CN104342409B - Recombinate the preparation method of ginseng superoxide dismutase - Google Patents
Recombinate the preparation method of ginseng 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 ginseng superoxide dismutase (recombinant ginseng superoxide dismutase, rgSOD) and the SOD nucleotide sequences of coding ginseng SOD, the carrier containing above-mentioned nucleotide sequence, the engineering cell containing aforementioned bearer and the method from engineering cell purifying rgSOD.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 rgSOD sterlings at low cost.Present invention additionally comprises preparing treatment and the application in the drug of people's aging-related disease or pathological symptom, food, health products and its cosmetics by restructuring ginseng superoxide dismutase prepared by the above method.
Description
Technical field
The nucleotide of the encoded signal peptide of restructuring ginseng superoxide dismutase is expressed the present invention relates to E. coli secretion
The nucleotide sequence of sequence and coding ginseng SOD, the carrier containing above-mentioned nucleotide sequence, the engineering containing aforementioned bearer are thin
Born of the same parents 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, EC1.15.1.1, SOD) is that one kind is widely present in
Metalloenzyme in animals and plants, microorganism.Superoxide radical (O in energy catalysis biological body2-) disproportionated reaction occurs, it is O in body2-
The natural remover (measure and its application study of sharp superoxide dismutases (SOD) activity of Li Ruizhen, Yang Qingjian, Chen Yi
Hainan Qiong Zhou college journals, on October 28th, 2004：34-36).So as to remove O2-, risen in the self-protection system of organism
Particularly important effect.SOD is a kind of active material for coming from life entity, can eliminate organism in metabolic processes
The harmful substance of generation.Constantly supplementing human body SOD has special-effect (Zhu Chinese, the Wang Zan Shun's aging machines of anti-aging
Hemocuprein foreign medical science disease of old people fascicles in reason research, 1985. (1):49~54；Literary firewood recklessly, Zhong Fusun, Korea Spro
The analysis biochemical drugs of lipid peroxide and hemocuprein are miscellaneous in the normal persons such as constitution, hyperlipemia, cardiovascular disease blood
Will, 1988 (2):48~50).Superoxide dismutase is Marn in 1938 et al. isolated super from ox red blood cell for the first time
Superoxide dismutase starts to count, people to SOD research oneself have the history of more than 70 years.McCord in 1969 etc. is sent out again
Existing this albumen, and it was found that their bioactivity has understood fully that it is catalyzed the property that disproportionated reaction occurs for superoxide anion
Matter, so being formally named as superoxide dismutase.SOD almost from people to cell, from animal to plant, there is its
In the presence of (Reis s.Rat liver sup eroxide dism utase purif icat ionand ager elat ed m
odificat ion.E uropan J.Biochem.1976,63:617~623), can be divided by its contained metal prothetic group difference
Three kinds, the first is the title (Cu.Zn-SOD) of cupric (Cu) zinc (Zn) metal prothetic group, a kind of most commonly seen enzyme, in green,
It is primarily present in body cell slurry；Second is the title (Mn-SOD) containing manganese (Mn) metal prothetic group, in purple, is present in
In the mitochondria and prokaryotic cell of eukaryocyte；The third is the title (Fe-SOD) of iron content (Fe) metal prothetic group, in yellowish-brown,
It is present in prokaryotic cell.
Ginseng superoxide dismutase (Cu/Zn types) 152 amino acid of full length protein, single peptide chain molecule amount are
15.3KD exists with dimeric forms.
With the development of bioscience technology, people have started gene engineering research expression SOD, but the table in Escherichia coli
Often exist up to SOD with inactive inclusion bodies, although people have attempted many refolding methods, eventually because it is of high cost,
The reasons such as renaturation yield is low are difficult to realize industrialization large-scale production requirement.Later, researcher had been inquired into is given birth to using eucaryon
Object yeast secreted expression obtains more being satisfied with as a result, current, the restructuring SOD of listing, is expressed with Escherichia coli intracellular
Or Yeast expression production, although using high density fermentation, expression is low, in addition the complicated purifying process thus brought
(needing reversed phase chromatography), yield is relatively low, this low-yield cannot meet the market demand.In addition to application escherichia expression system,
People also attempt to utilize the expression systems such as bacillus, brewer's yeast and mammalian cell production SOD.Using eucaryon table
The activity of SOD can be realized up to system, but regrettably because expression is low, the factors such as of high cost, at present almost without
People produces SOD using these expression systems.And the research of E. coli secretion expression system is most important one trend.
The present inventor is by repeatedly screening, it was found that one can be used for secreting, expressing ginseng superoxide dismutase proteins
Secretion signal peptide fragment coding gene (SEQ ID NO：1), with ginseng superoxide dismutase encoding gene (SEQ ID
NO：3) fusion forms signal peptide-ginseng superoxide dismutase encoding gene, after building prokaryotic expression carrier, is transferred to large intestine bar
In bacterium BL21 (DE3) cell, it is achieved thereby that the high-level secretory expression rgSOD of rgSOD, completes this hair on this basis
It is bright.
Present system optimization, the method for changing the engineered E. coli cell for obtaining SOD, utilize Escherichia coli base
Because secretory piece and the SOD amalgamation and expressions of expression obtain it is more satisfied as a result, and pass through the improvement of zymotechnique, simultaneously
Easy purifying process obtains high expression, high yield pulp1, a kind of rgSOD production methods of the great industrialization value of high activity.
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 ginseng SOD nucleotide sequences, the engineering cell containing aforementioned bearer and from the work
The method of journey cell purification rgSOD.
Inventors of the present invention have surprisingly found that in expression such as SEQ ID NO：Ginseng 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 ginseng SOD of high amalgamation and expression
Carrier, 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 ginseng 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 the secretory piece core that make to be secreted into cell membrane and plasmalemma gap after ginseng Expression of Superoxide Dismutase
Acid sequence, the nucleotide sequence SEQ ID NO：1 can encode SEQ ID NO：Amino acid sequence shown in 2, Er Qiesuo
State nucleotide sequence SEQ ID NO：1 and ginseng superoxide dismutase coding region sequence SEQ ID NO：3 fusions form nucleosides
Acid sequence SEQ ID NO：5.
In another aspect of the invention, a kind of expression vector is provided, it contains the above-mentioned nucleotides sequence of the present invention
Row.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 ginseng superoxide dismutase is provided, including
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 ginseng superoxides
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, ferments and lures
It leads temperature and is maintained at 16-25 DEG C, IPTG dosages are 0.05-1mM/L, and the pH value of induction period is 6-8.The escherichia coli host
Cell includes that synthesis ginseng superoxide dismutase can be induced with IPTG, and can be secreted into cell membrane and plasmalemma
The Bacillus coli cells in gap.
In another aspect of the present invention, to recombinating ginseng 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 leads to
Cross centrifugal method obtain clear liquid, (2) by saltout and/or ultrafiltration carry out preliminary purification after or directly by anion exchange,
Hydrophobic chromatography method obtains the sterling that purity reaches more than 95% (such as 95-99.9 %), the restructuring ginseng prepared by the present invention
SOD activity is in 5500U or more than 6000U.
In another aspect of the present invention, the present invention is provided the restructuring ginseng SOD prepared using the above method and is controlled in preparation
It treats and the purposes in the drug of human superoxide dismutase relevant disease or pathological symptom, food, health products and its cosmetics.
The restructuring ginseng 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.
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) place of the superoxide dismutase gene containing secreting signal peptide
Main 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.Large intestine bar
The hgSOD of bacterium expression is secreted into cell membrane and plasmalemma gap in the form of soluble, active；Product is not necessary to renaturation, can be with
Directly purified；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 ginseng SOD of the present invention.
The amino acid sequence of Fig. 4 natural ginseng SOD of the present invention.
Fig. 5 shows to encode the nucleotide sequence of recombined human ginseng SOD of the present invention.
The amino acid sequence of Fig. 6 present invention restructuring ginsengs SOD.
Fig. 7 is used to encode the amino acid of the nucleotide sequence of signal peptide of the present invention and the signal peptide of the present invention by its presumption
The corresponding diagram of sequence.
The nucleotide sequence and the present invention by its presumption that Fig. 8 is used to encode the restructuring ginseng SOD of the warm expression of the present invention
The corresponding diagram of the amino acid sequence of the restructuring ginseng SOD of warm expression.
Fig. 9 is the structure schematic diagram of expression plasmid pET-rgSOD of the present invention a kind of.
Figure 10 is rgSOD 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) rgSOD 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 rgSOD 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, place
Manage 2 it is small when, 15000rpm is centrifuged 10 minutes, takes supernatant, SDS-PAGE detection expressions.
Figure 13 is the expression electrophoretogram of the rgSOD in 6.5L fermentation tanks.Each swimming lane is as follows：It is taken before swimming lane 1, induction
Sample；Swimming lane 2, induction 20 sample when small.
Figure 14 is the electrophoretogram of rgSOD after purification.RgSOD is expressed in 6.5L fermentation tanks, 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, supernatant is taken, with anion-exchange chromatography, hydrophobic chromatography
Later protein SDS-PAGE electrophoretogram.
Specific embodiment
The substantive of the present invention is understood in order to provide, hereinafter describes the present invention's with different the level of detail
Some 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 ginseng superoxide dismutase is provided, wherein, it is described
Ginseng 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 ginseng 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 of the coding with special properties as herein defined
Or the nucleotide sequence of albumen.The term as used herein " nucleotide sequence " refers to oligonucleotide sequence or polynucleotide sequence
With its variant, homologue, segment and derivative (such as its part).The nucleotide sequence can be derived from genome or synthesis
Object 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, the separation
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 nucleotide for encoding the enzyme or albumen is prepared by synthesizing by ripe standard method
Sequence for another example, using synthetic oligonucleotide on automatic dna synthesizer, is then purified, anneals, connects and be cloned into suitably
Carrier in.
Therefore, the nucleotide sequence can be derived from the genome and synthesis source of mixing, the synthesis of mixing and cDNA
Source or mixing genome and cDNA sources, according to standard technique by connect from synthesis, genome or
The segment of cDNA is made as needed.The segment each connected corresponds to the different piece of entire nucleotide sequence.It is described
DNA sequence dna 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, for optional amplification
The primer of reaction；Or polynucleotides can be cloned into carrier.The length of the primer and other segments can be at least
12, such as at least 15,20, at least such as 25,30,35 or 40 nucleotide are preferably at least, and it is also covered by
Within term polynucleotides as used herein.
In general, preparing coding using recombinant DNA technology (DNA recombinated) has special properties as herein defined
Enzyme or albumen nucleotide sequence.Therefore, in the optional embodiment of the present invention, can use known in the art
Chemical method synthesize 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 to prepare by the available any method of those skilled in the art
Polynucleotides (such as DNA polynucleotide).They can also be cloned by standard technique.Usually using recombination method, such as make
Longer polynucleotides are prepared with PCR (PCR) clone technologies.This includes preparing positioned at required clone's
The pair of primers (such as from about 12 to 40 nucleotide) of lipid targeted sequence area flank makes what primer contact was obtained from ginseng
MRNA or cDNA carries out PCR under conditions of it can expand desired zone, separates the segment of amplification (such as
Pass through the purification reaction mixture on Ago-Gel), and recycle the DNA of amplification.Introducing, which can be designed, makes that it includes suitable
Restriction Enzyme recognition site, 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
Method such as prepares longer polynucleotides using PCR (PCR) clone technologies.This, which includes preparing, is located at institute
The pair of primers (such as from about 15 to 30 nucleotide) for the lipid targeted sequence area flank cloned is needed, makes primer contact from people
The mRNA or cDNA obtained in ginseng carries out PCR, separation amplification under conditions of it can expand desired zone
Segment (such as passing through the purification reaction mixture on Ago-Gel), and recycle the DNA of amplification.Introducing, which can be designed, makes it
Comprising suitable Restriction Enzyme recognition site, so as to the DNA clone that will expand into suitable cloning vector.Digestion position
The design of point can be according to the enzyme of vector cloning sites and coding with special properties as defined herein or the nucleotide of albumen
Sequence is set, and often adds in several protection bases in front, may be referred to the protection alkali of PROMEGA companies offer in detail
Base sets reference manual.
In the specific embodiment of the present invention, the ginseng 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
The nucleotide sequence of bright enzyme or albumen of the coding used with special properties as defined herein, and it is directly or indirectly
It is connected with promoter.In some embodiments, preferably construct includes at least the nucleotide sequence of the present invention or compiles
Enzyme or albumen of the code with special properties as defined herein and the nucleotide sequence being operationally connected with promoter.
Term " expression vector " is the construct for referring to express in vivo or in vitro.It includes the nucleosides of carrier in itself
The nucleotide sequence of acid sequence and the enzyme or albumen of the coding with special properties as defined herein that are used according to the present invention,
And it is directly or indirectly connected with strong promoter.Preferably, expression vector of the invention includes nucleotides sequence carrier in itself
The nucleotide sequence that row and the signal peptide-ginseng SOD are combined, expression vector be directed into biological (such as Escherichia coli place
Main 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 ginseng 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
It is transformed into above-mentioned suitable host cell, to express enzyme or egg with coding ginseng SOD activity as herein defined
In vain.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.
Once being transformed into selected host cell, carrier can not depend on the genome of host cell and replicate and function,
Or it can voluntarily be integrated into genome.
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.Institute
It can be pET 1-46 serial carriers (NOVAGEN companies), pGEX serial carriers, pUC serial carriers, pACYC series to state carrier
Carrier, pUB serial carriers, pE serial carriers, pAMB serial carriers and pIJ serial carriers.Preferably pET serial carriers, it is optimal
Elect pET22b as.
The detection of above-mentioned nucleotide sequence is completed by nucleotide sequence sequenator, while is given birth to by various molecules
Object software analyzes whether obtained gene is target gene.Common analysis software has GENTYX, Vector NTI,
GenDOC, DNAman, can also be carried out by means of BLAST and FASTA offline and/or on-line search and retrieval analysis to than
To nucleotide sequence and protein sequence.
Term as used herein " enzyme " and term " amino acid sequence " and/or term " polypeptide " and/or term " albumen "
It is synonymous.In some instances, term " amino acid sequence " is synonymous with term " peptide " and/or " enzyme ", alternative each other can use.
Term as used herein " restructuring ginseng SOD " is synonymous with term " the restructuring ginseng SOD of warm expression ".
This paper terms " signal peptide " are according to sequence and/or carrier used, by encoding ginseng by host cell expression
Ginseng SOD can be secreted or comprising in the cell caused by the nucleotide sequence of SOD.Signal sequence can be used for referring to
It leads coded sequence secretion and passes through specific cell membrane.The signal sequence can be natural for ginseng SOD coded sequences
Or external source.Can use can instruct expressed ginseng SOD into selected host cell (preferably e. coli bl21
Host cell) secretory pathway any signal coding sequence.
For either method of the present invention and/or application in coding ginseng SOD nucleotide sequence can encode it is natural
Ginseng SOD or ginseng SOD variants.For example, for the present invention coding ginseng SOD nucleotide sequence can be described in
It is a kind of.These documents of NCBI are incorporated herein by reference.
The term as used herein " ginseng SOD " preferably refer to have catalysis superoxide radical be converted into hydrogen peroxide and
Generate the enzyme of oxygen functionality.
Preferably, being for the ginseng 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
The nucleotide sequence of ginseng SOD selected by coding.Selected ginseng SOD can be in host cell as defined herein as fusion
Protein expression.
Present invention also contemplates that by the coding ginseng 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 points of ginseng superoxide dismutase for being particularly suitable for expressing in Bacillus coli cells
Secrete peptide fragment coded sequence.The sequence is filtered out according to principles such as e. coli codon preferences.That designs is special
For the coded sequence of rgSOD secreting, expressings full genome synthesis is carried out with conventional method or by PCR method realization.
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 art
Language " bacterium " and/or term " microorganism " and/or term " bacterial micro-organism " are synonymous, are replaced each other.Such as in background technology
The natural ginseng plant cell of the described native nucleotide sequence containing coding natural ginseng SOD enzymes 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 cell and/or wherein of the enzyme of special properties defined herein or the nucleotide sequence of albumen and/or product by its acquisition
Promoter can allow for coding to be listed in the enzyme of special properties as defined herein or the nucleotides sequence of albumen in the biology
Expression.Preferred nucleotide sequence is introduced in the genome of biology.Term " genetically modified organism " is not covered in itself day
It is subject in right environment and simultaneously the natural nucleotide code sequence of its natural promoter control being in together in itself natural surroundings
Row.Therefore, genetically modified organism of the invention is included comprising following any or combination biology：Coding has as fixed herein
The enzyme or the nucleotide sequence of albumen of the special properties of justice, construct defined herein, define carrier defined herein herein
Plasmid, fixed cell or its product herein.Have as defined herein for example, the genetically modified organism can also include coding
The enzyme of special properties or albumen and the nucleotide sequence for being activated sub- control, wherein the promoter originally not with again
Group ginseng superoxide dismutase encoding gene is connected.
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 ginseng SOD's or coding ginseng SOD of the present invention
Nucleotide sequence is available from including but not limited to the bacterial micro-organism of subordinate：Escherichia (Escherichia) divides
Ramibacterium (Mycobacterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus), de- Asia
Sulfuric 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
Belong to (Listeria), eisseria (Neisseria), Autoinducer category (Mesorhizobium), Lei Er Salmonellas
Belong to (Ralstonia), xanthomonas (Xanthomonas) and candida (Candida)；Preferably Escherichia
(Escherichia) is preferably Escherichia (Escherichia).
Expediently, the nucleotide sequence of the ginseng SOD or coding ginseng SOD of the present invention can be obtained from being preferably obtained from
In 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, some competent cells are prepared for so that external carrier is transferred to host
In cell.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 generations to have
The method of the genetically modified plants of enhancement of SOD expression, comprises the following steps：Utilize ginseng peroxide defined herein
Mutase (especially with the expression vector or construct for including ginseng superoxide dismutase defined herein) converts plant
Cell and the culture plant cell by converting go out plant.
In one embodiment, ginseng SOD according to the present invention can be available from being preferably obtained from large intestine bar
Bacterium BL21JZY001 (Classification And Nomenclatures：Escherichia coli, Escherichia Coli) ginseng SOD, the Escherichia coli bacterium
Strain JZY001 is used for the microbial preservation cloth of proprietary program by Jin Zi sources bio tech ltd of Jilin Province according to international recognition
Da Peisi treaties are 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, and preserving number is
CGMCC9743。
In certain embodiments of the present invention, to being suitble to the strain of bacterial micro-organism of the present invention, condition of culture, luring
Conducting bar part and expression fermentation condition etc. have carried out experimental research, and it is external each that the condition further includes dissolved oxygen, rotating speed and temperature etc.
Kind condition.
It, can culturing engineering cell under the suitable conditions after engineering cell is obtained.It is all to be suitable for Escherichia coli life
Long, expression culture medium can be used in the present invention.For example, suitable culture medium includes but is not limited to following culture medium：LB、
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 rgSOD 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
Chromatography technique is walked, increases substantially product yield, has obtained high expression quantity, specific activity height, purification is convenient, yield is high and character
Stable rgSOD products, substantially reduce production cost, are 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, uses IPTG
High expression engineering cell is selected in induction, shaking flask expression.
It, can culturing engineering cell under the suitable conditions after engineering cell is obtained.It is all to be suitable for Escherichia coli life
Long, expression culture medium can be used in the present invention.For example, suitable culture medium includes (but being 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
To OD600, with IPTG induced expressions, when induction 18-21 is small, rgSOD yield is up to 50-200mg/L.
In order to mass produce rgSOD, it is necessary on fermentation tank culturing engineering cell, rgSOD is expressed, therefore to large intestine
The pilot scale expression condition of bacillus BL21 (DE3) engineering cell optimizes, and for fermentation-scale from 1L to 300L, fermentability is in line
Property amplification.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 rgSOD 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
Feed supplement.
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, ginseng 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 rgSOD 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 rgSOD containing target protein.
Supernatant can by saltouing, ultrafiltration the methods of carry out preliminary purification after carry out chromatographic purifying again, also can directly carry out chromatographing pure
Change, described to saltout to be classified thick leach protein with 0-60% neutral sulphates ammonium, ultrafiltration is filtered with ultrafiltration membrane, such as with retaining
Molecular weight is that the ultrafiltration membrane of 10KD is filtered.
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 chromatographies
Technology, chromatography process compare, and the chromatography method of optimization includes：1. anion-exchange chromatography：Anion-exchange chromatography medium includes
But it is not limited to Q-Sepharose, DEAE-Sepharose.If the salinity of fermented sample is higher, influence to be situated between with ion exchange
The combination of matter then needs to reduce salinity before ion-exchange chromatography is carried out.Sample can use dilution, ultrafiltration, dialysis, gel mistake
The means such as filtering layer analysis are balanced the replacement of buffer solution, until, Ran Houshang similar with corresponding ion exchange column equilibrium liquid system
Sample carries out salinity or the gradient elution of pH；2. hydrophobic chromatography：Hydrophobic chromatoghaphy medium includes but not limited to Phenyl-
Sepharose、Butyl-Sepharose、Octyle-Sepharose.Sample is by adding NaCl, (NH4)2SO4Etc. modes carry
High salt concentration, then loading, is eluted by reducing salinity method.Removing hydrophobicity by hydrophobic chromatography has larger difference
Foreign protein.3. gel permeation chromatography hydrophobic chromatoghaphy medium includes but is not limited to Sephacryl, Superdex, Sephadex
Class.Buffer system or further consummate is replaced by gel permeation chromatography.
RgSOD 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 rgSOD after purification of conventional method.Certain stabilizer is selected,
The protective agents such as surfactant, antioxidant can be also added simultaneously and appropriate buffer solution is freeze-dried.Obtained product
Active loss is small, moisture is low, stability is good, the features such as being easy to preserve for a long time.RgSOD can also make liquid drugs injection, spray
Mist agent, 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 rgSOD are constructed, IPTG inductions are high
Level secretion expresses rgSOD, is not required to renaturation.
In another example of the present invention, optimize by fermentation parameter, improve the expression of rgSOD.
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 rgSOD 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 of rgSOD is made
Injection.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%
Enzyme or protein active.
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 ginseng super oxygens
The method of compound mutase, 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 shown in nucleotide sequence and/or SEQ ID NO.3 shown in SEQ ID NO.1
Nucleotide sequence shown in NO.5；
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
Cultivation stage and induction period, cultivation stage culture bacterial concentration reach OD600, and induction time 18-21hr ferments and lures
It leads temperature and is maintained 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 ginseng 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 pass through 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 ginseng 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 using the above method prepare ginseng SOD prepare treatment with
Purposes in the drug of human superoxide dismutase relevant disease or pathological symptom, food, health products and its cosmetics.It is described
Restructuring ginseng 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 view
Film lesion, Atopic dermatitis, pigmentation blackspot, age spot, skin allergy, hypertension, artery sclerosis, myocardial infarction, the heart
Myasthenia, cardiac arrhythmia, nose allergy, asthma, pulmonary emphysema, pulmonary fibrosis, adult respiratory distress, senile dementia, Parkinson
Family name'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 move
Plant, hepatitis, 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 this hair
It is bright rather than limit the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to routine
Condition, such as Sambrook etc., Molecular Cloning:A Laboratory guide (New York： ColdSpring Harbor Laboratory
Press, 1989)；Jan-Christer Janson etc., Protein Purification (John Wiley＆Sonss, Inc.,
1998) condition described in or according to the condition proposed by manufacturer.
Embodiment
The structure of embodiment 1.rgSOD coli expression carriers：
Secretory piece gene reference e. coli k12 strain secretory protein and SEQ ID NO：Memebrane protein shown in 2 (Fig. 2)
Signal peptide designs, and full genome synthesis, sequence such as SEQ ID NO：DNA fragmentation (Fig. 1) shown in 1.It is wherein used to encode
The amino acid sequence for the signal peptide of the present invention that the nucleotide sequence of signal peptide of the present invention is deduced with it is shown in Fig. 7.Simultaneously at its 5 ' end
Introduce NdeI restriction enzyme sites.Such as SEQ ID NO：The amino acid sequence (Fig. 4) of natural ginseng superoxide dismutase shown in 4 and
SEQ ID NO：Nucleotide sequence shown in 3 is known (Fig. 3), if the Genbank numbers of logging in of NCBI are respectively AF034630.1
And AAB87572, the acquisition of target gene is according to the amino acid sequence of natural ginseng superoxide dismutase, uses conventional gene
Synthetic method introduces secretory piece gene at 5 ' ends of the amino acid sequence of ginseng superoxide dismutase, and in ginseng super oxygen
3 ' ends of the amino acid sequence of object mutase introduce XhoI restriction enzyme sites, genes of SEQ ID NO：5 (Fig. 5) coding secretions peptide-
Ginseng superoxide dismutase amino acid sequence such as SEQ ID NO：(Fig. 6) shown in 6.For encoding the warm expression of the present invention
The amino acid sequence for recombinating the restructuring ginseng SOD of the warm expression of the present invention of the nucleotide sequence and its deduction of ginseng SOD corresponds to
Relation is shown in Fig. 8.
The acquisition building process of the structure of expression plasmid pET-rgSOD and high expression engineering cell is as shown in Figure 9.Synthesis
NdeI+XhoI restriction endonucleases (the derive from NEB Biolab) digestion of secretion peptide-superoxide dismutase full genome, while will carry
Body pET22b carries out digestion with identical NdeI+XhoI, then with NEB TAKARADNA ligases (solution 1,
TAKARA, DaLian, China) it connects in pET22b carriers, pET-rgSOD is obtained, sequencing confirms that DNA sequence dna is correct.So
Afterwards, by pET-rgSOD plasmids, convert into e. coli bl21 (DE3), under the conditions of 37 DEG C, with IPTG induction 4 it is small when after
Determine expression ginseng superoxide dismutase (Figure 10).
Influence of 2 temperature of embodiment to rgSOD 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 rgSOD 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 rgSOD expressions
Monoclonal is taken, is inoculated into LB primary seed solutions, cultivates 17-20hr；5 are inoculated in respectively in 1: 50 ratio
In 50mL culture tubes equipped with 5mL culture mediums, 37 DEG C are cultivated 4hr or so, are cooled to 20 DEG C, are separately added into IPTG, make culture medium
Middle 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 rgSOD expression quantity similar, both economical, cost-effective (Figure 12) is induced with the IPTG concentration of 0.1mM.
4. pilot scale fermentation condition of embodiment selects and 3000 fermentation tanks (6.5L) of optimization NBS BioFlo are by above-mentioned excellent
After change, rgSOD expression quantity is about 300mg/L zymotic fluids, and expression quantity substantially increases (Figure 13).
5000rpm is centrifuged 10 minutes at 4 DEG C of the fermentate of embodiment 5.rgSOD purifying embodiments 4, 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 surpasses
Filter is left 500ml or so to volume, adds in 10mM phosphate buffers (PB, pH7.4), continues 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 rgSOD solution (1L or so, the electricity of ultrafiltration concentration
Lead as 4000us/cm or so) loading cleaning：With the solution A cleaning chromatographic column gradient of 6CV (column volume, similarly hereinafter) after loading：Clearly
It is directly collected after washing 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 (pH 7.4), it is molten
Liquid 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 improves to 95% (Figure 14), yield the rgSOD sterlings 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
Hydrochlorate forms method and is combined with CN-inhibitor or SDS processing, is measured applied to Mn-SOD, and remolding sensitivity Cyte reduction methods carry
High several times, and specificity is strong, it is reproducible, it is easy to operate, specific apparatus and equipment is not required, is easy to practical application and pushes away
Extensively, it 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 standards, 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 according to SOD
Ability measures SOD vigor.
3 reagents：A liquid：Tri- rifle aminomethanes (Tris) of pH8.200.1mol/L-hydrochloride buffer (includes 1mmol/
L EDTA.2Na).It weighs 1.214g Tris and 37.2mg EDTA.2Na to be dissolved in 62.4mL 0.1mol/L hydrochloric acid solutions, use
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 in
A small amount of 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：At 25 DEG C or so, A is once added in 10mL cuvettes for mouse thymus cells rate determination
Liquid 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 items
Light absorption value, the difference between the two, that is, mouse thymus cells rate △ A after initial and 1min under part325(min-1) the definite △ of this experiment of
A325(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 work is individually recited just as each document
For 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 right
The present invention makes various changes or modifications, and 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, the list of the individual aspect as the present invention
One explanation.It will be understood by those skilled in the art that it is 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 pair in the scope of the present disclosure
It is apparent for those skilled in the art of this field.Such change and modification are intended to fall in appended claims
In the range of.The four corner limit that the disclosure is equal only by appended claims and with the scope of such claim
System.It should be appreciated that the disclosure is not limited to specific method, reagent, composition and biosystem, and certainly, the method, examination
Agent, composition and biosystem can change.It can also be appreciated that term used herein is only used for describing specific embodiment party
Formula, it is restricted to be not used to.
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.It will in right
Other embodiments are proposed in the range of asking.
Claims (9)
- A kind of 1. restructuring ginseng superoxide dismutase, which is characterized in that the ginseng superoxide dismutase amino acid sequence 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 as encoded SEQ ID NO:Shown in the SEQ ID NO.5 of 6 amino acid sequence.
- 3. a kind of restructuring ginseng superoxide dismutase, which is characterized in that the ginseng superoxide dismutase is compiled by expressing Code 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 ginseng 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 ginseng superoxide dismutase of amalgamation and expression, to fermented sample by 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 by centrifuge obtain it is 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 ginseng superoxides that purity reaches 95-99.9% The step of mutase.
- 9. restructuring ginseng superoxide dismutase prepared by method as claimed in claim 8 is preparing treatment and human superoxide Purposes in the drug of mutase relevant disease, food, health products and its cosmetics.
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