CN101525600A - Method for improving output of recombinant human Cu, Zn-SOD activated protein - Google Patents
Method for improving output of recombinant human Cu, Zn-SOD activated protein Download PDFInfo
- Publication number
- CN101525600A CN101525600A CN200910021725A CN200910021725A CN101525600A CN 101525600 A CN101525600 A CN 101525600A CN 200910021725 A CN200910021725 A CN 200910021725A CN 200910021725 A CN200910021725 A CN 200910021725A CN 101525600 A CN101525600 A CN 101525600A
- Authority
- CN
- China
- Prior art keywords
- sod
- rhcu
- mutant
- protein
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for improving the output of recombinant human Cu, Zn-SOD activated protein, which comprises the following steps: the site-directed mutagenesis to gene sequence for coding the human Cu, Zn-SOD protein is carried out; a human Cu, Zn-SOD recombinant expression carrier is built so as to convert host cells and improve the proportion of soluble recombinant protein expressed by the human Cu, Zn-SOD recombinant expression vector; and the site-directed mutagenesis adopts the way that the 6th cysteine residue and the 111th cysteine residue generates the site-directed mutagenesis to form hydrophilic amino acid residue. The 6th cysteine residue and the 111th cysteine residue are both mutated into hydrophilic serine to cause that the proportion of recombinant protein in schizomycete supernatant in the recombinant albumen total expression is improved to 80 percent from 40 percent, and the specific activity of mutant rhCu, Zn-SOD purified from the supernatant is equivalent to the specific activity of non-mutant rhCu, Zn-SOD purified in the supernatant.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the expression in prokaryotic cell prokaryocyte of site-directed point mutation, gene recombination, foreign gene and the purifying of target protein, be particularly related to a kind of raising recombinant human Cu, Zn-SOD (rhCu, Zn-SOD) method of activated protein output.
Background technology
1, the discovery of SOD and the mechanism of action
(Superoxide Dismutase SOD) has that the anti-width of cloth is penetrated, antitumor, oxidation resistant effect to superoxide-dismutase, is the medicinal widely enzyme of a kind of curative effect.This enzyme is to contain Cu protein in 1938 by what Mann and Keilin at first isolated a kind of blueness from ORBC at first, only recognizes at that time that it was a kind of protein.And it is subjected to extensive attention after having the specific biological activity of removing oxyradical in the organism since confirmations such as nineteen sixty-eight Fridovich.This enzyme extensively exists in each tissue of organism, the startup factor---ultra-oxygen anion free radical (O that can the catalysis aging
2 -) disproportionation reaction, and under catalatic effect, finally change nontoxic HO into
2And O
2, suppress the oxygenizement of free radical, thereby effectively prevent of the toxic action of superoxide radical negatively charged ion body.
2, the application of SOD
SOD can be applied to industries such as medicine, daily cosmetics, healthcare products and food as a kind of additive raw material anti-oxidant, anti-aging product.
Aspect medical applications, SOD can enhancing body Antiradiation injury ability, anti-aging, prevent and treat multiple disease, and humans and animals is had no side effect.SOD has good efficacy in the treatment of some tumours, inflammation, autoimmune disease, at present, SOD has begun to try out in diseases such as treatment of arthritis, lupus erythematosus as medicinal enzyme.Also oneself has breakthrough in the research in fields such as prevention of the radiotherapy of virus disease, autoimmune disorder, myocardial ischemia and ischemic reperfusion syndromes, senile cataract, cardiovascular disorder, Radiation sickness, cancer and cancer and human longevity.The appearance and the application of existing SOD preparation both at home and abroad.
Aspect daily cosmetics, in makeup, add SOD, can play sun-proof, anti-ageing, antiphlogistic effect, this series products is such as SOD honey, Kang Ni SOD etc.SOD can also join in toothpaste, collutory, the lozenge etc., and the prevention oral disease is had certain curative effect.
In healthcare products and food service industry, the same antioxidant that can be used as tinned pre-, fruit juice, beer etc. with other antioxidant of SOD prevents food spoilage and corruption that peroxidation causes; Also can be used as the good preservation agent of fruit, vegetables.
3, the structure of SOD and classification
Up to now, people oneself separate in the organisms such as bacterium, fungi, protozoon, algae, insect, fish, plant and Mammals and obtained SOD.Though naturally occurring SOD active centre ion difference, but there are the structure identity of height and the conservative property of evolution in the catalytic activity position, and promptly the active centre metal ion all is and 3 or 4 Histidines (His), imidazolyl (Mn-SOD contains an aspartic acid carboxyl coordination) and 1 H
2The O molecule is the tetrahedral coordination of distored tetragonal pyramid or distortion.SOD can be divided into two families: Cu on structure, Zn-SOD is a first family, and Mn-SOD and Fe-SOD are second family.According to the difference of metal prothetic group, SOD can be divided into 5 types at least: Cu, Zn-SOD, Mn-SOD, Fe-SOD, Ni-SOD and Fe, Zn-SOD.Wherein, Cu, Zn-SOD distributes the widest, extensively is present in blood, the liver of animal, in the organisms such as the leaf of spinach, Rosa roxburghii.Cu, Zn-SOD remove biological intravital superoxide anion single-mindedly in human body, be the major protein of oxyradical in the balance body, are the key breakthrough mouths of SOD structural research.
Compare the Cu of different sources, the aminoacid sequence of Zn-SOD can find that their homology is all very high; Some amino acid is also very conservative, and is all constant in all sequences, and this is hinting that there is confidential relation in these amino acid and active centre.People Cu, (hCu Zn-SOD) is made up of 2 subunits Zn-SOD, and each subunit contains a Cu
2+With a Zn
2+, Cu
2+Relevant with catalytic activity, Zn
2+With to keep molecular structure relevant, mainly be present in the tenuigenin, also see in the peroxysome.
4, existing Cu, the Zn-SOD product
It all is Cu that the SOD product of having developed at present divides extremely greatly, and Zn-SOD mainly is a separation and Extraction the blood, liver from animal.The Orgotein that produces as Germany is for separating the water-soluble Cu that gets by animal erythrocyte, liver etc., and Zn-SOD can be used as the non-steroid antiphlogistic drug of inflammation such as premature infant's the toxicide of oxygen intoxication disease and treatment of arthritis etc.Some investigators have also studied respectively from garlic, mulberry leaf, sea-buckthorn, Root and stem of Cholla and have extracted Cu in recent years in addition, the technology of Zn-SOD.
Cu, the animal of Zn-SOD is extracted and mainly contains following step: the preparation of hemolysate, selective thermal sex change, ultrafiltration and concentration, acetone precipitation, column chromatography, lyophilize.Though the said extracted method can obtain Cu effectively from the tissue of animal and plant, Zn-SOD exists following a few class defective.One, raw materials for production are limited, the cost height; Two, production needs a large amount of organic solvents, pollutes big; Three, product purity of Huo Deing and specific activity are all very low; Four, the Cu of Huo Deing, Zn-SOD has the foreign protein immunogenicity for human body, the anaphylaxis that causes as inevitable some the allos SOD of the active factor of medicine, healthcare products and skin care product.Above-mentioned these defectives make Cu, and Zn-SOD is very limited in application facet.Therefore, utilize gene engineering method to produce recombinant human Cu, (rhCu Zn-SOD) just becomes the effective way of setting up in a wide range the enzyme source, reduce cost and obtain the non-immunogenicity product to Zn-SOD.
5, rhCu, the progress of Zn-SOD
By to hCu, the Zn-SOD analysis of gene sequences finds that this enzyme contains 4 Cys residues, lay respectively at 6,57,111 and 146, wherein the disulfide linkage of Cys57 and Cys146 formation is hCu, a pair of intrachain disulfide bond among the Zn-SOD, and Cys6 and Cys111 maintenance-SH group are unbound state.
Utilize gene engineering method to produce rhCu at present, Zn-SOD mainly concentrates on two aspects, is by to Cu on the one hand, and Zn-SOD modifies and improves its stability; Be to utilize different expression systems to improve its output on the other hand.
To Cu, Zn-SOD modifies and improves its stable aspect, with natural hCu, and the Cys of Zn-SOD
6With or Cys
111The amino-acid residue of changing into other may increase its stability, and the fact has also proved this point.As Kanematsu with rhCu, the Cys among the Zn-SOD
6Change Ala into
6, this mutator gene is behind expression in escherichia coli, and the purified back of expression product identifies that with natural hCu, Zn-SOD phase specific activity is consistent, and the Tm value has improved 10 ℃.Oblige beautiful grade on this basis with hCu, the Cys of Zn-SOD
6Change Ala into
6, and the sudden change hCu that in the E.coli cell, obtains expressing, Zn-SOD, its SOD enzymic activity is more stable.Zhou Zanhu etc. pass through hCu, the 111st the active Cys in nonactive center converts the Ala of relative inertness in the Zn-SOD gene, in nontoxic synechococcus, express the hCu after improveing, Zn-SOD, expression amount is to account for 3.61% of frond soluble proteins, have corresponding enzyme and live, and than natural hCu, the thermostability of Zn-SOD improves.
Utilizing different expression systems to improve aspect its output, people such as Hallewell report hCu, the nucleotide sequence of the cDNA of Zn-SOD, and make it in intestinal bacteria, obtain to efficiently express, the expressed proteins solubility is 5%; They have also studied hCu, the expression of Zn-SOD gene in yeast, and the hCu of its generation, Zn-SOD are soluble, the normal enzyme of tool is than living.Synthetic such as Zhao Minshun Cu, Zn-SOD gene, and in E.coli, carried out effective expression, expression amount accounts for 13% of bacterial protein.Institute of Basic Medical Sciences, the Academy of Medical Sciences and Navy General Hospital Molecular Biology Research Lab are with hCu, and in intestinal bacteria, expression rate is up to 50% in gene clone for Zn-SOD.The beautiful grade of obliging obtains hCu with the RT-PCR method, and Zn-SOD also makes it to express, and has obtained 38% and 50% high expression level rate and expressed proteins respectively and has had SOD enzymic activity is arranged.
Therefore to hCu, Zn-SOD recombinates exactly in order to obtain the activated protein of high expression level, overcomes the defective of traditional technology, is expressing on the successful basis, and the ratio that the activated protein of expression accounts for total protein is very important to the influence of reorganization scheme.And above-described to hCu, Zn-SOD modifies or selects different expression vectors, though obtain to have active recombinant protein, the output of the final activated protein that obtains is not high.
Summary of the invention
The problem that the present invention solves is to utilize gene engineering method to obtain the rhCu of biologically active, Zn-SOD albumen, the output of raising activated protein; The preparation purifying process of activated protein is provided simultaneously, reduces production costs, be rhCu, widespread use and the large-scale industrial production of Zn-SOD lay the foundation.
The present invention is achieved through the following technical solutions:
A kind of raising recombinant human Cu, the method for Zn-SOD activated protein output is to coding people Cu, the proteic gene order of Zn-SOD is carried out rite-directed mutagenesis, make up people Cu, the Zn-SOD recombinant expression vector improves the ratio of its expressed solubility recombinant protein with transformed host cell;
Described rite-directed mutagenesis is: people Cu, Zn-SOD proteic the 6th and the 111st cysteine residues rite-directed mutagenesis are hydrophilic amino-acid residue.
Described people Cu, Zn-SOD proteic the 6th and the 111st the equal rite-directed mutagenesis of cysteine residues are serine residue; Described structure people Cu, the nucleotide sequence that the Zn-SOD recombinant expression vector is introduced is shown in SEQ ID NO.2.
Above-mentioned raising recombinant human Cu, the method for Zn-SOD activated protein output may further comprise the steps:
A, by site-directed point mutation, the rhCu that will encode, 1 of Zn-SOD protein 11 and 6 s' Cys residue sports the Ser residue: at first make up rhCu, the 111st mutant rhCu of Zn-SOD, Zn-SOD
Cys6, Ser111, at mutant rhCu, Zn-SOD
Cys6, Ser111The basis on make up rhCu, the 6th, 111 mutant rhCu of Zn-SOD, Zn-SOD
Ser6, Ser111According to the said mutation site, designed 5 primers, be respectively:
P1：gtacatatgg cgacgaaggc cgtgtg 26
P2：gtacatatgg cgacgaaggc cgtgagcgtg ctgaag 36
P3：cgagtcgact tattgggcga tcccaattac 30
P4：aatgatggaa tggtctcctg agagcgag 28
P5：gaccattcca tcattggccg cacactg 27
Wherein, P1 and P2 contain the restriction enzyme site of Nde I, and P3 contains the restriction enzyme site of terminator codon and Sal I;
Primer is right before and after with P1, P4 and P3, P5 serving as respectively, people Cu, the Zn-SOD gene is a template, carry out twice PCR, products therefrom template and primer each other carries out PCR for the third time, with P1, P3 serve as again at last before and after primer right, PCR rite-directed mutagenesis products therefrom is that template is carried out PCR the 4th time for the third time, the final Cys that obtains 111 sports the mutant gene of Ser, and its nucleotide sequence is shown in SEQ IDNO.1;
Primer is right before and after with P2, P3 serving as, the gene order shown in the SEQ ID NO.1 is that template is carried out PCR, obtains the mutant gene that the 6th, 111 Cys sports Ser, and its nucleotide sequence is shown in SEQ IDNO.2;
B, restriction enzyme site, Sal I restriction enzyme site by Nde I are cloned into construction of expression vector among the prokaryotic expression carrier pET22b with above-mentioned two kinds of mutant genes respectively, transformed competence colibacillus cell respectively, picking mono-clonal overnight incubation; Extract plasmid, identify affirmation insertion fragment through Sal I and Nde I double digestion;
C, picking single colony inoculation of recombinating contains in the LB nutrient solution of Amp100mg/mL in 10mL, 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB nutrient solution that contains Amp100mg/mL, cultivate 3.5h to logarithmic growth mid-term at 37 ℃ of shaking tables, adding final concentration is the IPTG of 1mmol/L, continue to cultivate 4h, centrifugal collection thalline;
The thalline of d, collection split bacterium and centrifugal after, supernatant adds the (NH of massfraction 30%
4)
2SO
4Precipitation, adding copper sulfate to final concentration again is 1mmoL/L, dialyses then to the Tris-Hcl damping fluid of 0.01M, pH8.0, crosses the DEAE post and carries out purifying, adopts the Tris-Hcl wash-out target protein of the 0.01M, the pH8.0 that contain 0.2M NaCl, collects elution peak.
Beneficial technical effects of the present invention:
The present invention is with natural hCu, and the gene order of Zn-SOD is inserted in the pET22b prokaryotic expression carrier, makes up natural hCu, the recombinant expression vector of Zn-SOD; With natural hCu, the gene order of Zn-SOD is a template in addition, and to hCu, Zn-SOD does rite-directed mutagenesis, makes up the hCu of sudden change, the Zn-SOD recombinant expression vector.Above-mentioned expression vector not only can efficiently express in BL21 (DE3), and with mutant not relatively, mutant (account for tropina total amount 50%) under the constant situation of overall expression amount, splitting recombinant protein in the bacterium supernatant accounts for the overall ratio of expressing of recombinant protein and brings up to 80% by 40%, and the mutant rhCu of purifying from supernatant, the not sudden change rhCu of purifying in the specific activity of Zn-SOD and the supernatant, Zn-SOD's is suitable.
The rhCu of purifying from supernatant relatively, Zn-SOD and from precipitation the rhCu of purifying, the specific activity of Zn-SOD, the rhCu of discovery purifying from supernatant, the specific activity of Zn-SOD is the rhCu of purifying from precipitation, and therefore 4～5 times of Zn-SOD increase rhCu, the expression ratio of Zn-SOD albumen in splitting the bacterium supernatant can significantly improve rhCu, the active yield of Zn-SOD.
The rhCu that the supernatant expression amount of determining rolls up, the aminoacid sequence general formula of Zn-SOD mutant is: hCu, 6,111 cysteine mutation Serine of Zn-SOD natural acid sequence, perhaps 6,111 cysteine mutation is other hydrophilic amino acids.Wherein 6,111 two sports the wetting ability Serine and makes that splitting recombinant protein in the bacterium supernatant accounts for the overall ratio of expressing of recombinant protein and bring up to 80% by 40%, and the mutant rhCu of purifying from supernatant, the not sudden change rhCu of purifying in the specific activity of Zn-SOD and the supernatant, Zn-SOD's is suitable.
On the other hand, preparation purifying process provided by the invention, the activated protein volume production amount that obtains after the fermentation is increased, and the purifying process in downstream is simplified greatly, reduced rhCu effectively, the Zn-SOD production cost has improved its output, be rhCu, widespread use and the large-scale industrial production of Zn-SOD are laid a good foundation.
Description of drawings
Fig. 1 is three kinds of rhCu, the double digestion qualification result of Zn-SOD mutant expression vector: wherein, swimming lane 1 is DL2000; Swimming lane 2 is the pET22b-rhSOD double digestion; Swimming lane 3 is pET22b-rhSOD
Cys6, Ser111Double digestion; Swimming lane 4 is pET22b-rhSOD
Ser6, Ser111Double digestion; Swimming lane 5 is a control vector.
Fig. 2 is rhCu, Zn-SOD mutant and rhCu, and the abduction delivering sds gel electrophoresis qualification result of Zn-SOD: wherein swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is not induced for pET22b-rhSOD/BL21; Swimming lane 3 is induced for pET22b-rhSOD/BL21IPTG; Swimming lane 4 is pET22b-rhSOD
6, 111/ BL21IPTG induces.
Fig. 3 is the Western Blot qualification result of rhCu-ZnSOD mutant and rhCu-ZnSOD: wherein, swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is not induced for pET22b-rhSOD/BL21; Swimming lane 3 is induced through IPTG for pET22b-rhSOD/BL21; Swimming lane 4pET22b-rhSOD
6,111/ BL21 induces through IPTG.
Fig. 4 is rhCu, and Zn-SOD mutant and rhCu, Zn-SOD split bacterium protein precipitation sds gel electrophoresis result: swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 splits the bacterium precipitation for inducing pET22b-rhSOD/BL21; Swimming lane 3 is for inducing pET22b-rhSOD
6,111/ BL21 splits the bacterium precipitation.
Fig. 5 is rhCu, and Zn-SOD mutant and rhCu, Zn-SOD split bacterium supernatant Protein S DS gel electrophoresis result: swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 splits the bacterium supernatant for inducing pET22b-rhSOD/BL21; Swimming lane 3 is for inducing pET22b-rhSOD
6,111/ BL21 splits the bacterium supernatant.
Fig. 6 is rhCu, Zn-SOD and rhCu, Zn-SOD
6,111The evaluation of purification result: swimming lane 1 is the molecular weight of albumen standard; Swimming lane 2 is rhCu, Zn-SOD
6,111Purification result; Swimming lane 3 is rhCu, the Zn-SOD purification result.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
In order to solve the rhCu that utilizes genetically engineered to obtain biologically active, Zn-SOD albumen improves the output of activated protein, and the scheme that the present invention adopts is to improve the ratio that soluble proteins accounts for total protein, implements by following scheme:
1, at first determine to increase rhCu, the ratio of Zn-SOD in splitting the bacterium supernatant can significantly improve rhCu, the active yield of Zn-SOD.
With hCu, the Zn-SOD gene clone is gone among the prokaryotic expression carrier pET22b, makes it obtain effective expression after inducing in intestinal bacteria, and expression amount accounts for about 50% of thalline total amount.Split bacterium through N,O-Diacetylmuramidase, the ratio of target protein in splitting the bacterium supernatant account for catalogue protein content 40%, the ratio in splitting the bacterium precipitation account for catalogue protein content 60%.From last cleer and peaceful precipitation, carry out purifying simultaneously, all obtain purity greater than 90% rhCu, Zn-SOD.The determination of activity result shows, the rhCu of purifying from supernatant, the specific activity of Zn-SOD is 13000U/mg, purifying rhCu from precipitation, the specific activity of Zn-SOD is 3200U/mg, and the specific activity of the target protein of purifying is 4 times of specific activity of the target protein of purifying from split bacterium precipitation from split the bacterium supernatant.That is to say under the recombinant protein situation of expressing same amount, compare the shared ratio height of activated protein in the soluble recombining albumen with sedimentary recombinant protein.So improve rhCu, the ratio that Zn-SOD expresses in the supernatant (solubility) at thalline can improve rhCu, the active yield of Zn-SOD greatly.
2, pass through rhCu, Zn-SOD and its mutant solubility target protein account for the ratio comparative analysis that target protein is expressed total amount, determined the rhCu that the supernatant expression amount rolls up, the aminoacid sequence general formula of Zn-SOD mutant is: hCu, 6,111 cysteine mutation of Zn-SOD natural acid sequence is the hydrophilic amino acid Serine, or 6,111 cysteine mutation is other hydrophilic amino acids.
Adopt the point mutation technology to hCu, it is hydrophilic serine residue that 6 of Zn-SOD and 111 halfcystines carry out rite-directed mutagenesis, obtains 111 mutation variants (called after rhCu, Zn-SOD
111), 6 and 111 double-mutants (called after rhCu, Zn-SOD
6,111) serial genes; With rhCu, Zn-SOD, rhCu, Zn-SOD
6,111Gene inserts in the pET22b prokaryotic expression carrier, and transformed into escherichia coli efficiently expresses it.
Split to expressing thalline that the target protein ratio compares analysis in the bacterium supernatant, find 6,111 sport Serine and can significantly improve the ratio of purpose product in splitting the bacterium supernatant, and with mutant not relatively, under the constant situation of the overall expression amount of recombinant protein (account for tropina total amount 50%), 6,111 two sudden changes account for the overall ratio of expressing of recombinant protein to the recombinant protein that splits the bacterium supernatant and bring up to 80% by 40%, and the rhCu of purifying from supernatant, the rhCu/Zn-SOD that do not suddenly change of the specific activity of Zn-SOD mutant and purifying from supernatant quite.
The analysis The above results draws: make rhCu, Zn-SOD is expressed in the mutant general formula that ratio improves in the supernatant, the activated protein ultimate production improves and is: hCu, and 6,111 cysteine mutation of Zn-SOD natural acid sequence is a serine residue;
Because the 6th, the mutant serine of 111 halfcystines do not cause the active variation of recombinant protein, illustrate that the 6th, 111 is not to influence hCu, the critical sites of Zn-SOD; In view of in order to improve the solubility of recombinant protein, infer the cysteine mutation of working as the 6th, 111 and can realize identical technique effect equally for other hydrophilic amino-acid residue.
3, with gene engineering method to above-mentioned rhCu, the Zn-SOD mutant efficiently expresses, and carried out evaluation, purifying and active the detection, has obtained the preparation technology of the rhCu/Zn-SOD mutant of high resolution, high reactivity output.
Below above-mentioned embodiment is elaborated:
1. hCu, the obtaining of Zn-SOD gene
According to natural hCu among the gene Bank, the gene order of Zn-SOD is carried out full gene and is synthesized, and its gene order is as follows:
atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60
ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120
gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180
gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240
catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300
gaagattctg tgatctcgct ctcaggagac cattgcatca ttggccgcac actggtggtc 360
catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacgggaaac 420
gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa 465
2. rhCu, the structure of the expression system of Zn-SOD and mutant thereof
By with hCu, the Cys that Zn-SOD is the 6th, 111 sports Ser, makes up hCu, the mutant of Zn-SOD, the mutational site be respectively (Cys6, Ser111) and (Ser6, Ser111).According to the said mutation site, designed 5 primers, be respectively:
P1：gtacatatgg cgacgaaggc cgtgtg 26
P2：gtacatatgg cgacgaaggc cgtgagcgtg ctgaag 36
P3：cgagtcgact tattgggcga tcccaattac 30
P4：aatgatggaa tggtctcctg agagcgag 28
P5：gaccattcca tcattggccg cacactg 27
Wherein, P1 and P2 contain the restriction enzyme site of Nde I, and P3 contains the restriction enzyme site of terminator codon and Sal I.Above primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
Primer is right before and after with P1, P4 and P3, P5 serving as respectively, and rhCu, Zn-SOD gene are template, carry out twice PCR, and products therefrom template and primer each other carries out PCR for the third time.Primer is right before and after with P1, P3 serving as again at last, and the PCR products therefrom is that template is carried out PCR the 4th time for the third time, obtains rhCu, Zn-SOD
Cys6, Ser111Mutant gene.Its four PCR reaction conditionss are respectively:
A. 5×buffer 4μL
Primer P1 0.5 μ L
Primer P4 0.5 μ L
pET22b-rh Cu，Zn-SOD 1μL
dNTP 3μL
ddH
2O 10μL
20μL
B. 5×buffer 4μL
Primer P3 0.5 μ L
Primer P5 0.5 μ L
pET22b-rh Cu，Zn-SOD 1μL
dNTP 3μL
ddH
2O 10μL
20μL
Obtain the big or small purpose band about 300bp (product A), 100bp (product B) that is respectively; With its each other template and primer carry out PCR reaction for the third time, its PCR reaction conditions is
C. 2×TaqPCRMasterMix 10μL
ddH
2O 8uL
20μL
Obtaining product C, is that template is carried out the 4th PCR reaction with it.
D. 2×Premix Taq 10μL
Primer P1 0.5 μ L
Primer P3 0.5 μ L
ddH
2O 8uL
20μL
By above-mentioned four PCR reaction, obtain rhCu, Zn-SOD
Cys6, Ser111Mutant gene, its gene order is shown in SEQ ID NO.1.
With P2, P3 serves as that the front and back primer is right, hCu, Zn-SOD
Cys6, Ser111Gene is that template is carried out the PCR rite-directed mutagenesis, obtains hCu, Zn-SOD
Ser6, Ser111Mutant gene.Its PCR reaction conditions is:
2×TaqPCRMasterMix 10μL
Primer P2 0.5 μ L
Primer P3 0.5 μ L
pET22b-rh Cu，Zn-SOD
Ser6，Ser111 1μL
ddH
2O 8μL
20μL
Gained hCu, Zn-SOD
Ser6, Ser111Gene order is shown in SEQ ID NO.2.
Restriction enzyme site by Nde I, Sal I restriction enzyme site are respectively with hCu, and Zn-SOD and 2 kinds of mutant genes are cloned among the prokaryotic expression carrier pET22b, and above-mentioned recombinant vectors is transformed BL21 (DE3) competent cell, picking mono-clonal overnight incubation respectively; Extract plasmid, identify through Sal I and Nde I double digestion agarose gel electrophoresis, as shown in Figure 1, contrast swimming lane 1 and 5, swimming lane 2,3,4 is cut evaluation through enzyme and is all obtained to expect the insertion fragment of 460bp size, above-mentioned vector construction is described and transforms successfully.
To introducing the hCu of pET22b, Zn-SOD and mutant thereof check order respectively, the hCu that introduces, Zn-SOD and natural hCu, the gene order of Zn-SOD is identical, (Cys 6, Ser111) with (Ser6, gene order Ser111) is respectively shown in SEQ ID NO.1, SEQ ID NO.2 for 2 mutant.Its mutational site is respectively 111 single-points, 6 and 111 two points.
With hCu, Zn-SOD and 2 mutant (Cys 6, and Ser 111) thereof and (Ser6, Ser111) recombinant plasmid difference called after pET22b-rhSOD, pET22b-rhSOD
Ser6, Cys111, and pET22b-rhSOD
Ser6, Ser111HCu, Zn-SOD and its mutant (Ser6, Ser111) gained engineering bacteria difference called after pET22b-rhSOD/BL21, pET22b-rhSOD
6,111/ BL21.
3. rhCu, the expression of Zn-SOD and mutant thereof
Picking rhCu, single colony inoculation of Zn-SOD and its mutant engineering bacteria contains in the nutrient solution of LB of 100mg/mL penbritin (Amp) in 10mL, 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB nutrient solution that contains Amp, and 37 ℃ of shaking tables are cultivated 3.5h to logarithmic growth (OD in mid-term
600Be 0.4～0.6), adding final concentration is the IPTG of 1mmol/L, continues to cultivate 4h.Centrifugal collection thalline.
4. the evaluation of expression product
Polyacrylamide gel electrophoresis (SDS-PAGE)
Get a little thalline and add 50 μ L water and 50 μ L2 * load sample damping fluid, mixing.Boil 5min in the boiling water, the centrifugal 5min of 12000rpm gets 4.5 μ L supernatant application of samples and concentrates glue, voltage 160V, electrophoresis 1h in 15% separation gel 6%.With 0.5% Coomassie brilliant blue R-250 vibration dyeing 1h, decolouring is preserved until the dried glue of the clear back preparation of background.The result as shown in Figure 2, swimming lane 3,4 has newborn electrophoretic band near 20.1KD, the expression vector of above-mentioned thalline all obtains expressing.
Gel scanning analysis expression amount, the result shows rhCu, Zn-SOD, rhCu, Zn-SOD
6,111Overall expression amount accounts for 50% of tropina total amount.
Immunoblotting reaction
After SDS-PAGE finishes, press the Bio-Rad description of product, gel is near negative electrode one side, nitrocellulose membrane (NC film) is put (25mmol/L Tris in the transfering buffering liquid near anode one side, 192mmol/LGlycine, 20% methyl alcohol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/L pH7.4TBS with washings TBST, 0.4%Tween20) room temperature is washed 3 times, immerse in the confining liquid (TBST that contains 2%BSA) 1h under 37 ℃ of conditions, washings (TBST) room temperature is washed 3 times, adds mouse anti human SOD monoclonal antibody, hatches 1h for 37 ℃, the TBST room temperature is washed film 3 times, it is anti-to add goat anti-mouse igg-AP two, hatches 1h for 37 ℃, and the TBST room temperature is washed film 3 times, wash 3 times with TBS again, the NC film immerses in the colour developing liquid, room temperature lucifuge colour developing 5min, distilled water flushing termination reaction.The result as shown in Figure 3, the albumen after inducing (swimming lane 3,4) all can specificly combine with mouse anti human SOD monoclonal antibody specificity, and colour developing band position conforms to expected results, and rhCu is described, Zn-SOD and mutant thereof obtain expression after inducing.
5. express the cracking and the expression product interpretation of result of thalline
Get rhCu, each 1 gram of the fermentation thalline of Zn-SOD and mutant thereof joins among the 10mL cracking Buffer, and 4 ℃ of stirrings obtain bacteria suspension, add the 0.5mg N,O-Diacetylmuramidase then; 4 ℃ were stirred 20 minutes, added the 7mg Sodium desoxycholate, and stirring at room adds MgCl to thickness
2To final concentration be 10mmoL/L, add DNase I 5 μ L, be stirred to not thickness.Get the solution after 50 μ L split bacterium, 4 ℃, 15, the centrifugal 30min of 000rpm, sucking-off supernatant add 50 μ L, 2 * load sample damping fluid, mixing; Split the bacterium deposit sample and add 50 μ L water, add 50 μ L, 2 * load sample damping fluid mixing behind the suspendible again.Boil 5min in the boiling water, the centrifugal 5min of 12000rpm.The sample that makes is got 4.5 μ L supernatants and is carried out SDS-PAGE.With 0.5% Coomassie brilliant blue R-250 vibration dyeing 1h, decolouring is until the dried glue of the clear back preparation of background, result such as Fig. 4, shown in Figure 5; Target protein rhCu, Zn-SOD all have distribution in the cleer and peaceful precipitation on splitting bacterium.
Gel scanning analysis rhCu, Zn-SOD and mutant thereof split on the bacterium ratio of target protein in the cleer and peaceful precipitation.The result shows rhCu, and the ratio of Zn-SOD in splitting the bacterium supernatant is 40% of protein expression total amount, rhCu, Zn-SOD
6,111Ratio in splitting the bacterium supernatant is 80% of a protein expression total amount.
6. rhCu, the proteic purifying of Zn-SOD
From last cleer and peaceful precipitation, carry out purifying simultaneously, all obtain purity greater than 90% rhCu, Zn-SOD.The determination of activity result shows, the rhCu of purifying from supernatant, the specific activity of Zn-SOD is 13000U/mg, purifying rhCu from precipitation, the specific activity of Zn-SOD is 3200U/mg, and the specific activity of the target protein of purifying is 4 times of specific activity of the target protein of purifying from split bacterium precipitation from split the bacterium supernatant.
RhCu, Zn-SOD and rhCu, Zn-SOD
6,111Split the bacterium supernatant respectively behind 30% ammonium sulfate precipitation, add copper sulfate to final concentration 1mmoL/L, dialyse then to the Tris-Hcl damping fluid of 0.01M, pH8.0, cross the DEAE post and carry out purifying, adopt 0.2M NaCl, 0.01M pH8.0Tris-Hcl wash-out target protein is collected elution peak.
To the rhCu of purifying, Zn-SOD carries out SDS-PAGE and specific activity is measured.SDS-PAGE result as shown in Figure 6, the target protein peak shown in the swimming lane 2,3 is near the 20.1KD, conforms to expected results.
7. rhCu, the vitality test of Zn-SOD and different mutants thereof
According to GB/T5009.171-2003, adopt the Marklund method of revising to carry out the rhCu of purifying, Zn-SOD and rhCu, Zn-SOD
6,111Vitality test.
Reagent:
A liquid: pH 8.2,0.1moL/L Tris-HCl (containing 1mmoL/L EDTA2Na).Take by weighing 1.2114gTris and 37.2mg EDTA2Na, be dissolved in the 62.4mL 0.1moL/L HCl solution, distilled water is settled to 100mL;
B liquid: 4.5mmoL/L pyrogallol solution.Take by weighing pyrogallol (AR) 56.7mg and be dissolved in earlier in the 10mmoL/L hydrochloric acid soln, be settled to 100mL with it again.
Method:
A. about 25 ℃, in the 10mL colorimetric cylinder, add A liquid 2.35mL successively, distilled water 2.00mL, B liquid 0.15mL adds behind the B liquid mixing immediately, pick up counting, behind the 1min in the autoxidation speed light absorption value of 325nm place mensuration pyrogallol;
B. about 25 ℃, in the 10mL colorimetric cylinder, add A liquid 2.35mL successively, distilled water 1.8mL, SOD solution 20uL to be measured, B liquid 0.15mL adds behind the B liquid mixing immediately, picks up counting, in the light absorption value of 325nm place working sample, getting 1/2 the value that light absorption value is about the autoxidation speed of pyrogallol is that sample determination value substitution following formula calculates behind the 1min:
Wherein: the U/mL-SOD enzyme activity unit
Δ A
325-pyrogallol autoxidation speed
Δ A '
325-SOD enzyme liquid suppresses pyrogallol autoxidation speed
V-SOD enzyme liquid amasss (mL)
The extension rate of D-SOD enzyme liquid
4.5-reaction cumulative volume (mL)
Calculation result is:
RhCu, Zn-SOD vigor (U/mL)=4000U/mL
RhCu, Zn-SOD
6,111Vigor (U/mL)=11000U/mL
8. specific activity calculates
Dye method (Yang Angang etc., " Biochemistry and Molecular Biology experimental technique ", Higher Education Publishing House) detects the protein concentration of the target protein of purifying, calculates rhCu, Zn-SOD and rhCu, Zn-SOD with vigor of measuring and protein concentration
6,111Specific activity.
Reagent:
The Xylene Brilliant Cyanine G dye liquor: take by weighing 0.1g Xylene Brilliant Cyanine G G-250 and be dissolved in 95% ethanol, add 100mL85% phosphoric acid, thin up is to 1000mL.
Method:
Typical curve is drawn: add 0 respectively in test tube, the 0.1mg/mL bovine serum albumin standardized solution (available from Xi'an boat ancient cooking vessel state Bioisystech Co., Ltd) of 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, water is supplied 1.0mL, add 5.0mL Xylene Brilliant Cyanine G dye liquor, room temperature is placed 5min behind the mixing, in the colorimetric estimation of 595nm place, the typical curve of making is: y=0.0272x+0.1221 (wherein y is the absorbancy of sample at the 595nm place, and x is protein content ug);
Add enzyme liquid 0.8mL to be measured in test tube respectively, water is supplied 1.0mL, adds 5.0mL Xylene Brilliant Cyanine G dye liquor, and room temperature is placed 5min behind the mixing, at 595nm place colorimetric estimation light absorption value, calculates protein concentration and albumen specific activity.Concrete outcome sees the following form:
| Sample | rhCu，Zn-SOD | rhCu，Zn-SOD 6，111 |
| Protein concentration (mg/mL) | 0.56 | 1.2 |
| The unit volume protein-active+(U/mL) | 4100 | 13000 |
| Albumen specific activity (U/mg) | 7321 | 10833 |
| The target protein gross activity (U) that the 1g wet thallus obtains | 8.2 ten thousand | 260,000 |
The result shows the rhCu that purifying obtains from supernatant, Zn-SOD
6,111Vigor be rhCu, 3.2 times of Zn-SOD.From rhCu, Zn-SOD
6,111The total activity of the target protein that obtains in the mutant supernatant will be higher than the not rhCu of sudden change, Zn-SOD far away.
The nucleotides sequence tabulation
＜110〉Shaanxi Institute of Microbiology
＜120〉a kind of raising recombinant human Cu, the method for Zn-SOD activated protein output
<160>2
<210>1
<211>465
<212>DNA
＜213〉synthetic
<400>1
atggcgacga aggccgtgag cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60
ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120
gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180
gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240
catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300
gaagattctg tgatctcgct ctcaggagac cattccatca ttggccgcac actggtggtc 360
catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacgggaaac 420
gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa 465
<210>2
<211>465
<212>DNA
＜213〉synthetic
<400>2
atggcgacga aggccgtgag cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60
ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120
gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180
gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240
catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300
gaagattctg tgatctcgct ctcaggagac cattccatca ttggccgcac actggtggtc 360
catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacgggaaac 420
gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa 465
Claims (4)
1, a kind of raising recombinant human Cu, the method for Zn-SOD activated protein output is characterized in that, to coding people Cu, the proteic gene order of Zn-SOD is carried out rite-directed mutagenesis, makes up people Cu, the Zn-SOD recombinant expression vector improves the ratio of its expressed solubility recombinant protein with transformed host cell;
Described rite-directed mutagenesis is: people Cu, Zn-SOD proteic the 6th and the 111st cysteine residues rite-directed mutagenesis are hydrophilic amino-acid residue.
2, raising recombinant human Cu as claimed in claim 1, the method for Zn-SOD activated protein output is characterized in that, people Cu, Zn-SOD proteic the 6th and the 111st the equal rite-directed mutagenesis of cysteine residues are serine residue.
3, raising recombinant human Cu as claimed in claim 2, the method for Zn-SOD activated protein output is characterized in that, makes up people Cu, the nucleotide sequence that the Zn-SOD recombinant expression vector is introduced is shown in SEQ IDNO.2.
4, raising recombinant human Cu as claimed in claim 2, the method for Zn-SOD activated protein output is characterized in that, may further comprise the steps:
A, by site-directed point mutation, the rhCu that will encode, 1 of Zn-SOD protein 11 and 6 s' Cys residue sports the Ser residue: at first make up rhCu, the 111st mutant rhCu of Zn-SOD, Zn-SOD
Cys6, Ser111, at mutant rhCu, Zn-SOD
Cys6, Ser111The basis on make up rhCu, the 6th, 111 mutant rhCu of Zn-SOD, Zn-SOD
Ser6, Ser111According to the said mutation site, designed 5 primers, be respectively:
P1：gtacatatgg cgacgaaggc cgtgtg 26
P2：gtacatatgg cgacgaaggc cgtgagcgtg ctgaag 36
P3：cgagtcgact tattgggcga tcccaattac 30
P4：aatgatggaa tggtctcctg agagcgag 28
P5：gaccattcca tcattggccg cacactg 27
Wherein, P1 and P2 contain the restriction enzyme site of Nde I, and P3 contains the restriction enzyme site of terminator codon and Sal I;
Primer is right before and after with P1, P4 and P3, P5 serving as respectively, people Cu, the Zn-SOD gene is a template, carry out twice PCR, products therefrom template and primer each other carries out PCR for the third time, with P1, P3 serve as again at last before and after primer right, PCR rite-directed mutagenesis products therefrom is that template is carried out PCR the 4th time for the third time, the final Cys that obtains 111 sports the mutant gene of Ser, and its nucleotide sequence is shown in SEQ IDNO.1;
Primer is right before and after with P2, P3 serving as, the gene order shown in the SEQ ID NO.1 is that template is carried out PCR, obtains the mutant gene that the 6th, 111 Cys sports Ser, and its nucleotide sequence is shown in SEQ IDNO.2;
B, restriction enzyme site, Sal I restriction enzyme site by Nde I are cloned into construction of expression vector among the prokaryotic expression carrier pET22b with above-mentioned two kinds of mutant genes respectively, transformed competence colibacillus cell respectively, picking mono-clonal overnight incubation; Extract plasmid, identify affirmation insertion fragment through Sal I and Nde I double digestion;
C, picking single colony inoculation of recombinating contains in the LB nutrient solution of Amp100mg/mL in 10mL, 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB nutrient solution that contains Amp100mg/mL, cultivate 3.5h to logarithmic growth mid-term at 37 ℃ of shaking tables, adding final concentration is the IPTG of 1mmol/L, continue to cultivate 4h, centrifugal collection thalline;
The thalline of d, collection split bacterium and centrifugal after, supernatant adds the (NH of massfraction 30%
4)
2SO
4Precipitation, adding copper sulfate to final concentration again is 1mmoL/L, dialyses then to the Tris-Hcl damping fluid of 0.01M, pH8.0, crosses the DEAE post and carries out purifying, adopts the Tris-Hcl wash-out target protein of the 0.01M, the pH8.0 that contain 0.2M NaCl, collects elution peak.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100217258A CN101525600B (en) | 2009-03-27 | 2009-03-27 | Method for improving output of recombinant human Cu, Zn-SOD activated protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100217258A CN101525600B (en) | 2009-03-27 | 2009-03-27 | Method for improving output of recombinant human Cu, Zn-SOD activated protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101525600A true CN101525600A (en) | 2009-09-09 |
| CN101525600B CN101525600B (en) | 2012-07-04 |
Family
ID=41093671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100217258A Active CN101525600B (en) | 2009-03-27 | 2009-03-27 | Method for improving output of recombinant human Cu, Zn-SOD activated protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101525600B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342408A (en) * | 2014-06-18 | 2015-02-11 | 吉林省金梓源生物科技有限公司 | Preparation method of recombinant cordyceps sinensis superoxide dismutase (rcSOD) |
| CN104342409A (en) * | 2014-06-17 | 2015-02-11 | 吉林省金梓源生物科技有限公司 | Preparation method of recombinant ginseng superoxide dismutase (rgSOD) |
| CN115896048A (en) * | 2022-12-22 | 2023-04-04 | 河北纳科生物科技有限公司 | Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof |
-
2009
- 2009-03-27 CN CN2009100217258A patent/CN101525600B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342409A (en) * | 2014-06-17 | 2015-02-11 | 吉林省金梓源生物科技有限公司 | Preparation method of recombinant ginseng superoxide dismutase (rgSOD) |
| CN104342409B (en) * | 2014-06-17 | 2018-05-25 | 吉林金梓源生物科技股份有限公司 | Recombinate the preparation method of ginseng superoxide dismutase |
| CN104342408A (en) * | 2014-06-18 | 2015-02-11 | 吉林省金梓源生物科技有限公司 | Preparation method of recombinant cordyceps sinensis superoxide dismutase (rcSOD) |
| CN104342408B (en) * | 2014-06-18 | 2018-05-25 | 吉林金梓源生物科技股份有限公司 | Recombinate the preparation method of Cordyceps militaris superoxide dismutase |
| CN115896048A (en) * | 2022-12-22 | 2023-04-04 | 河北纳科生物科技有限公司 | Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof |
| CN115896048B (en) * | 2022-12-22 | 2023-08-22 | 河北纳科生物科技有限公司 | Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101525600B (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| CN104524564B (en) | A kind of crucian Simplex Virus combination vaccine preparation and preparation method and application | |
| CN108559739A (en) | The mannase PMan5A mutant and its gene and application that heat resistance improves | |
| CN110117602B (en) | Maitake mushroom UDP-glucose pyrophosphorylase and application thereof | |
| CN112852650B (en) | Saccharomyces cerevisiae engineering bacterium for high yield of santalene and santalol and construction method and application thereof | |
| KR20110070977A (en) | Method for producing biological heme iron, and iron supplementing composition containing the heme iron produced by same | |
| CN104593388B (en) | Crucian herpesvirus disease JDORF25 vaccine as well as preparation method and application thereof | |
| CN100529085C (en) | Preparation method of recombinant human plasminogen Kringle 5(hk5) | |
| CN101525600B (en) | Method for improving output of recombinant human Cu, Zn-SOD activated protein | |
| JPH04501807A (en) | Urate oxidase active protein, recombinant gene encoding it, expression vector, microorganism and transformed cell | |
| CN112574980B (en) | Recombinant alginate lyase with thermal stability and high enzyme activity and application thereof | |
| CN106318940A (en) | High-temperature-resistant SOD protein, and cloning and purifying method thereof | |
| CN110452931A (en) | A kind of method of squalene content in raising yeast | |
| JP4996934B2 (en) | Production method of recombinant lysozyme | |
| CN104946606B (en) | A kind of heat-resistant of genetic engineering transformation is against SOD and its encoding gene and application | |
| CN103865814A (en) | High-yield ganoderan engineering strain Rmust and construction method thereof | |
| CN101914476B (en) | Deep sea elastase gene as well as preparation method and application thereof | |
| CN115141841A (en) | Pichia pastoris mutant strain and application thereof in production of alginate lyase | |
| CN111808836B (en) | Heat-resistant mutant enzyme of pullulanase I and preparation method and application thereof | |
| CN111171144B (en) | Preparation and application of antibody for resisting porcine epidemic diarrhea virus | |
| CN108203697B (en) | Yeast engineering bacterium of fish natural killer cell enhancement factor and application thereof | |
| CN107354137A (en) | A kind of selenoprotein of high activity and preparation method thereof | |
| CN104911155B (en) | Using the heat-resistant of genetic engineering transformation against SOD and its encoding gene and application | |
| CN115896048B (en) | Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof | |
| CN104152465A (en) | Lilium regale cytochrome b5 gene LrCyt-b5 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230308 Address after: 712000 Room 109, Building 7A, West Yungu Phase II, Fengxi New Town, Xixian New District, Xianyang City, Shaanxi Province Patentee after: Shaanxi Keju Meikang Biotechnology Co.,Ltd. Address before: 710043 Shaanxi province Xi'an Xiying Road No. 76 Patentee before: MICROBIOLOGY INSTITUTE OF SHAANXI |