The structure of embodiment 1.rcSOD coli expression carriers：
Secretory piece gene reference e. coli k12 strain secretory protein and SEQ ID NO：Memebrane protein shown in 2 (Fig. 2) is believed
The design of number peptide, and full genome synthesis, sequence such as SEQ ID NO：DNA fragmentation (Fig. 1) shown in 1.Wherein it is used for code book
The amino acid sequence for the signal peptide of the present invention that the nucleotide sequence of invention signal peptide is deduced with it is shown in Fig. 7.Draw simultaneously at its 5 ' end
Enter NdeI restriction enzyme sites.Such as SEQ ID NO：The amino acid sequence (Fig. 4) of Natural C.militaris superoxide dismutase shown in 4 and
SEQ ID NO：Nucleotide sequence shown in 3 (Fig. 3) is known, if the Genbank numbers of logging in of NCBI are respectively AY195841.1
And AAO40743.1, the acquisition of target gene is according to the amino acid sequence of Natural C.militaris superoxide dismutase, with routine
Gene synthesis introduces secretory piece gene at 5 ' ends of the amino acid sequence of Cordyceps militaris superoxide dismutase, and in pupa worm
3 ' ends of the amino acid sequence of careless superoxide dismutase introduce XhoI restriction enzyme sites, genes of SEQ ID NO：5 coding secretions
Peptide-Cordyceps militaris superoxide dismutase amino acid sequence such as SEQ ID NO：(Fig. 5 and Fig. 6) shown in 6.Melt for encoding the present invention
With the ammonia of the restructuring Cordyceps militaris SOD of the warm expression of the present invention of the nucleotide sequence and its deduction of the restructuring Cordyceps militaris SOD of expression
Base acid sequence correspondence is shown in Fig. 8.
The acquisition building process of the structure of expression plasmid pET-rcSOD and high expression engineering cell is as shown in Figure 9.Synthesis
Secrete NdeI+XhoI restriction endonucleases (the derive from NEB Biolab) digestion of peptide-superoxide dismutase full genome, while by carrier
PET22b carries out digestion with identical NdeI+XhoI, then with NEB TAKARADNA ligases (solution 1, TAKARA,
DaLian, China) it connects in pET22b carriers, pET-rcSOD is obtained, sequencing confirms that DNA sequence dna is correct.Then, by pET-
RcSOD plasmids are converted into e. coli bl21 (DE3), under the conditions of 37 DEG C, with IPTG induction 4 it is small when after determine expression pupa
Cordyceps sinensis superoxide dismutase (Figure 10).
Influence of 2 temperature of embodiment to rcSOD expressions
Monoclonal is taken, is inoculated into LB primary seed solutions, cultivates 17-20hr；3 50mL culture tubes are taken, by 1: 50 ratio
Example is inoculated in respectively in the 50mL culture tubes of three 5mL culture mediums, culture 4hr or so, 1mM/LIPTG inductions, inducing temperature point
Wei not be 16 DEG C, 25 DEG C, 37 DEG C, thalline is collected, with lysozyme under the conditions of 37 DEG C, when processing 2 is small, 15000rpm centrifuges 10 points
Clock takes supernatant, SDS-PAGE detection expressions.The experimental results showed that in shaking flask express rcSOD 16 DEG C, 25 DEG C it is all several
Almost, however 37 DEG C of expression secretory proteins are relatively fewer (Figure 11).
Influence of the embodiment 3IPTG concentration to rcSOD expressions
Monoclonal is taken, is inoculated into LB primary seed solutions, cultivates 17-20hr；5 dresses are inoculated in respectively in 1: 50 ratio
Have in the 50mL culture tubes of 5mL culture mediums, 37 DEG C are cultivated 4hr or so, are cooled to 20 DEG C, are separately added into IPTG, make in culture medium
Concentration induces for 0.6mM, 0.4mM, 0.2mM, 0.1mM, collects thalline, with lysozyme under the conditions of 37 DEG C, when processing 2 is small,
15000rpm is centrifuged 10 minutes, takes supernatant, SDS-PAGE detection expressions.The experimental results showed that several gradient concentration IPTG
It induces rcSOD expression quantity similar, both economical, cost-effective (Figure 12) is induced with the IPTG concentration of 0.1mM.
Influence of 4 induction time of embodiment to rcSOD expressions.Monoclonal is taken, is inoculated into LB primary seed solutions, is trained
Support 5hr；It is inoculated in respectively in 5 50mL culture tubes equipped with 5mL culture mediums in 1: 50 ratio, 37 DEG C are cultivated 4hr or so, cold
But to 25 DEG C, the IPTG of 0.1mM concentration is separately added into, when induction 8,10,16,18,20,30 is small respectively, thalline is collected, uses bacteriolyze
Enzyme is under the conditions of 37 DEG C, and when processing 2 is small, 15000rpm is centrifuged 10 minutes, takes supernatant, SDS-PAGE detection expressions.Experiment
The result shows that 20 hourly output highests (Figure 13) of induction.
5 pilot scale fermentation condition of embodiment selects and 3000 fermentation tanks (6.5L) of optimization NBS BioFlo pass through above-mentioned optimization
Afterwards, rcSOD expression quantity is about 300mg/L zymotic fluids, and expression quantity substantially increases (Figure 14).
5000rpm is centrifuged 10 minutes at 4 DEG C of the fermentate of rcSOD purifying embodiment 4 in embodiment 6, collects thalline, use is molten
Bacterium enzyme dissolves thalline, and lyase usage amount is 1mg/g thalline, when 37 DEG C of warm bath 2 are small.Removal thalline is centrifuged from 15000rpm, is received
Collect clarified solution.Using Millipore ultrafilters, ultrafiltration membrane shuts off molecular weight for 10KD, and when ultrafiltration leaves and takes phegma.Supernatant ultrafiltration
It is left 500ml or so to volume, adds in 10mM phosphate buffers (PB, pH7.4), continue ultrafiltration；This program repeatedly, until
The conductance of gentle 10mM PB (pH7.4) buffer solution of conductivity water of sample is on close level.
Anion-exchange chromatography：Chromatography media：Q Sepharose F.F. (Phamarcia) buffer solution：Solution A：10mM
PBS (pH7.4), solution B：10mM PB+1M NaCl (pH7.4) loading：By rcSOD solution (1L or so, the conductance of ultrafiltration concentration
For 4000us/cm or so) loading cleaning：With the solution A cleaning chromatographic column gradient of 6CV (column volume, similarly hereinafter) after loading：After cleaning
Directly collected with 30% solution B elution target protein：Collect the protein chromatographic under 30%B elutions.
Hydrophobic chromatography：Phenyl-sepharose (Phamarcia) buffer solution：Solution A：10mM PBS (pH7.4), solution
C：10mM PB+3M NaCl (pH7.4) loading：1 obtained sample will be chromatographed and add in solid NaCl to 3.0M, loading gradient：
100%A directly cleans collection：The destination protein sample collected under 100%A is washed is chromatographed by this 2 step, i.e. anion exchange layer
After analysis, hydrophobic chromatography, purity is improved to 95% (Figure 15 and Figure 16), rcSOD sterling of the yield more than 50%, sterling yield
200mg/L cultures.
The vigour-testing method of SOD
The assay NBT photoreduction of improvement is simple and practicable, more practical.Chemoluminescence method and Nitrite formation method are uncomfortable
For measuring Mn-SOD, but react extremely sensitive for Cu/Zn-SOD.Cyte reduction methods are steady for Mn-SOD vitality test results
It is fixed, it is reproducible.But specificity and sensitivity are not ideal enough, and need specific apparatus, and practical application is restricted.Nitrous acid
Salt forms method and is combined with CN-inhibitor or SDS processing, is measured applied to Mn-SOD, and remolding sensitivity Cyte reduction methods improve number
Times, and specificity is strong, and it is reproducible, it is easy to operate, specific apparatus and equipment is not required, is easy to practical application and popularization, is
One of presently preferred assay method.Under normal conditions, SOD enzyme activity measure can only apply indirect activity measuring method.According to SOD
National standard, our company is using improvement Marklund methods, i.e. mouse thymus cells method.
1 definition：At 25 DEG C inhibit pyrogallol from oxygen talk about rate 50% when required SOD amounts be a unit of activity.
2 principles：In alkaline conditions, autoxidation can occur for pyrogallol, can inhibit mouse thymus cells energy according to SOD
Power measures SOD vigor.
3 reagents：A liquid：Tri- rifle aminomethanes (Tris) of pH8.20 0.1mol/L-hydrochloride buffer (includes 1mmol/
L EDTA.2Na).It weighs 1.214g Tris and 37.2mg EDTA.2Na to be dissolved in 62.4mL0.1mol/L hydrochloric acid solutions, with steaming
Distilled water is settled to 100ml.B liquid：4.5mmol/L pyrogallol hydrochloric acid solutions.Pyrogallol (A.R) 56.7mg is weighed to be dissolved on a small quantity
10mmol/L hydrochloric acid is dissolved in, and is settled to 100mL.
4 instruments：Ultraviolet-visible spectrophotometer, secret acidometer, accuracy 0.01pH, centrifuge, 10mL cuvettes,
10mL centrifuge tubes, glass mortar.
The preparation of 5 samples：
6 analytical procedures：Mouse thymus cells rate determination at 25 DEG C or so, once adds in A liquid in 10mL cuvettes
2.35mL, distilled water 2mL, B liquid 0.15mL.It adds in B liquid to mix immediately and be poured into cuvette, be measured respectively in 325 wavelength conditions
Light absorption value after lower initial and 1min, the difference between the two, that is, mouse thymus cells rate △ A325(min-1) the definite △ A of this experiment of325
(min-1) it is 0.060.SOD determinations of activity sample-adding program list 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：Fluid sample calculates as follows:
In formula：
U/mL：SOD enzyme activity units
△A325：Mouse thymus cells rate
△A’325：Sample liquid or SOD enzyme solutions inhibit mouse thymus cells rate
V：Added enzyme solution or sample liquid volume, unit are milliliter (mL)
D：The extension rate of enzyme solution or sample liquid
4.5：Reaction solution total volume, unit are milliliter (mL)
Result of calculation retains 3 effective digitals.
Solid sample calculation formula is as follows：
In formula：
U/g：SOD enzyme activity units
△A325：Mouse thymus cells rate
△A’325：Sample liquid or SOD enzyme solutions inhibit mouse thymus cells rate
V：Added enzyme solution or sample liquid volume, unit are milliliter (mL)
V1：Sample liquid total volume, unit are milliliter (mL)
D：The extension rate of enzyme solution or sample liquid
m：Sample quality, unit are gram (g)
4.5：Reaction solution total volume, unit are milliliter (mL)
Result of calculation retains three effective digitals.
All documents referred to are incorporated herein by reference, and conduct is individually recited just as each document
With reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can be to this hair
Bright to make various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.
The present invention is not limited to particular implementation described in this application, as the single of individual aspect of the invention
Explanation.It will be understood by those skilled in the art that carry out various modifications in the case of spirit and scope can not departed from and
Change.As described above, in addition to enumerating herein, the functionally equivalent purposes in the scope of the present disclosure is for this
It is apparent for the those skilled in the art in field.Such change and modification are intended to fall in scope of the appended claims
It is interior.The four corner that the disclosure is equal only by appended claims and with the scope of such claim is limited.It should
Work as understanding, the disclosure is not limited to specific method, reagent, composition and biosystem, certainly, the method, reagent, combination
Object and biosystem can change.It can also be appreciated that term used herein is only used for describing specific embodiment, it is not used to
It is restricted.
All patents referred herein or reference, patent application, earlier application and publication are by quoting and full text
It is incorporated herein, including all attached drawing and form so that they are not contradicted with the clearly teaching of this specification.In claim
In the range of propose other embodiments.
Claims (9)
- A kind of 1. restructuring Cordyceps militaris superoxide dismutase, which is characterized in that the Cordyceps militaris superoxide dismutase amino acid Sequence is as shown in SEQ ID NO.6.
- 2. separated nucleic acid molecules, which is characterized in that the nucleotide sequence of the separated nucleic acid molecules is such asEncode SEQ ID NO:Shown in the SEQ ID NO.5 of 6 amino acid sequence.
- 3. a kind of restructuring Cordyceps militaris superoxide dismutase, which is characterized in that the Cordyceps militaris superoxide dismutase passes through table Up to coding SEQ ID NO:Nucleotide sequence shown in the SEQ ID NO.5 of 6 amino acid sequence obtains.
- 4. a kind of expression vector, which is characterized in that the expression vector contains the nucleotide sequence shown in SEQ ID NO.5.
- 5. expression vector as claimed in claim 4, which is characterized in that the expression vector is pET22b.
- 6. a kind of engineering cell, which is characterized in that the engineering cell be by convert the expression vector described in claim 4 into Enter the bacterial cell obtained after host cell.
- 7. engineering cell as claimed in claim 6, which is characterized in that the engineering cell is thin for e. coli bl21 (ED3) Born of the same parents.
- A kind of 8. method of Prepare restructuring Cordyceps militaris superoxide dismutase, which is characterized in that the described method includes:A) the step of nucleotide sequence shown in SEQ ID NO.5 being cloned into expression vector pET22b；B) the step of expression vector pET22b being transferred to host cell E. coli BL21 (ED3) cell；C) host cell E. coli BL21 (ED3) cell is subjected to incubation step, condition of culture includes：Culture is divided into culture rank Section and induction period, cultivation stage culture bacterial concentration reach OD600, induction time 18-21hr, and fermentation and inducing temperature are protected It holds at 16-25 DEG C, induction period IPTG, concentration was in 0.1-0.2mM/L；D) the step of isolating and purifying out the restructuring Cordyceps militaris superoxide dismutase of amalgamation and expression, passes through fermented sample centrifugation And/or filter type collects thalline, with lysozyme 1mg/g thalline, under the conditions of 37 DEG C, when processing 2 is small, then passes through centrifugation acquisition Clear liquid；E) by saltout and/or ultrafiltration carry out any preliminary purification step, it is described saltout for 0-60% neutral sulphates ammonium classification slightly carry Albumen, ultrafiltration are filtered with ultrafiltration membrane, and the ultrafiltration membrane is the ultrafiltration membrane that molecular cut off is 10KD；F) by anion exchange, hydrophobic chromatography method, so as to obtain the restructuring Cordyceps militaris super oxygen that purity reaches 95-99.9% The step of object mutase.
- 9. restructuring Cordyceps militaris superoxide dismutase prepared by method as claimed in claim 8 is preparing treatment and people's super oxygen Purposes in the drug of object mutase relevant disease, food, health products and its cosmetics.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410535820.0A CN104342408B (en) | 2014-06-18 | 2014-10-12 | Recombinate the preparation method of Cordyceps militaris superoxide dismutase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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