CN101280015A - Preparation of soluble human selenium-containing single-chain abzyme - Google Patents
Preparation of soluble human selenium-containing single-chain abzyme Download PDFInfo
- Publication number
- CN101280015A CN101280015A CNA2008100505566A CN200810050556A CN101280015A CN 101280015 A CN101280015 A CN 101280015A CN A2008100505566 A CNA2008100505566 A CN A2008100505566A CN 200810050556 A CN200810050556 A CN 200810050556A CN 101280015 A CN101280015 A CN 101280015A
- Authority
- CN
- China
- Prior art keywords
- chain antibody
- chain
- gene
- primer
- abzyme
- 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
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method to prepare soluble human selenium-containing catalytic single-chain antibodies, belonging to biotechnology field. The method includes the following steps: with glutathione derivatives as target antigen, filtrating the recombinant phage display human single-chain antibody library through immune affinity selection method to obtain single-chain antibodies B3 and D8; assembling the single-chain antibodies to secretory procaryon or eucaryon expression vector; translating colon bacillus or yeast cell; expressing and purifying the single-chain antibody proteins in procaryon or eucaryon; introducing GPX catalytic group SeCys at the substrate combining sites of the antibodies through chemical mutation method or directly expressing the soluble single-chain antibody proteins which contain GPX catalytic groups at the substrate combining sites with auxotrophic colon bacillus through genetic mutation techniques to endue the antibodies with GPX catalytic activity. The method of the invention is simple and the human selenium-containing catalytic single-chain antibodies prepared with the method are of high catalytic activity; the proteins expressed by the antibodies are soluble; therefore the method is good for large-scale production and is of broad application prospect in biological pharmacy.
Description
Technical field
The invention belongs to biological technical field, the technology that particularly a kind of using gene engineering combines with chemical mutation prepares the method for gsh human selenium-containing single-chain abzyme.
Background technology
The substrate that contains the Selenoperoxidase (GPX) of selenium is gsh (GSH), and catalytic group is seleno-cysteine (SeCys).In vivo, the same superoxide-dismutase of GPX (SOD), catalase (CAT) have constituted body anti-oxidative defense system together.GPX plays an important role in this system, it is reductive agent with GSH, decompose intravital hydrogen peroxide and all kinds of hydroperoxide, thereby can remove activity in vivo oxygen (ROS), prevent lipid peroxidation, the various diseases that treatment is caused by active oxygen is as ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different with other antioxidase, GPX is except removing the ROS, and the lipid peroxide of can also degrading prevents the cell peroxide injury, and the function of the protection cell of this uniqueness makes it occupy the position of particularly important in the antioxidase system.Yet, because quite limited, the poor stability in source of natural GPX causes the research of its stand-in to receive much concern.
Small molecule mimetics mainly contains PZ51 (ebselen), AL3823A, and the BXT series product, their weakness is that vigor is low, only is about the thousandth of natural GPX.The abzyme technology of preparing that the mid-80 occurs is that the manual simulation of GPX has opened up new way, according to the zymetology principle, utilize immunology and chemical knowledge, designed a kind of technological line of new generation abzyme, and successfully prepared several and have the selenium-containing abzyme (Chinese patent 94102481.4 and 96112628.0) of very high GPX vigor.It is the mouse source that the selenium-containing abzyme amino acid of these high vigor is formed, and can be used as the antigenic stimulation human body and produces human antimouse antibody, has therefore limited the use of such medicine.In order to address this problem, the preparation human selenium-containing single-chain abzyme is a best bet, and the success of phage people antibody library is configured to this imagination possibility is provided.
The preparation method of Chinese patent 99104234.4 disclosed people source strand selenium-containing abzymes is: the design of haptens-GSH derivative is with synthetic; Derivative with GSH is the directly special single-chain antibody of screening GSH from phage display people antibody library of target antigen; In intestinal bacteria with the single chain antibody protein of inclusion body formal representation non-activity; Sex change, renaturation, purification of single stranded antibody protein; With chemical mutation (modification) method the catalytic group SeCys of GPX is introduced the single-chain antibody variable region, thereby at the existing substrate binding site in single-chain antibody variable region catalytic group is arranged again, the result produces the Se-contained humanized abzyme with GPX vigor.The method of the human selenium-containing single-chain abzyme of this method preparation has following shortcoming: the inclusion body protein of expressing in (1) preparation process is a kind of no proper space conformation, is in the inactive protein molecule under the precipitation state, and needing just can have activity through denaturation renaturation.The inclusion body protein renaturation is a difficulty and complicated process, and only this step of renaturation will be lost a large amount of single chain antibody proteins, causes this legal system to be equipped with the abzyme productive rate to descend; (2) long, complex operation of the cycle of preparation abzyme is because of renaturing inclusion bodies is to make the protein molecular of non-activity recover active slow process gradually under suitable condition; (3) Zhi Bei abzyme activity is low, only is 28.8-256U/ μ mol, is lower than order of magnitude level of natural enzyme.
Summary of the invention
The technical problem to be solved in the present invention is, the method that provides a kind of affine triage techniques of antibody library immunity, genetic engineering technique, chemical mutation technology of application standard to combine prepares the method for gsh human selenium-containing single-chain abzyme, reaches to improve the abzyme activity, can directly apply to human body, simplify step, shorten preparation cycle, improve the purpose of abzyme productive rate.
General thought of the present invention is, screening earlier has the human single chain variable fragments antibody of GPX substrate binding site, determine its aminoacid sequence by checking order, the human single chain variable fragments antibody genome of this aminoacid sequence of will encoding then installs on protokaryon or the eucaryon secreted expression carrier, transformed into escherichia coli or yeast cell, express the soluble single-chain antibody protein, with the chemical mutation method catalytic group of GPX is incorporated into the substrate binding site of single-chain antibody again; Or directly be expressed in the soluble single-chain antibody protein that substrate binding site contains the GPX catalytic group with transgenation technology and auxotroph intestinal bacteria.Thereby on this single-chain antibody the substrate binding site of existing GPX, catalytic group is arranged again, the result gives the catalytic activity of the high GPX of this single-chain antibody, has just produced the human selenium-containing single-chain abzyme of high vigor.
Specifically the preparation method is, utilizes the phage antibody display technique, is target antigen with the gsh derivative, by the affine sieve method of immunity reorganization phage display people single-chain antibody library is screened.Separate and amplification GSH specific single-chain antibody gene.Said gene is assembled on secretor type protokaryon or the carrier for expression of eukaryon transformed into escherichia coli or yeast cell, great expression and purification of single stranded antibody protein in protokaryon or eucaryon.With the hydroxyl on Serine (Ser) residue in the phenylmethylsulfonyl fluoride activation single-chain antibody, handle with sodium hydrogen selenide again, then Ser is mutated into SeCys, the result gives the catalytic activity of antibody with GPX at the catalytic group SeCys of the substrate binding site introducing GPX of antibody; Or directly be expressed in the soluble single-chain antibody protein that substrate binding site contains the GPX catalytic group with transgenation technology and auxotroph intestinal bacteria, give the catalytic activity of antibody with GPX.
The present invention is a target antigen with the gsh derivative, by the affine sieve method of immunity reorganization phage display people single-chain antibody library is screened.The human single chain variable fragments antibody that is used to prepare human selenium-containing single-chain abzyme that obtains, the small peptide of being made up of 15 amino acid connects, and aminoacid sequence comprises the light chain and the variable region of heavy chain of immunoglobulin (Ig); Concrete single-chain antibody B3 and the aminoacid sequence of single-chain antibody D8 be respectively,
The aminoacid sequence of B3:
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWM50GWINAGNGNTKYS63QKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVAL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGS200SSGNTASLTITGAQAEDEADYYCNSRDSSGHPSVFGGGTKLTVLG245；
The aminoacid sequence of D8:
MARVQLVQSGAEVKKTGSSVKVSCKASGYTLTYRYLHWVRQAPGQALEWM50GWITPFNGNANYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAVYYCAR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVAL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGS200SSGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGGGTKLTVLG244。
The first method of preparation soluble human selenium-containing single-chain abzyme: separate the amplification single-chain antibody gene, be assembled on the Procaryon secreted expression carrier, transformed into escherichia coli, express the soluble single-chain antibody protein, with the chemical mutation method catalytic group of GPX is incorporated into the substrate binding site of single-chain antibody again.Detailed process is as follows.
A kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, the process of the expression and purification of soluble single-chain antibody, the introducing of catalytic group,
The screening of described people's single-chain antibody is the gsh diester that the method synthesizing series haptens-S-dinitrobenzene according to prior art (seeing patent 96112628.0 and 99104234.4) replaces.With the synthetic haptens is target antigen, and the immune affine sieve method by standard is screened reorganization phage display people single-chain antibody library, obtain can with GSH and derivative bonded human single chain variable fragments antibody B3 or D8.
The expression and purification of described soluble single-chain antibody, be to keep under the constant prerequisite of aminoacid sequence, according to the single-chain antibody B3 of screening or the gene order design primer of D8, with the phage single-chain DNA that contains single-chain antibody gene is template, with primer and polymerase chain reaction (PCR) amplification single-chain antibody gene.Cut the single-chain antibody gene and the prokaryotic expression carrier pPELB of purifying with endonuclease, single-chain antibody gene is assembled on the pPELB carrier by restriction enzyme site.The prokaryotic expression carrier transformed into escherichia coli competent cell that will contain single-chain antibody gene, the screening positive transformant, with isopropyl-(IPTG) abduction delivering, single chain antibody protein is secreted in the pericentral siphon chamber of thalline with soluble form.Collect thalline, ultrasonication under the low temperature, centrifugal removal bacterial sediment.With GSH affinitive layer purification single chain antibody protein.Promptly get the pure product of single chain antibody protein after the dialysis freeze-drying.Verify as a band through electrophoresis, molecular weight is 31000 dalton.
The introducing of described catalytic group is the substrate binding site that the catalytic group SeCys of GPX is incorporated into single-chain antibody with disclosed chemical mutation method (seeing patent 99104234.4).Promptly, handle with sodium hydrogen selenide again, then Ser is mutated into SeCys with the hydroxyl on Serine (Ser) residue in the phenylmethylsulfonyl fluoride activation single-chain antibody.Because GSH is the substrate of GPX, SeCys is the catalytic group of GPX, thus on this single-chain antibody the substrate binding site of existing GPX, catalytic group is arranged again, the result produces the human selenium-containing single-chain abzyme of high vigor.
The concrete grammar that catalytic group is introduced can be: 0.4-1.5 * 10
-5The pure product of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 2.0-6.0 * 10
-5The mmol/L phenylmethylsulfonyl fluoride, 15-30 ℃ of vibration 1-3h.The logical about 20min of high pure nitrogen is with oxygen in the eliminating system.Add 0.5-3.0 * 10
-4Mol/L sodium hydrogen selenide (NaHSe) solution, 30-40 ℃ of reaction 25-40h under nitrogen protection.Take out, uncapping makes its abundant oxidation.The centrifugal 15min of 10000g, supernatant liquor with the SephadexG25 desalination after freeze-drying promptly get human selenium-containing single-chain abzyme with GPX vigor, its GPX vigor is 1005-1321U/ μ mol, reaches the order of magnitude level of natural GPX.
In the expression and purification process of soluble single-chain antibody, described primer is that 5 ' end primer adds initiator codon and NcoI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; Or antibody library consensus primer 5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 '.Described endonuclease can be NcoI and NotI.
Said screening positive transformant, can be the conversion after product to be coated on 2 * TY solid medium (contain 100 μ g/mL penbritins and 34 μ g/mL paraxin), 37 ℃ of overnight incubation, select well-grown mono-clonal bacterium colony on flat board, determine to contain the positive transformant of single-chain antibody gene by dna sequencing once more.
Said abduction delivering can be positive transformant to be inoculated in 2ml 2 * TY liquid nutrient medium (contain 100 μ g/mL penbritins and 34 μ g/mL paraxin), and 37 ℃ of shaking culture are to OD
600Be 0.4-1.0.Adding final concentration is the IPTG of 0.5-1mmol/L, 30 ℃ of following abduction delivering 4-8h.
Said centrifugal removal bacterial sediment can be with bacterial cell disruption liquid at low temperatures, and the centrifugal 15-30min of 8000-12000g removes bacterial sediment, obtains supernatant liquor.
The second method of preparation soluble human selenium-containing single-chain abzyme: separate the amplification single-chain antibody gene, be assembled on the eucaryon secreted expression carrier, transformed yeast cell, express the soluble single-chain antibody protein, with the chemical mutation method catalytic group of GPX is incorporated into the substrate binding site of single-chain antibody again.Detailed process is as follows.
A kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, the expression and purification of soluble single-chain antibody, and the process of the introducing of catalytic group,
The screening process of people's single-chain antibody is identical with first method.
The expression and purification of described soluble single-chain antibody, be to keep under the constant prerequisite of aminoacid sequence, according to the single-chain antibody B3 of screening or the gene order design primer of D8,5 ' end primer adds initiator codon and EcoRI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; With the phage single-chain DNA that contains positive single-chain antibody gene is template, with this primer and pcr amplification single-chain antibody gene, cut single-chain antibody gene and secretor type Yeast expression carrier PIC9K with EcoRI and NotI enzyme, by the EcoRI/NotI restriction enzyme site, single-chain antibody gene is assembled on the PIC9K; Use the BglII single endonuclease digestion, make the expression vector linearizing that contains single-chain antibody gene, the carrier that will contain single-chain antibody gene transforms the pichia spp competent cell, under methanol induction, and expressing human source single chain antibody protein in the pichia spp cell; Single chain antibody protein is expressed justacrine in substratum with soluble form, collects culture supernatant, after concentrating, with gsh affinitive layer purification single chain antibody protein, promptly gets the pure product of single chain antibody protein after the dialysis freeze-drying, carries out the introducing of catalytic group again.
The careful step of soluble single-chain antibody expression and purge process can be: keeping under the constant prerequisite of aminoacid sequence, according to the gene order design primer of single-chain antibody.With the phage single-chain DNA that contains single-chain antibody gene is template, with primer and round pcr amplification single-chain antibody gene.Separate single-chain antibody gene with agarose gel electrophoresis, reclaim test kit purification of single stranded antibody gene with DNA glue.Cut the single-chain antibody gene and the secretor type Yeast expression carrier PIC9K of purifying with EcoRI and NotI enzyme,, single-chain antibody gene is assembled on the PIC9K by the EcoRI/NotI restriction enzyme site.Make the expression vector PIC9K linearizing that contains single-chain antibody gene with the BglII single endonuclease digestion, transform pichia spp GS115 competent cell, the yeast cell after the conversion is coated with culture plate, is inverted in 30 ℃ of incubators and cultivates 2-3 days.Picking mono-clonal from the culture plate is determined to contain the positive transformant of single-chain antibody gene by dna sequencing.Positive transformant is inoculated in the BMGY substratum, and 30 ℃ of concussions are cultivated, and reach 2-6 to OD600, and room temperature centrifugal (3000rpm) 5min abandons supernatant, and with isopyknic BMMY substratum re-suspended cell precipitation, 30 ℃ of concussion cultivations are expressed with methanol induction.In inducing process, every 24h replenishes a methyl alcohol to final concentration 0.5%, replenishes the sterilization ultrapure water simultaneously, and the nutrient solution cumulative volume is remained unchanged.Continuous induction was cultivated 7 days, and every 24h gets the 1ml nutrient solution, and centrifugal thalline, supernatant are used for the SDS-PAGE analysis of protein, the high expression level bacterial strain of screening positive clone.Get the high expression level bacterial strain, cultivated 3 days with an amount of methanol induction after the enlarged culturing, single chain antibody protein is expressed justacrine in substratum with soluble form, collect culture supernatant, concentrate 20 times through 10k molecular weight ultra-filtration membrane, with GSH affinitive layer purification single chain antibody protein (use pH7.5,50mmol/L Tris-Cl balance is with this buffer solution elution target protein that contains 10mmol/L GSH).Promptly get the pure product of single chain antibody protein after the dialysis freeze-drying.Verify as a band through electrophoresis, molecular weight is 31000 dalton.
The process of the introducing of catalytic group is identical with first method.
The third method of preparation soluble human selenium-containing single-chain abzyme is to introduce catalytic group by transgenation method rather than chemical mutation method.Detailed process is as follows.
A kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, and the transgenation method is introduced catalytic group, the process of the expression and purification of abzyme.
The screening process of people's single-chain antibody is identical with first method.
Described transgenation method is introduced catalytic group, be to keep under the constant prerequisite of aminoacid sequence, gene order design primer according to single-chain antibody, with the phage single-chain DNA that contains single-chain antibody gene is template, with primer and pcr amplification single-chain antibody gene, cut the single-chain antibody gene and the prokaryotic expression carrier pPELB of purifying with endonuclease, single-chain antibody gene is assembled on the pPELB carrier by restriction enzyme site.Keeping under the constant prerequisite of other aminoacid sequence, design sports Ser the rite-directed mutagenesis primer of halfcystine (Cys), with rite-directed mutagenesis primer and quick rite-directed mutagenesis test kit (Invitrogen company product, press test kit specification sheets operation), the sequence encoding mutant of the Serine of the 0-32 in the single-chain antibody gene that is structured on the prokaryotic expression carrier is become the codon (TGC) (0 is meant the activity that also has GPX under the situation that no Ser suddenlys change) of Cys.Determine to suddenly change successfully by dna sequencing, and do not have other unexpected transgenation generation, the result has introduced catalytic group by transgenation at the substrate binding site of single-chain antibody.
The expression and purification of described abzyme, be will contain the carrier of single-chain antibody gene of sudden change transform the competent cell of auxotrophic strain-BL21 (DE3) CysE, be coated with the nutrient agar plate that contains Cys, the screening positive transformant.With positive transformant spread cultivation support after, in the substratum that contains SeCys and essential somatomedin and nutrient substance, induce through IPTG, auxotrophy bacterial strain-BL21 (DE3) CysE can synthesize SeCys with the codon of Cys, the last human selenium-containing single-chain abzyme that contains SeCys at the substrate binding site of GSH that directly gives expression to, the single-chain antibody enzyme is expressed justacrine in the pericentral siphon chamber of thalline with soluble form.Collect thalline, ultrasonication under the low temperature, centrifugal removal bacterial sediment.With GSH affinitive layer purification single-chain antibody enzyme.Promptly get the pure product of single-chain antibody enzyme after the dialysis freeze-drying.Verify as a band through electrophoresis, molecular weight is 31000 dalton.Because GSH is the substrate of GPX, SeCys is the catalytic group of GPX, thereby produces the human selenium-containing single-chain abzyme of high vigor.
Introduce in the catalytic group process in the transgenation method, described primer can be that 5 ' end primer adds initiator codon and NcoI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; Or antibody library consensus primer 5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 '.Said endonuclease can be NcoI and NotI.
Introduce in the catalytic group process in the transgenation method, described rite-directed mutagenesis primer, can be to design two rite-directed mutagenesis primers of complete isometric complementary with its neighbour amino acid whose gene order according to the position of the Ser that will sport Cys, the long 35-50bp of primer is the center with the codon (TGC) of Cys.
Said centrifugal removal bacterial sediment can be with bacterial cell disruption liquid at low temperatures, and the centrifugal 15-30min of 8000-12000g removes bacterial sediment, obtains supernatant liquor.
The present invention also has the 4th kind of method: any amino acid of the complementary antigen determining area of single-chain antibody (CDR) is carried out the chain reorganization or replaces with other amino acid with transgenation or synthesis method, other is constant, also available above-mentioned three kinds of methods prepare the single-chain antibody enzyme, and the vigor of enzyme is improved.
The present invention has following characteristics:
(1) the present invention uses simple antibody library screening, genetically engineered and chemical mutation technology, and the preparation method is simple;
(2) the human selenium-containing single-chain abzyme vigor height of the inventive method preparation has reached the order of magnitude level of natural GPX vigor, and Cell Biology Experiment has confirmed that the myocardial cell of hydrogen peroxide damage is had protective effect;
(3) human selenium-containing single-chain abzyme of the inventive method preparation can be used for human body, can not cause immune response, has broad application prospects aspect bio-pharmaceuticals;
(4) secreted expression carrier of the present invention's use, can with single chain antibody protein with soluble form express justacrine to colibacillary pericentral siphon chamber or zymic external, the targeting signal peptide of pilot protein secreting, expressing is excised automatically by thalline, expressed albumen is solubility, space conformation and activity with native protein, do not need renaturation, the productive rate that can avoid the renaturing inclusion bodies process to cause descends and inactivation, thereby with short production cycle.
These advantages all help scale operation, for from now on practical application lays a solid foundation, solve the natural GPX insufficient problem of originating.
Embodiment:
Embodiment 1:
With existing technology (seeing the embodiment 1 of patent 99104234.4) synthesizing glutathion dibutylester, get the gsh dibutylester of 50 μ g, after methyl-sulphoxide (DMSO) dissolving, be coated on the immune flat board, the residue site is sealed with BSA.Gsh dibutylester with the bag quilt is a target antigen, with the immune affine sieve method of standard phage display people single-chain antibody library is carried out 5 and take turns screening, use the further screening positive clone of ELISA method at last again, obtain the specific single-chain antibody B3 higher with GSH avidity, full gene and aminoacid sequence are determined in order-checking.
Extract phage DNA, as template, adopt antibody library consensus primer (5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 ') to do pcr amplification single-chain antibody B3 gene with the phage DNA that contains single-chain antibody B3 gene.The PCR product is carried out electrophoresis on 1.2% sepharose, use the single-chain antibody B3 gene that DNA glue reclaims the amplification of test kit purifying.
B3 gene behind the purifying with behind restriction enzyme Nco I and the Not I double digestion, is connected on the carrier pPELB carrier that same enzyme cuts, will connects product (pPELB-B3) transformed into escherichia coli Rosetta (or BL21).To transform after product is coated on 2 * TY solid medium and (contains 100 μ g/mL penbritins and 34 μ g/mL paraxin), 37 ℃ of overnight incubation, select well-grown mono-clonal bacterium colony on flat board, after cultivating in a small amount on 2ml 2 * TY liquid nutrient medium, determine to contain the positive transformant of single-chain antibody gene by dna sequencing.Positive transformant is inoculated in 2ml 2 * TY liquid nutrient medium (contains 100 μ g/mL penbritins and 34 μ g/mL paraxin), 37 ℃ of shaking culture are to OD
600Be 0.6.Adding final concentration is the IPTG of 1mmol/L, 30 ℃ of following abduction delivering 4h.Single chain antibody protein is expressed justacrine in the pericentral siphon chamber of thalline with soluble form, with the high expression level bacterial strain of denaturing polyacrylamide gel electrophoresis (SDS-PAGE) screening great expression single chain antibody protein.The high expression level inoculation (is contained 100 μ g/mL penbritins, 34 μ g/mL paraxin) in 1L 2 * TY substratum, 37 ℃ of shaking culture are to OD
600For 0.6. adds final concentration is the IPTG (isopropyl-) of 1mmol/L, 30 ℃ of following abduction delivering 4h.The centrifugal 10min of 6000rpm collects thalline and is resuspended in (pH 7.4 for 300mmol/L NaCl, 20mmol/LTris, the 1mmol/L phenylmethylsulfonyl fluoride) in the solution, ultrasonication under the ice bath.At 4 ℃, the centrifugal 15min of 8000g obtains supernatant liquor with liquid.By specification is handled GSH affinity post, and (50mmol/LTris, pH7.5) balance add supernatant liquor on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein to use damping fluid.With elutriant dialysis and freeze-drying, promptly get human single chain variable fragments antibody.Verify as a band through electrophoresis, molecular weight is 31KD.
1.2 * 10
-5The pure product of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 5.0 * 10
-5The mmol/L phenylmethylsulfonyl fluoride, room temperature vibration 3h.The logical about 20min of high pure nitrogen is with oxygen in the eliminating system.Add 2.0 * 10
-4Mol/L sodium hydrogen selenide (NaHSe) solution, 37 ℃ of reaction 40h under nitrogen protection.Take out, uncapping makes its abundant oxidation, the centrifugal 15min of 10000g.Freeze-drying promptly got human selenium-containing single-chain abzyme after supernatant liquor was used the SephadexG25 desalination, and its GPX vigor is 1321u/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 2:
According to the synthetic haptens of the method for patent 99104234.4-gsh dibutylester, get the gsh dibutylester of 50 μ g, after methyl-sulphoxide (DMSO) dissolving, be coated on the immune flat board, the residue site is sealed with BSA.Gsh dibutylester with the bag quilt is a target antigen, with the immune affine sieve method of standard phage display people single-chain antibody library is carried out 5 and take turns screening, use the further screening positive clone of ELISA method at last again, obtain the specific single-chain antibody B3 higher with GSH avidity, full gene and aminoacid sequence are determined in order-checking.
Keeping under the constant prerequisite of aminoacid sequence, according to the gene order of single-chain antibody B3 design primer (5 '-CGGAATTCATGGCCCGGGT-3 ' and 5 '-GTGCGGCCGCACCTAGGA-3 '), 5 ' end primer has added initiator codon (ATG) and EcoRI restriction enzyme site, and 3 ' end primer has added the NotI restriction enzyme site in the terminator codon downstream.Extract phage DNA, as template, adopt above-mentioned primer to do the pcr amplification single-chain antibody gene with the phage DNA that contains single-chain antibody B3 gene.The PCR product is carried out electrophoresis on 1.2% sepharose, use the single-chain antibody gene that DNA glue reclaims the amplification of test kit purifying.
Single-chain antibody gene behind the purifying with behind restriction enzyme EcoRI and the NotI double digestion, is connected on the secretor type Yeast expression carrier PIC9K that same enzyme cuts.Make expression vector (PIC9K-B3) linearizing that contains single-chain antibody gene with the BglII single endonuclease digestion, transform pichia spp GS115 competent cell.Yeast cell after the conversion is coated with MD and MM culture plate, is inverted in 30 ℃ of incubators and cultivates 2～3 days.Can on MD and MM, all grow what be that the quick type of methyl alcohol utilization can only grow on MD is the slow type of methyl alcohol utilization.
10 clones of each picking on MD and the MM culture plate after cultivating in a small amount, carry out the positive transformant that dna sequencing determines to contain single-chain antibody gene in the 2mlBMGY substratum.Positive transformant is inoculated in respectively in the 10ml BMGY substratum, and (225rpm) 24h are cultivated in 30 ℃ of concussions, reach 2-6 to OD600, room temperature centrifugal (3000rpm) 5min abandons supernatant, with the BMMY substratum re-suspended cell precipitation of equal-volume (10ml), abduction delivering is cultivated in 30 ℃ of concussions.In inducing process, every 24h replenishes a methyl alcohol to final concentration 0.5%, replenishes the sterilization ultrapure water simultaneously, and the nutrient solution cumulative volume is remained unchanged.Continuous induction was cultivated 7 days, and every 24h gets the 1ml nutrient solution, and centrifugal thalline, supernatant are used for the SDS-PAGE analysis of protein, screening high expression level bacterial strain.Get the high expression level bacterial strain, shook in the bottle inducing culture 3 days, collect the nutrient solution supernatant, after 10k molecular weight ultra-filtration membrane concentrates 20 times at the 5L that contains the 1L nutrient solution.With GSH affinity column purification of single stranded antibody (use pH7.5,50mmol/L Tris-Cl balance is with this buffer solution elution target protein that contains 10mmol/L GSH).Collection has the elutriant of single-chain antibody, the dialysis desalination, and freeze-drying promptly gets human single chain variable fragments antibody.Verify as a band through electrophoresis, molecular weight is 31KD.
0.4 * 10
-5The pure product of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 2.0 * 10
-5The mmol/L phenylmethylsulfonyl fluoride, 30 ℃ of vibration 1.5h.The logical about 20min of high pure nitrogen is with oxygen in the eliminating system.Add 0.5 * 10
-4Mol/L sodium hydrogen selenide (NaHSe) solution, 40 ℃ of reaction 25h. take out under nitrogen protection, and uncapping makes its abundant oxidation.Freeze-drying promptly got human selenium-containing single-chain abzyme Sec-B3 after the centrifugal 15min. supernatant liquor of 10000g was used the SephadexG25 desalination, and its GPX vigor is 1235U/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 3:
With existing technology (seeing the embodiment 1 of patent 99104234.4) synthesizing glutathion dibutylester, get the gsh dibutylester of 50 μ g, after methyl-sulphoxide (DMSO) dissolving, be coated on the immune flat board, the residue site is sealed with BSA.Gsh dibutylester with the bag quilt is a target antigen, with the immune affine sieve method of standard phage display people single-chain antibody library is carried out 5 and take turns screening, use the further screening positive clone of ELISA method at last again, obtain the specific single-chain antibody B3 higher with GSH avidity, full gene and aminoacid sequence are determined in order-checking.
Extract phage DNA, as template, adopt antibody library consensus primer (5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 ') to do pcr amplification single-chain antibody B3 gene with the phage DNA that contains single-chain antibody B3 gene.The PCR product is carried out electrophoresis on 1.2% sepharose.Use the single-chain antibody B3 gene that DNA glue reclaims the amplification of test kit purifying.B3 gene behind the purifying with behind restriction enzyme Nco I and the Not I double digestion, is connected on the carrier pPELB carrier that same enzyme cuts.Keeping under the constant prerequisite of other aminoacid sequence, designing two rite-directed mutagenesis primers of complete isometric complementary according to the amino acid whose gene order near No. 63 Serines, the long 35-50bp of primer is the center with the codon (TGC) of Cys.With these two complete complementary primers and quick rite-directed mutagenesis test kit (Invitrogen company, press the operation of test kit specification sheets), the encoding sequence (TCA) that is structured in No. 63 Serine of single-chain antibody gene on the prokaryotic expression carrier pPelB is mutated into the codon (TGC) of Cys, determine to suddenly change successfully by dna sequencing, and do not have other unexpected transgenation generation.
Carrier (pPelB-B3) with the single-chain antibody gene that sudden change is housed transforms auxotroph e. coli bl21 (DE3) CysE, is coated with the nutrient agar plate that contains Cys, the screening positive transformant.With positive transformant be inoculated in 1.2L M9 express in the substratum (in the M9 substratum, adding each 100 μ g/m1 of 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and 19 seed amino acids except Cys) to OD600 be 0.1, be cultured to OD600 and be about 0.3-1, the IPTG that adds final concentration 0.1-1mM, the paraxin of adding final concentration 10 μ g/ml behind the 10min.Adding paraxin medium centrifugal after five minutes, centrifugal 5 minutes of low temperature 6000rpm, wash twice with the physiological salt solution of ice bath, each 1.2L that uses, use production substratum (in the M9 substratum, adding each the 50-400 μ g/ml of 19 seed amino acids except Cys) resuspended then, and in substratum, replenish Rifampin and the 600 μ M DL-SeCys of final concentration 400 μ g.Continue to cultivate 2-12h, the single-chain antibody enzyme is expressed in the pericentral siphon chamber of thalline with soluble form.(6000rpm is 10min) also with isopyknic bufferT (50mM Tris buffer, pH7.5 contains 1mM EDTA) washed twice to collect thalline.With the resuspended bacterial sediment of BufferT, add the PMSF (phenylmethylsulfonyl fluoride) of final concentration 1mM, the ultrasonication thalline, 4 ℃, the centrifugal 30min of 12000rpm collects supernatant.By specification is handled GSH affinity post, and (50mmol/L Tris, pH7.5) balance add supernatant liquor on the GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein to use damping fluid.With elutriant dialysis and freeze-drying, promptly get the human single chain variable fragments antibody enzyme.Verify as a band through electrophoresis, molecular weight is 31KD.Its GPX vigor is 1289U/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 4:
With the gsh diisoamyl ester is that target antigen screens, and screening method and embodiment 1 are identical, obtain single-chain antibody D8.The introducing of the expression of antibody, purifying and catalytic group is also identical with embodiment 1.Get human selenium-containing single-chain abzyme Sec-D8, its GPX vigor is 1005U/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 5
With the gsh diisoamyl ester is that target antigen screens, and screening method and embodiment 1 are identical, obtain single-chain antibody D8.The introducing of the expression of antibody, purifying and catalytic group and embodiment 2 are identical, and just the design of gene amplification primer will be according to the gene order decision of D8.Get human selenium-containing single-chain abzyme Sec-D8, its GPX vigor is 1098U/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 6
With the gsh diisoamyl ester is that target antigen screens, and screening method and embodiment 1 are identical, obtain single-chain antibody D8.The introducing of the expression of antibody, purifying and catalytic group and embodiment 3 are identical, just position and the decision of the gene order near it of the transgenation primer design concrete Ser that will will suddenly change according to D8.Obtain human selenium-containing single-chain abzyme Sec-D8, its GPX vigor is 1065U/ μ mol, reaches the order of magnitude level of natural GPX.
Claims (7)
1, a kind of human single chain variable fragments antibody that is used to prepare human selenium-containing single-chain abzyme is characterized in that, the small peptide of being made up of 15 amino acid connects, and aminoacid sequence comprises the light chain and the variable region of heavy chain of immunoglobulin (Ig); The aminoacid sequence of human single chain variable fragments antibody B3 and human single chain variable fragments antibody D8 is respectively,
The aminoacid sequence of B3:
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWM50GWINAGNGNTKYS63QKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVAL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGS200SSGNTASLTITGAQAEDEADYYCNSRDSSGHPSVFGGGTKLTVLG245；
The aminoacid sequence of D8:
MARVQLVQSGAEVKKTGSSVKVSCKASGYTLTYRYLHWVRQAPGQALEWM50GWITPFNGNANYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAVYYCAR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVAL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGS200SSGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGGGTKLTVLG244。
2, a kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, the expression and purification of soluble single-chain antibody, and the process of the introducing of catalytic group is characterized in that,
The screening of described people's single-chain antibody, be that the gsh diester that replaces with haptens-S-dinitrobenzene is a target antigen, by the affine sieve method of immunity reorganization phage display people single-chain antibody library is screened, obtain can with gsh and derivative bonded human single chain variable fragments antibody B3 or D8;
The expression and purification of described soluble single-chain antibody is to keep under the constant prerequisite of aminoacid sequence, according to the single-chain antibody B3 of screening acquisition or the gene order design primer of D8; With the phage single-chain DNA that contains single-chain antibody gene is template, with primer and PCR amplification single-chain antibody gene; Cut single-chain antibody gene and secretor type prokaryotic expression carrier pPELB with endonuclease, single-chain antibody gene is assembled on the pPELB carrier by restriction enzyme site; The prokaryotic expression carrier transformed into escherichia coli competent cell that will contain single-chain antibody gene, the screening positive transformant; Behind the isopropyl-abduction delivering, single chain antibody protein is secreted in the pericentral siphon chamber of thalline with soluble form; Collect thalline, ultrasonication under the low temperature, centrifugal removal bacterial sediment; With gsh affinitive layer purification single chain antibody protein, promptly get the pure product of single chain antibody protein after the dialysis freeze-drying; Carry out the introducing of catalytic group again.
3, according to the preparation method of the described soluble human selenium-containing single-chain abzyme of claim 2, it is characterized in that, described primer is that 5 ' end primer adds initiator codon and NcoI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; Or 5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 '; Described endonuclease is NcoI and NotI.
4, a kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, the expression and purification of soluble single-chain antibody, and the process of the introducing of catalytic group is characterized in that,
The screening of described people's single-chain antibody, be that the gsh diester that replaces with haptens-S-dinitrobenzene is a target antigen, by the affine sieve method of immunity reorganization phage display people single-chain antibody library is screened, obtain can with gsh and derivative bonded human single chain variable fragments antibody B3 or D8;
The expression and purification of described soluble single-chain antibody, be to keep under the constant prerequisite of aminoacid sequence, according to the single-chain antibody B3 of screening acquisition or the gene order design primer of D8,5 ' end primer adds initiator codon and EcoRI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; With the phage single-chain DNA that contains positive single-chain antibody gene is template, with primer and PCR amplification single-chain antibody gene, cut single-chain antibody gene and secretor type Yeast expression carrier PIC9K with EcoRI and NotI enzyme, by the EcoRI/NotI restriction enzyme site, single-chain antibody gene is assembled on the PIC9K; Use the BglII single endonuclease digestion, make the expression vector linearizing that contains single-chain antibody gene, the carrier that will contain single-chain antibody gene transforms the pichia spp competent cell, under methanol induction, expressing human source single chain antibody protein in the pichia spp cell, single chain antibody protein is expressed justacrine in substratum with soluble form, collect culture supernatant, after concentrating,, promptly get the pure product of single chain antibody protein after the dialysis freeze-drying with gsh affinitive layer purification single chain antibody protein; Carry out the introducing of catalytic group again.
5, a kind of preparation method of soluble human selenium-containing single-chain abzyme has the screening of people's single-chain antibody, and the transgenation method is introduced catalytic group, the process of the expression and purification of abzyme,
The screening of described people's single-chain antibody gene, be that the gsh diester that replaces with haptens-S-dinitrobenzene is a target antigen, by the affine sieve method of immunity reorganization phage display people single-chain antibody library is screened, obtain can with gsh and derivative bonded human single chain variable fragments antibody B3 or D8;
Described by transgenation introducing catalytic group, be to keep under the constant prerequisite of aminoacid sequence, according to the single-chain antibody B3 of screening acquisition or the gene order design primer of D8; With the phage single-chain DNA that contains single-chain antibody gene is template, with primer and PCR amplification single-chain antibody gene, cut single-chain antibody gene and secretor type prokaryotic expression carrier pPELB with endonuclease, single-chain antibody gene is assembled on the pPELB carrier by restriction enzyme site; Keeping under the constant prerequisite of other aminoacid sequence, design sports Ser the rite-directed mutagenesis primer of Cys, with the rite-directed mutagenesis primer and fast the rite-directed mutagenesis test kit sequence encoding mutant that will be structured in 0-32 Serine in the single-chain antibody gene on the prokaryotic expression carrier become the codon of Cys, determine to suddenly change successfully by dna sequencing, the result has introduced catalytic group by transgenation at the substrate binding site of single-chain antibody;
The expression and purification of described abzyme, it is the competent cell that the carrier that will contain the single-chain antibody gene of sudden change transforms auxotrophy bacterial strain-BL21 (DE3) CysE, be coated with the nutrient agar plate that contains Cys, the screening positive transformant, with positive transformant spread cultivation support after, in the substratum that contains SeCys and essential somatomedin and nutrient substance, induce through isopropyl-, auxotrophy bacterial strain-BL21 (DE3) CysE can synthesize SeCys with the codon of Cys, the last human selenium-containing single-chain abzyme that contains SeCys at the substrate binding site of gsh that directly gives expression to, the single-chain antibody enzyme is expressed justacrine in the pericentral siphon chamber of thalline with soluble form; Collect thalline, ultrasonication under the low temperature, centrifugal removal bacterial sediment with gsh affinitive layer purification single-chain antibody enzyme, promptly gets the pure product of single-chain antibody enzyme after the dialysis freeze-drying.
6, according to the preparation method of the described soluble human selenium-containing single-chain abzyme of claim 5, it is characterized in that, described primer is that 5 ' end primer adds initiator codon and NcoI restriction enzyme site, and 3 ' end primer adds the NotI restriction enzyme site in the terminator codon downstream; Or 5 '-CAGGAAACAGCTATGAC-3 ' and 5 '-GAATTTTCTGTATGAGG-3 '; Described endonuclease is NcoI and NotI.
7, according to the preparation method of claim 5 or 6 described soluble human selenium-containing single-chain abzymes, it is characterized in that, described rite-directed mutagenesis primer, be to design two rite-directed mutagenesis primers of complete isometric complementary with its neighbour amino acid whose gene order according to the position of the Ser that will sport Cys, the long 35-50bp of primer is the center with the codon of Cys.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810050556A CN101280015B (en) | 2008-04-01 | 2008-04-01 | Preparation of soluble human selenium-containing single-chain abzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810050556A CN101280015B (en) | 2008-04-01 | 2008-04-01 | Preparation of soluble human selenium-containing single-chain abzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101280015A true CN101280015A (en) | 2008-10-08 |
| CN101280015B CN101280015B (en) | 2012-09-05 |
Family
ID=40012701
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810050556A Expired - Fee Related CN101280015B (en) | 2008-04-01 | 2008-04-01 | Preparation of soluble human selenium-containing single-chain abzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101280015B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898073A (en) * | 2013-07-18 | 2014-07-02 | 吉林大学 | Glutathione peroxidase GPX1 mutant containing more serine and preparation method of mutant |
| CN104059894A (en) * | 2013-04-24 | 2014-09-24 | 吉林大学 | Method for preparing recombinant glutathione peroxidase by use of eukaryotic secretory expression system |
| CN113214370A (en) * | 2021-02-07 | 2021-08-06 | 武汉大学 | Preparation method of single selenoprotein of saccharomyces cerevisiae |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063484C (en) * | 1994-03-09 | 2001-03-21 | 吉林大学 | Prepn. of selenium-bearing antibody enzyme using monoclonal antibody |
| CN1062600C (en) * | 1996-09-10 | 2001-02-28 | 中国科学院长春应用化学研究所 | Preparation of selenic antibody enzyme with activity higher than that of natural enzyme |
| CN1274007A (en) * | 1999-05-13 | 2000-11-22 | 中国科学院长春应用化学研究所 | Process for preparing Se-contained humanized abzyme |
-
2008
- 2008-04-01 CN CN200810050556A patent/CN101280015B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059894A (en) * | 2013-04-24 | 2014-09-24 | 吉林大学 | Method for preparing recombinant glutathione peroxidase by use of eukaryotic secretory expression system |
| CN103898073A (en) * | 2013-07-18 | 2014-07-02 | 吉林大学 | Glutathione peroxidase GPX1 mutant containing more serine and preparation method of mutant |
| CN103898074A (en) * | 2013-07-18 | 2014-07-02 | 吉林大学 | Glutathione peroxidase GPX1 mutant containing more than two catalysis groups and preparation method thereof |
| CN103898073B (en) * | 2013-07-18 | 2015-12-02 | 吉林大学 | A kind of Selenoperoxidase GPX1 mutant containing more multifibres propylhomoserin and preparation method thereof |
| CN113214370A (en) * | 2021-02-07 | 2021-08-06 | 武汉大学 | Preparation method of single selenoprotein of saccharomyces cerevisiae |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101280015B (en) | 2012-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103320406B (en) | High-activity glutathion peroxidase GPX 1 mutant and its preparation method | |
| CN103864914B (en) | The preparation method of high-purity interleukin II 4 inclusion body | |
| CN103224915A (en) | Gene engineering method for preparing a recombinant glutathion peroxidase | |
| CN103555729B (en) | Trail dna sequence, expression and the application of a kind of transformation | |
| CN101280015B (en) | Preparation of soluble human selenium-containing single-chain abzyme | |
| CN112695054A (en) | Construction method and application of high-expression chitinase endophytic fungus Liquidambaris phomopsis | |
| CN103710367B (en) | A kind of recombined human kallikrein 1 and encoding gene thereof and preparation method | |
| CN103193887B (en) | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein | |
| CN107236039A (en) | Recombinate Wzt albumen rabbit anteserum polyclonal antibodies and preparation method thereof | |
| CN105018482A (en) | Hybridized tRNA (transfer ribonucleic acid) and application thereof to glutathione peroxidase preparation | |
| CN109852623B (en) | Preparation method, purification method and application of PEG-modified recombinant humanized urate oxidase | |
| CN1206351C (en) | Preparing process and application of lysozyme protein | |
| CN1865286A (en) | Double function epidermal growth factor and its preparation method and uses | |
| CN104651329A (en) | Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant | |
| CN103898075A (en) | Novel high activity glutathione peroxidase (GPX) mutant and preparation method thereof | |
| CN102485890B (en) | With Pichia sp. excreting and expressing recombinant human protein disulfide isomerase | |
| CN102321665B (en) | Method for expressing human humanin protein in peanut seed | |
| CN107354137A (en) | A kind of selenoprotein of high activity and preparation method thereof | |
| CN1940066A (en) | Production of expression vermis kinase tr-gene plant and its products | |
| CN103497927B (en) | The recombinant BCG viable bacteria bacterial strain of expression-secretion S. aureus L-forms enterotoxin albumen, live bacterial vaccines and construction process thereof and application | |
| CN103881988A (en) | High-vitality glutathione peroxidase GPX3 (glutathione peroxidase) mutant and preparation method thereof | |
| CN114891125B (en) | Long-acting recombinant interleukin-18binding protein and production method and application thereof | |
| CN104059902B (en) | Beta-amylase-trehalose synthase fused enzyme and expression gene and application thereof | |
| CN104059894A (en) | Method for preparing recombinant glutathione peroxidase by use of eukaryotic secretory expression system | |
| CN103756984B (en) | High vigor Selenoperoxidase GPX2 mutant and preparation method 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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 Termination date: 20130401 |