CN101280015B - Preparation of soluble human selenium-containing single-chain abzyme - Google Patents
Preparation of soluble human selenium-containing single-chain abzyme Download PDFInfo
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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), katalase (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 like ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different with other antioxidase, GPX is except removing the ROS, and the MDA of can also degrading prevents the cell peroxide injury, and the function of the protection cell of this uniqueness makes it in the antioxidase system, occupy the position of particularly important.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, is merely about the per mille 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 HAMA, 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 haptin-GSH verivate is with synthetic; Verivate 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 existing substrate binding site has catalytic group again in the single-chain antibody variable region, 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 deposition 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 under suitable condition, recover active slow process gradually; (3) the abzyme activity of preparation is low, is merely 28.8-256U/ μ mol, is lower than natural enzyme one one magnitude level.
Summary of the invention
The technical problem that the present invention will solve 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, reach improve abzyme active, 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; Confirm its aminoacid sequence through 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 auxotrophy 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 verivate, through 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 PMSF 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 auxotrophy intestinal bacteria, give the catalytic activity of antibody with GPX.
The present invention is a target antigen with the gsh verivate, through 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 Tegeline; Concrete single-chain antibody B3 and the aminoacid sequence of single-chain antibody D8 be respectively,
The aminoacid sequence of B3:
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEW
M50GWINAGNGNTKYS63QKFQGRVTITRDTSASTAYMELS?SLRSEDTAVYYC
AR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSV
AL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFS
GS200SSGNTASLTITGAQAEDEADYYCNSRDSSGHPSVFGGGTKLTVLG245；
The aminoacid sequence of D8:
MARVQLVQSGAEVKKTGSSVKVSCKASGYTLTYRYLHWVRQAPGQALE
WM50GWITPFNGNANYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAVYYC
AR100RGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSV
AL150GQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFS
GS200SSGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGGGTKLTVLG244。
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 following.
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 substituted gsh diester of method synthesizing series haptin-S-dinitrobenzene according to prior art (seeing patent 96112628.0 and 99104234.4).With the synthetic haptin is target antigen, and the immune affine sieve method through standard is screened reorganization phage display people single-chain antibody library, obtain can with GSH and verivate 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 through 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 article 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 use the hydroxyl on Serine (Ser) residue in the PMSF activation single-chain antibody, handle with sodium hydrogen selenide again, then Ser is mutated into SeCys.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 article of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 2.0-6.0 * 10
-5The mmol/L PMSF, 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 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 cultures; Select well-grown mono-clonal bacterium colony on flat board, confirm to contain the positive transformant of single-chain antibody gene through 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.
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 following.
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; Through 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 article 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 through 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 confirmed to contain the positive transformant of single-chain antibody gene through 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 deposition, 30 ℃ of concussion cultivations are expressed with methanol induction.In inducing process, every 24h replenishes methyl alcohol a to final concentration 0.5%, replenishes the sterilization ultrapure water simultaneously, and the nutrient solution TV 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, collects 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 article 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 through transgenation method rather than chemical mutation method.Detailed process is following.
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; According to the gene order design primer of single-chain antibody, be template with the phage single-chain DNA that contains single-chain antibody gene, 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 through 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 products; 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.Confirm to suddenly change successfully through dna sequencing, and do not have other unexpected transgenation generation, the result has introduced catalytic group through transgenation at the substrate binding site of single-chain antibody.
The expression and purification of described abzyme is the competent cell that the carrier of the single-chain antibody gene that contains sudden change is transformed auxotrophic strain-BL21 (DE3) CysE, is 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 growth factor and nutrient substance; Induce through IPTG; Auxotrophy property bacterial strain-BL21 (DE3) CysE can directly give expression to the human selenium-containing single-chain abzyme that contains SeCys at the substrate binding site of GSH at last with the synthetic SeCys of the codon of Cys, and 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 article 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.
The present invention also has the 4th kind of method: any amino acid in the complementary antigen determining district 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 of the inventive method preparation is high, 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 immunoreation, aspect bio-pharmaceuticals, has broad application prospects;
(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 by thalline automatically, 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.With the gsh dibutylester that encapsulates is 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 confirmed 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, Application of DNA glue reclaims the single-chain antibody B3 gene of test kit purifying amplification.
With the B3 gene behind the purifying with restriction enzyme Nco I and Not I double digestion after, be connected on the carrier pPELB carrier that same enzyme cuts, will connect 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 cultures; Select well-grown mono-clonal bacterium colony on flat board; After cultivating in a small amount on 2ml 2 * TY liquid nutrient medium, confirm to contain the positive transformant of single-chain antibody gene through 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 PMSF) in the solution, ultrasonication under the ice bath.At 4 ℃, the centrifugal 15min of 8000g obtains supernatant with liquid.By specification is handled GSH affinity post, and (50mmol/LTris, pH7.5) balance add supernatant 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 article of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 5.0 * 10
-5The mmol/L PMSF, 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.Supernatant with the SephadexG25 desalination after freeze-drying promptly get human selenium-containing single-chain abzyme, its GPX vigor is 1321u/ μ mol, reaches the order of magnitude level of natural GPX.
Embodiment 2:
According to the synthetic haptin 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.With the gsh dibutylester that encapsulates is 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 confirmed 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, Application of DNA glue reclaims the single-chain antibody gene of test kit purifying amplification.
With the single-chain antibody gene behind the purifying with restriction enzyme EcoRI and NotI double digestion after, be 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 in the 2mlBMGY substratum, cultivating in a small amount, carry out the positive transformant that dna sequencing confirms to contain single-chain antibody gene.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 deposition of equal-volume (10ml); Abduction delivering is cultivated in 30 ℃ of concussions.In inducing process, every 24h replenishes methyl alcohol a to final concentration 0.5%, replenishes the sterilization ultrapure water simultaneously, and the nutrient solution TV 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 article of mmol/L single-chain antibody are dissolved in the oxygen-free water, add 2.0 * 10
-5The mmol/L PMSF, 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.The centrifugal 15min. supernatant of 10000g with the SephadexG25 desalination after freeze-drying promptly get human selenium-containing single-chain abzyme Sec-B3, 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.With the gsh dibutylester that encapsulates is 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 confirmed 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.Application of DNA glue reclaims the single-chain antibody B3 gene of test kit purifying amplification.With the B3 gene behind the purifying with restriction enzyme Nco I and Not I double digestion after, be 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; Confirm to suddenly change successfully through 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 auxotrophy 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/ml 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 uses 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, replenishes 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 (PMSF) 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 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 the position of the transgenation primer design concrete Ser that will will suddenly change according to D8 with it near the gene order decision.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 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 Tegeline; Concrete human single chain variable fragments antibody is human single chain variable fragments antibody B3 and human single chain variable fragments antibody D8, and its aminoacid sequence is respectively,
The aminoacid sequence of B3:
MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELS?SLRSEDTAVYYCARRGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGHPSVFGGGTKLTVLG；
The aminoacid sequence of D8:
MARVQLVQSGAEVKKTGSSVKVSCKASGYTLTYRYLHWVRQAPGQALEWMGWITPFNGNANYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAVYYCARRGAYQNGPANWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVFGGGTKLTVLG。
2. the preparation method of a soluble human selenium-containing single-chain abzyme has the expression and purification of soluble single-chain antibody, and the process of the introducing of catalytic group is characterized in that,
The expression and purification of described soluble single-chain antibody is under the constant prerequisite of the human single chain variable fragments antibody B3 that keeps claim 1 or human single chain variable fragments antibody D8 aminoacid sequence, according to the gene order design primer of single-chain antibody B3 or 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 through 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 article 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, 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. the preparation method of a soluble human selenium-containing single-chain abzyme has the expression and purification of soluble single-chain antibody, and the process of the introducing of catalytic group is characterized in that,
The expression and purification of described soluble single-chain antibody; Be under the constant prerequisite of the human single chain variable fragments antibody B3 that keeps claim 1 or human single chain variable fragments antibody D8 aminoacid sequence; Gene order design primer according to single-chain antibody B3 or 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; Through 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 article 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. the preparation method of a soluble human selenium-containing single-chain abzyme has the transgenation method to introduce catalytic group, the process of the expression and purification of abzyme,
Describedly introducing catalytic group through transgenation, is under the constant prerequisite of the human single chain variable fragments antibody B3 that keeps claim 1 or human single chain variable fragments antibody D8 aminoacid sequence, according to the gene order design primer of single-chain antibody B3 or 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 through 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; Confirm to suddenly change successfully through dna sequencing, the result has introduced catalytic group through 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 property 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 growth factor and nutrient substance; Induce through isopropyl-; Auxotrophy property bacterial strain-BL21 (DE3) CysE can directly give expression to the human selenium-containing single-chain abzyme that contains SeCys at the substrate binding site of gsh at last with the synthetic SeCys of the codon of Cys, and 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 article 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, 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 according to the position of the Ser that will sport Cys with its neighbour amino acid whose gene order, the long 35-50bp of primer is the center with the codon of Cys.
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| CN1093405A (en) * | 1994-03-09 | 1994-10-12 | 吉林大学 | Chemical mutation has the Monoclonal Antibody selenium-containing abzyme of substrate binding site |
| CN1176307A (en) * | 1996-09-10 | 1998-03-18 | 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 |
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| CN1093405A (en) * | 1994-03-09 | 1994-10-12 | 吉林大学 | Chemical mutation has the Monoclonal Antibody selenium-containing abzyme of substrate binding site |
| CN1176307A (en) * | 1996-09-10 | 1998-03-18 | 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 |
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| 李杰帅.人源抗GSH单链抗体的筛选及表达.《中国优秀硕士学位论文全文数据库，基础学科辑》.2007,(第3期),A006-80. * |
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