CN103305537A - Superoxide dismutase from pig blood cells and preparation method thereof - Google Patents
Superoxide dismutase from pig blood cells and preparation method thereof Download PDFInfo
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- CN103305537A CN103305537A CN2013101194601A CN201310119460A CN103305537A CN 103305537 A CN103305537 A CN 103305537A CN 2013101194601 A CN2013101194601 A CN 2013101194601A CN 201310119460 A CN201310119460 A CN 201310119460A CN 103305537 A CN103305537 A CN 103305537A
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Abstract
The invention provides superoxide dismutase from pig blood cells and a preparation method thereof. The nucleotide sequence of the superoxide dismutase is shown in a figure 1; the amino acid sequence of the superoxide dismutase is shown in a figure 2 in the specification; the vector of the nucleotide molecules is yeast plasmid; the cells of the nucleotide molecules are converted from the vector; and the nucleotide molecular cells of the superoxide dismutase contain nucleic acid molecules or pichia pastoris converted from the vector. The method can be used for preparing a recombinant production strain which can effectively express and secret Cu, Zn-SOD to realize production industrialization of Cu, Zn-SOD (superoxide dismutase) to obtain a Cu, Zn-SOD product with good pH stability, excellent thermal stability and protease hydrolysis capability.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of superoxide-dismutase that derives from the pig blood cell and preparation method thereof.
Background technology
1938, Mann and Keilin from ORBC, isolate first a kind of blue cuproprotein (superoxide dismutase, Hemocuprein).1969, MeCord and Fridovich found this from the red corpuscle purifying and the cuproprotein that contains that comes has removing ultra-oxygen anion free radical (O
2 -.) function, with its called after superoxide-dismutase (Superoxide Dismutase, EC1.15.1.1 are called for short SOD).
SOD is a metalloid enzyme that extensively is present in the organism, mainly can be divided into Cu, Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD by the difference of metal prothetic group.It is by the following reaction of catalysis
With unnecessary in the organism and the extremely strong ultra-oxygen anion free radical disproportionation of cytoclasis power produced hydrogen peroxide and oxygen, the hydrogen peroxide subsequently catalase (CAT) in the body or peroxidase (POD) minute is taken off, thereby removes O
2 -.Therefore the oxidative stress that causes plays an important role in the running balance of the generation of ultra-oxygen anion free radical and elimination in keeping organism.Oneself conducts extensive research people it, and it is maximum a kind of of research report in present 2000 plurality of enzymes, also is when one of neontology Some Questions To Be Researched.Research to it has a very important role aspect the Free Radical Biology, is subject to the great attention of bioid educational circles, doctor's (medicine) educational circles, chemical circles.In clinical application, that SOD has is anti-ageing, radioprotective, anti-curing oncoma, cell signaling, treatment myocardial ischemia and ischemia-reperfusion syndrome, treat the effect of the aspects such as autoimmune disorder and some cardiovascular disorder.In addition, SOD also shows widely application prospect as the effective constituent of protective foods and makeup.
At present, the preparation of SOD source mainly contains animal, plant and microorganism.SOD product major part is in the market extracted from animal blood.The reasons such as, purification difficult limited owing to starting material, cause the purity of SOD low, output is few, particularly frequently report along with mad cow disease all over the world, bird flu, foot and mouth disease and by pernicious transmissible diseases such as synzoic SARS, the risk of production animal source blood products strengthens, in addition, increasing of product purity requirement also increased production cost.The SOD of natural microbial and plant origin also few because of its kind, expression amount is low, enzyme molecular weight is large, with animal and human body in the homology of SOD low, thereby also be difficult to be widely used.Therefore, seeking the SOD foreign gene of the animal-origin of high-quality, it is reasonably transformed, make up cheap, efficient expression system, is one of effective way that breaks through traditional preparation method.
Summary of the invention
The object of the present invention is to provide a kind of superoxide-dismutase that derives from the pig blood cell and preparation method thereof; A kind of sod gene of animal-origin specifically, and with the method for its clonal expression in yeast cell; Solve above-mentioned the deficiencies in the prior art part, construct the high efficient expression of energy and secrete Cu, the recombinant production strain of Zn-SOD, realize Cu, the production industrialization of Zn-SOD obtains to have good pH stability, the Cu of good thermostability and protease inhibitor hydrolysis ability, the Zn-SOD product.
The invention provides a kind of superoxide-dismutase, this superoxide-dismutase is copper-zinc superoxide dismutase (Cu, Zn-SOD), and the nucleotide sequence of this superoxide-dismutase as shown in Figure 1; The aminoacid sequence of this superoxide-dismutase as shown in Figure 2.
The invention provides the recombinant vectors of the nucleic acid molecule of described superoxide-dismutase, this recombinant vectors is yeast plasmid (being preferably pPIC9k-SOD).
The invention provides the host cell of the encoding sequence of described superoxide-dismutase, this host cell selects yeast cell; The cell of described encoding sequence is transformed and is got by described carrier.The cell of the nucleic acid molecule of described superoxide-dismutase is the pichia spp that comprises described nucleic acid molecule or transform with described carrier.
The invention provides the preparation method of described superoxide-dismutase, the method step is as follows: Cu, and the acquisition of Zn-SOD gene: separation and Extraction mRNA from pig blood is cDNA with the mRNA reverse transcription, uses pcr amplification again; Get the PCR product after amplification finishes and carry out electrophoresis detection, and the Cu in the recovery gel, the Zn-SOD target gene; The cDNA for preparing carries out subclone: the gained goal gene is carried out dna sequencing, and compare in ncbi database, obtain Cu, the Zn-SOD gene.
The invention provides the construction process of recombinant vectors of the nucleic acid molecule of described superoxide-dismutase, this construction process is: adopt described Cu, the Zn-SOD encoding sequence is connected with Not I double digestion and equally is connected the pPIC9k carrier with Not I double digestion through the Xho I and is connected through the Xho I, obtains yeast recombinant expression vector pPIC9k-SOD.
The invention provides the structure of host cell of the encoding sequence of described superoxide-dismutase, the construction process of this host cell is with the described recombinant vectors structure that is converted; Described host cell selects yeast cell (being preferably pichia spp), selects pichia spp to make up reconstitution cell and expresses Cu, Zn-SOD.
The invention provides the expression of described superoxide-dismutase, cultivation comprises Cu, and the cell of Zn-SOD encoding sequence or described cell through transforming are induced its expression, the results expression product.By comprising Cu, the yeast fermentation of Zn-SOD encoding sequence is produced Cu, Zn-SOD, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography have obtained the target protein of pure enzyme form.
Describe in detail:
The present invention relates to the Cu that from the pig blood cell, clones, Zn-SOD gene.
The present invention relates to from pig blood, clone Cu, the encoding sequence of Zn-SOD.One of embodiment, the present invention extracts the genomic dna of pig blood cell, and the method by the good screening active ingredients of genomic library construction is cloned into coding Cu, the full length sequence of Zn-SOD.In one of embodiment, described encoding sequence comprises nucleotide sequence as shown in Figure 1, is referred to as Cu, Zn-SOD.In one of embodiment, described encoding sequence is the nucleotide sequence shown in the Nucleotide 1 to 459 among Fig. 1.
The invention still further relates to and comprise described Cu, the recombinant vectors of Zn-SOD encoding sequence, for example by the recombinant vectors of various this areas expression vector preparation commonly used, wherein, described encoding sequence does not comprise the endogenous signal peptide sequence of its source microorganism.In one of embodiment, with not with the Cu of the present invention of endogenous signal coding sequence, the Zn-SOD encoding sequence is connected with Not I double digestion through the Xho I and is connected the pPIC9k carrier with Not I double digestion with the Xho I and is connected, and obtains yeast recombinant expression vector pPIC9k-SOD.
The present invention also prepares and comprises Cu of the present invention, the cell of Zn-SOD encoding sequence.In one of embodiment, described cell is with the foregoing invention recombinant vectors structure that is converted.The preferred various cells that are beneficial to the gene product fermentative production of described cell, this type of cell has been well known and commonly used, for example yeast cell.In one of embodiments of the present invention, select Pichia pastoris GS115 construction expression Cu, the reconstitution cell of Zn-SOD.
The present invention also provides expression Cu, and the method for Zn-SOD comprises: cultivate the described the present invention of preamble and comprise Cu, the cell of Zn-SOD encoding sequence or described cell through transforming, induce its expression, gather in the crops expression product, can also comprise to feasibility the step of purifying expression product.In one of embodiment, the present invention is by comprising Cu of the present invention, and Cu, Zn-SOD are produced in the yeast of Zn-SOD encoding sequence (for example Pichia pastoris GS115) fermentation, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography obtain the target protein of pure enzyme form.
The present invention utilizes genetic engineering means to prepare can high efficient expression and secrete Cu, and the recombinant production strain of Zn-SOD has realized Cu, the production industrialization of Zn-SOD, and obtained the Cu of high-quality, the Zn-SOD product.The present invention identifies by zymologic property, carried out optimum temperature, Optimun pH, pH stability, the thermostability of enzyme and than the analysis of the physico-chemical properties such as vigor, prove Cu of the present invention, Zn-SOD has good pH stability, good thermostability and protease inhibitor hydrolysis ability.
Description of drawings
Fig. 1 derives from the nucleotide sequence of the SOD of pig blood cell;
Fig. 2 derives from the aminoacid sequence of the SOD of pig blood cell;
The recon structure iron of the SOD of Fig. 3 pig blood cell on pPIC9k;
The optimal reactive temperature of the SOD of Fig. 4 Pichia anomala expression pig blood cell;
The optimal reaction pH of the SOD of Fig. 5 Pichia anomala expression pig blood cell;
The thermostability of the SOD of Fig. 6 Pichia anomala expression pig blood cell;
The pH stability of the SOD of Fig. 7 Pichia anomala expression pig blood cell;
Enzyme activity in the fermentation engineering of the SOD of Fig. 8 Pichia anomala expression pig blood cell;
The molecular weight of the SOD of Fig. 9 Pichia anomala expression pig blood cell;
The SOD of Figure 10 Pichia anomala expression pig blood cell is to stomach en-and tryptic resistance.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment
Experiment material and reagent
1, thalline and carrier
Pichia pastoris GS115 and expression vector pPIC9k are all available from Invitrogen company (Carlsbad, CA, USA).
2, enzyme and other biochemical reagents
DEPC, restriction enzyme, DNAMaker, protein Maker are all available from Fermentas(MBI), the SOD detection kit is built up bio-engineering research institute available from Nanjing, and other conventional reagent are that worker or import are given birth in Shanghai, are all analytical pure.
3, substratum
The substratum that uses: the LB substratum, YPD, YPAD, BMDY, BNNY, MM, MD substratum are all with reference to Invitrogen pichia spp operational manual.
4, used Measurement for Biochemistry is routine techniques in this area among the present invention.In following examples, unless specified otherwise, all experimental implementation are all carried out according to the related Sections in following laboratory manual or the document or part, comprising: [U.S.] J. Sha nurse Brooker etc., molecular cloning experiment guide; Zhao Yongfang etc., Measurement for Biochemistry principle and application thereof (second edition); Zhu Jian etc., Biochemistry Experiment [M].
5, all involved enzyme work, enzyme activity, enzymic activity all refer to the SOD enzymic activity among the present invention, all adopt SOD detection kit (building up bio-engineering research institute available from Nanjing), and measure and calculate according to the method described in its specification sheets.
Embodiment 1Cu, the acquisition of Zn-SOD gene
(1) separation and Extraction of mRNA
Get normal pig fresh blood 5ml, adding 0.4ml concentration is the EDTA solution of 15g/L, prevent blood coagulation, draw 0.25ml with micropipet, processing water according to the ratio of 1:1 with DEPC dilutes, get fresh blood after the 0.25ml dilution in the 1.5ml centrifuge tube, add 1mlTRIzol solution (
Reagent, Invitrogen
TMCat NO.15596-026), and by specification method operation extract total RNA.
Get 100 times of 1ul dilutions and carry out detection by quantitative, get necessary amount and be used for reverse transcription (RT), remaining amount adds the long-pending ethanol of triploid and mixes, in-80 ℃ of storages.
(2) the first chain of cDNA is synthetic
RT-PCR is to be cDNA with the mRNA reverse transcription first, uses pcr amplification again.CDNA refers to take mRNA as template, the complementary DNA that forms under the effect of ThermoScript II (complementary DNA is called for short cDNA).CDNA the first chain synthetic has two key factors, the one, template mRNA, the 2nd, ThermoScript II.The synthetic application Shen of this cDNA the first chain can lottery industry reverse transcription test kit, and operates by explanation.
(3) PCR obtains goal gene
The pig SOD sequences Design upstream and downstream primer P1 and the P2 that deliver according to NCBI.The upstream and downstream primer contains respectively Xho I and Not I restriction enzyme site, gives birth to the worker by Shanghai synthetic, and primer sequence is as follows:
P1：5’-GGATAAAGAAATTATCATGAAAAC-3’
P2：5’-TATAAACTTCTTTCATAATACTGC-3’
This research PCR program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 2min, cyclic amplification 30 times; Last 72 ℃ are extended 10min.Get the PCR product after amplification finishes and carry out electrophoresis detection, and the target gene in the recovery gel.
(4) cDNA subclone
The double-stranded cDNA for preparing is inserted on the carrier system pPIC9k, obtain recombinant plasmid pPIC9k-SOD(as shown in Figure 3), with competent cell conversion method transformation receptor bacterium GS115, containing 37 ℃ of overnight incubation on the LB flat board of 100mg/ml Amp and 0.5%X-gal.The mono-clonal of picking blueness (blue spot screening) colony inoculation contains in the LB liquid nutrient medium of 100mg/ml Amp to 2ml, 37 ℃ of 200rpm cultivate 3-6h, the centrifugal 10min of 10000rpm collects thalline, extract plasmid, enzyme cuts back to close goal gene (plasmid extraction and glue reclaim E.Z.N.A.Plasmid Mini Kit I and the E.Z.N.A.Gel Extraction Kit test kit of using respectively OMEGA company) for subsequent use.The gained goal gene is carried out determined dna sequence (Invitrogen company), the encoding sequence of resulting SOD has 462bp(Fig. 1 thus), wherein the 460-462 position is terminator codon GGT, 1-459 position coding does not contain the maturation protein of signal peptide, and this maturation protein contains 153 amino acid (Fig. 2).Determine tentatively that according to the result of sequence analysis in ncbi database resulting SOD is Cu, Zn-SOD, and verified that from dna level insertion point, direction and the sequence of foreign gene are correct.
Resulting pPIC9k-SOD among the embodiment 1 is cut through Sac I enzyme, obtain linearization plasmid pPIC9k-SOD1.
Get the linear recombinant plasmid dna 50ug that builds, directly join still in sub-zero competent cell (Pichia pastoris GS115); Adding 1.0ml contains solution II (40%(w/v) cetomacrogol 1000 of the salmon sperm dna of 5ug/ml, 0.2M N, and the N-bicine N-, pH8.35); More than 30 ℃ of water bath heat preservation 1h, every 15min mixing gently once; 42 ℃ of insulation 10min; The centrifugal 5min of room temperature 3000 * g, supernatant discarded is with solution III (0.15M NaCl, 10mM N, N-bicine N-, pH8.35) the Eddy diffusion thalline of 1.0ml; The centrifugal 5min of room temperature 3000 * g removes the 800ul supernatant, with remaining 200ul supernatant Eddy diffusion thalline; 200ul bacterium liquid is coated with the YPD flat board, and (YP and 20%D sterilize separately, are down flat plate and add 20%D by 1:9 in YP before; The screening resistance is 80ug/ml Amp), be inverted to cultivate 3-4 days for 30 ℃, resistant panel grow for content has positive colony of recombinant plasmid, and carry out the recon evaluation.
Get Pichia pastoris GS115 bacterial strain positive colony that recombinant plasmid pPIC9k-SOD1 transforms, be inoculated in the 150mlYPD nutrient solution, 30 ℃ of 250rpm concussions are cultured to approximately 20h of OD600nm=0.3-0.5(), then be inoculated in 3L fermentation minimum medium (26.2ml/ phosphoric acid, 0.80g/L calcium sulfate, 18.7g/L vitriolate of tartar, 15.5g/L sal epsom, 4.17g/L potassium hydroxide, Glucose40g/L) in, in the 5L fermentor tank, ferment.
In initial period-thalli growth stage, the ammoniacal liquor with 25% in the fermenting process is regulated pH, makes it maintain 6.5, and the speed stream with 4.0ml/h adds PTM1(30mM copper sulfate, 0.54mM ferrous sulfate, 1.6mM vitamin H, 0.19M sulfuric acid), carry out continuous flow feeding.Stir and aerated culture 20-24h, dissolved oxygen drops to gradually and is lower than 100% in the thalli growth process, until carbon source exhausts, dissolved oxygen rises to gradually again and is higher than 80%, and this moment, the bacterium weight in wet base can reach 83g/L.
Enter carbon source and feed the stage, add the 25%(w/v that contains with the distilled water configuration with the flow velocity stream of 25ml/h) glucose and the solution of 12ml/L PTM1, continuous flow adds 4-6h, and adjusting air flow, dissolved oxygen is maintained about in the of 20%, and to the latter stage in this stage, the bacterium weight in wet base can reach 192g/L.
At induction period, add the methyl alcohol that contains 12ml/L PTM1 with the flow velocity stream of 20-30ml/h, make the highest 0.3%(v/v that surpasses of the final concentration of methyl alcohol in the substratum), and regulate air flow, dissolved oxygen is maintained about in the of 20%.Every 8-16h sampling 10ml, the centrifugal 5min of 10000rpm collects supernatant liquor and measures the SOD vigor in the fermenting process of induction period, and the result as shown in Figure 8.During fermentation 160h, wet bacterium is heavy can to reach 341g/L, and the expression level of SOD (enzyme activity with fermented liquid supernatant represents) can reach 3785U/ml, illustrates that the sod gene of pig blood cell derived has all obtained expressing in pichia spp and accumulation.
The purifying of embodiment 3 restructuring SOD
The embodiment 2 prepared centrifugal 10min of fermentation culture 10000rpm are removed thalline, get supernatant liquor as crude enzyme liquid, be that the external compression type hollow fiber ultrafiltration membrane of 8000Da carries out ultrafiltration with molecular weight cut-off, removing the impurity of crude enzyme liquid small molecular, and with its concentrated 3-5 doubly.
The concentrated solution of top gained crude enzyme liquid is placed ice bath, slowly add while stirring ammonium sulfate to 85%, the centrifugal 15min of 13000rpm, get precipitation, again dissolve with damping fluid, placing molecular weight cut-off is the dialysis tubing of 8000Da, with pH8.0,10mM Tris-HCl is extracellular fluid dialysis, and the volume ratio of extracellular fluid dialysis and interior liquid is greater than 50,4 ℃ of dialysis 12-16h, extracellular fluid dialysis is changed once every 4h in the centre, after having dialysed, gets dialyzed solution and concentrates with vacuum rotary evaporator, after carrying out lyophilize again, place-20 ℃ cryogenic refrigerator to preserve stand-by.
Get above the 20mg resulting lyophilized powder in centrifuge tube, add 2ml pH8.0,10mM Tris-HCl damping fluid, it is fully dissolved after, upper DEAE Sepharose Fast Flow anion column.Use first pH8.0,10mM Tris-HCl damping fluid balance pillar, then stream adds sample, and with 5 column volumes of 0-0.5M NaCl gradient elution of same buffer configuration, flow velocity is 1ml/min again, collects every pipe 3ml with Fraction Collector.Then to the measured in solution SOD vigor in the collection tube and protein electrophoresis analysis.
Peak Activity after collection of ions exchange separates after concentrated, desalination, the freeze-drying, is used pH7.0 again, 1.5 column volumes of 20mM PBS buffer solution elution, flow velocity is 0.25ml/min, presses the peak and collects, then to the sample determination SOD vigor collected and carry out protein electrophoresis.
After purifying was finished, the SOD specific activity of pig blood gene source was brought up to pure enzyme 3963U/mg from the 275U/mg of crude enzyme liquid, and purification is 14.4, and yield is 12.1%.SDS-PAGE result (Fig. 9) shows, the SOD albumen behind the SOD purifying of pig blood gene source only has single band, and molecular weight all is about 18kDa.
The zymologic property analysis of embodiment 4 restructuring SOD
Optimal reactive temperature be determined at that (30 ℃-80 ℃) carry out enzymatic reaction under phosphoric acid salt (pH7.0) buffer system and the differing temps.THERMAL STABILITY is carried out enzyme assay again for to process 10-60min under differing temps.Enzymatic reaction optimum temperuture measurement result (Fig. 4) shows, the optimal reactive temperature of SOD is 37 ℃.Be incubated 60min in 30 ℃-60 ℃ scope, residual enzyme activity all can maintain (Fig. 6) more than 90%.At 70 ℃ of insulation 60min, residual enzyme activity is about 80%, and at 80 ℃ of insulation 60min, residual enzyme activity is about 75%, and at 90 ℃ of insulation 60min, residual enzyme activity still can reach about 51%.Illustrate that pig blood gene source SOD has preferably thermostability.
In pig blood gene source SOD enzyme solution, add respectively 0.05ml trypsin 0.1mg/ml, with the configuration of pH7.0PBS damping fluid) and stomach en-(0.1mg/ml, with pH2.0 glycine-HCl damping fluid configuration) in 37 ℃ of processing 30-240min, measure again the SOD activity after the dilution.Behind trypsin treatment 240min, the residual enzyme activity of SOD still maintains 90%, without significantly loss; Behind pepsin 240min, the SOD residual enzyme activity is (Figure 10) about 48%, illustrates that pig blood gene source SOD has preferably protease inhibitor hydrolysis ability.
Claims (10)
1. superoxide-dismutase that derives from the pig blood cell, it is characterized in that: this superoxide-dismutase is copper-zinc superoxide dismutase Cu, Zn-SOD, this superoxide-dismutase has nucleotide sequence shown in Figure 1.
2. superoxide-dismutase according to claim 1, it is characterized in that: described superoxide-dismutase has aminoacid sequence shown in Figure 2.
3. the recombinant vectors of the nucleic acid molecule of a claim 1 or 2 described superoxide-dismutases, it is characterized in that: this recombinant vectors is yeast plasmid.
4. the recombinant vectors of the nucleic acid molecule of superoxide-dismutase according to claim 3, it is characterized in that: described recombinant vectors is pPIC9k-SOD.
5. the host cell of the encoding sequence of a claim 1 or 2 described superoxide-dismutases, it is characterized in that: this host cell is transformed by recombinant vectors and obtains, and described host cell is yeast cell.
6. the host cell of the encoding sequence of superoxide-dismutase according to claim 5, it is characterized in that: described host cell is pichia spp.
7. the preparation method of a claim 1 or 2 described superoxide-dismutases, it is characterized in that: the method step is:
Separation and Extraction mRNA from pig blood is cDNA with the mRNA reverse transcription, uses pcr amplification again; Get the PCR product after amplification finishes and carry out electrophoresis detection, and the Cu in the recovery gel, the Zn-SOD target gene; The gained target gene is carried out dna sequencing, and in ncbi database, compare, obtain Cu, the Zn-SOD gene.
8. the construction process of the recombinant vectors of the nucleic acid molecule of a superoxide-dismutase claimed in claim 3, it is characterized in that: this construction process is: adopt described Cu, the Zn-SOD encoding sequence is connected with Not I double digestion and equally is connected the pPIC9k carrier with Not I double digestion through the Xho I and is connected through the Xho I, obtains yeast recombinant expression vector pPIC9k-SOD.
9. the structure of the host cell of the encoding sequence of a superoxide-dismutase claimed in claim 5 is characterized in that: this construction process is with the described recombinant vectors structure that is converted; Described host cell selects yeast cell, selects pichia spp to make up reconstitution cell and expresses Cu, Zn-SOD.
10. the expression of a claim 1 or 2 described superoxide-dismutases is characterized in that: cultivate and comprise Cu, the cell of Zn-SOD encoding sequence or described cell through transforming are induced its expression, the results expression product;
Be specially by comprising Cu, the yeast fermentation of Zn-SOD encoding sequence is produced Cu, Zn-SOD, and by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography have obtained the target protein of pure enzyme form.
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| CN105920591A (en) * | 2016-04-29 | 2016-09-07 | 安徽农业大学 | Preparation method for bright mung bean superoxide dismutase liposome |
| CN109295066A (en) * | 2018-10-26 | 2019-02-01 | 宁德市富发水产有限公司 | 5 like of Larimichthys crocea antibacterial peptide piscidin and the preparation method and application thereof |
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| CN105920591A (en) * | 2016-04-29 | 2016-09-07 | 安徽农业大学 | Preparation method for bright mung bean superoxide dismutase liposome |
| CN105920591B (en) * | 2016-04-29 | 2019-08-13 | 安徽农业大学 | A kind of preparation method of bright mung bean superoxide dismutase liposome |
| CN109295066A (en) * | 2018-10-26 | 2019-02-01 | 宁德市富发水产有限公司 | 5 like of Larimichthys crocea antibacterial peptide piscidin and the preparation method and application thereof |
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Application publication date: 20130918 |