CN1468862A - Technological process of producing recombinant human histiotype plasminogen activator TNK mutant - Google Patents
Technological process of producing recombinant human histiotype plasminogen activator TNK mutant Download PDFInfo
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Abstract
The present invention discloses the technological process of producing recombinant human histiotype plasminogen activator TNK mutant. TNK-tPA gene containing said mutant sites is synthesized via full length gene synthesis process and expressed in Chinese hamster ovary cell (CHO-DHFR), and through cloning and screening high-level expressed TNK-tPA engineering cell strain is obtained. By means of sustained culture of the engineering cell in cell culturing bioreactor, collection of the supernatant and serial separation and purification in chromatographic column, rhTNK-tPA product is obtained. The destination gene of the present invention has expression level in cell as high as 18000 IU/106 cell/d, and the prepared rhTNK-tPA product has single strand rate as high as 80 % and purity up to 95 %. As for industrial scale, the present invention has relatively simple requirement in apparatus, low cost and simple technological requirement.
Description
Technical field
The present invention relates to the method for a kind of production tr-PA mutant, particularly relate to a kind of zooblast that utilizes and produce the recombinant human histiotype plasminogen activator TNK mutant processing method of (being called for short rhTNK-tPA).
Background technology
Tissue-type plasminogen activator's (Tissue Plasminogen activitor is called for short t-PA) is one of important component of blood coagulation and molten fine system in the blood of human body, it starts molten fine system by the Profibrinolysin that activates the thrombus surface, plays an important role in keeping blood coagulation and molten fine balance.T-PA is synthetic by vascular endothelial cell, new synthetic t-PA precursor is made up of 562 amino-acid residues, in translation, secretion and the ripening process of polypeptide, the signal peptide and the leading peptide of N end are cut, sophisticated t-PA molecule contains 527 amino-acid residues, molecular weight is 67-72KD, is made up of the wall scroll peptide chain, contains 17 disulfide linkage.In molten fine process, the t-PA molecule is cut by proteolytic enzyme in the Arg275-Ile276 position, forms active higher duplex molecule, and 275 amino-acid residues of N end form heavy chains, and 252 amino acid of C end form light chains, is linked to each other by a disulfide linkage between light, the heavy chain.Heavy chain contains 4 structural domains, they respectively with some functional domain height homology of some other plasma proteins, described 4 structural domains are respectively: 1, finger-type district (claiming the F district again), form by 4～46 amino acids residues, with the finger-type district homology of Zeta protein (fibronectin), fibre-bearing protein binding site.2, epidermal growth factor subarea (claiming the E district again) is made up of 47～85 amino acids residues, and sequence and Urogastron homology contain the cell-membrane receptor binding site.This district mediation t-PA combines with liver cell and vascular endothelial cell, and there is material impact the transformation period.3, K1 district is made up of 92～173 amino acids residues.4, K2 district is made of the fibre-bearing protein binding site 180-261 amino acids residue.Light chain and trypsinase height homology contain the serine protease structural domain, form the active centre by His322, Asp371 and Ser478, and the catalysis Profibrinolysin changes into plasmin.The t-PA molecule contains 4 possible glycosylation site: Asn117, Asn184, Asn218, Asn448.Natural type t-PA molecule contains other three the glycosyl sites except that Asn218.The t-PA molecule can be divided into two types according to glycosylated mode: the I type is three equal glycosylations in site, and the II type is then in Asn184 site sugar basedization, and the two content in vivo is basic identical.
T-PA interacts with following 4 kinds of different proteins in vivo at least: 1, Profibrinolysin, and it is the substrate of t-PA, when no scleroproein, only there is lower avidity t-PA and it.2, scleroproein, the t-PA molecule has higher avidity with it, and can improve about 100 times at the protease activity that combines back t-PA with it.3, plasminogen activator inhibitor PAI, t-PA combine the back protease activity to be suppressed with it.4, t-PA acceptor is present in liver cell and vascular endothelial cell surface, and mediation t-PA removes in vivo fast.Because t-PA and scleroproein has high affinity and its activity can be improved greatly by scleroproein, therefore, it only activates near the Profibrinolysin the thrombus in certain concentration range, make thrombolysis, and can not cause the bleeding tendency of whole body, so t-PA is more safe and effective than other plasminogen activator.
Tr-PA utilizes gene engineering method above-mentioned native protein to be transformed and the mutant or the mosaic that obtain.Wherein, the mutational site of TNK-tPA mutant and function corresponding thereof are respectively:
● with T103 (Threonine) site mutation is N103 (l-asparagine), make it become glycosylation site by non-glycosylated site, reduced the removing speed of t-PA in blood plasma, prolonged the transformation period, but this sudden change has also reduced t-PA and fibrinous binding ability.
● N117 (l-asparagine) site mutation is Q117 (glutamine), removes the glycosylation function in 117 sites, and this change has been repaired the N103 site mutation to the narrow spectrum disadvantageous effect of scleroproein, and also reduces the removing speed of t-PA in blood plasma.
● K296 (Methionin), H297 (Histidine), R298 (arginine), R299 (arginine) site mutation are A296 (L-Ala), A297 (L-Ala), A298 (L-Ala), A299 (L-Ala), strengthen t-PA to fibrinous binding ability, the most important thing is to make t-PA that its inhibitor PAI-1 is had antagonistic action.
Above-mentioned TNK-tPA mutant is compared with natural type, and the antagonistic ability of inhibitor PAI-1 has been strengthened 80 times; With the scleroproein binding ability be 10～14 times of natural type; Plasma clearance speed has reduced by 4 times.
At present, adopt intestinal bacteria system expression t-PA deletion mutant usually, this technology is by the DNA recombinant technology, lacks the partial function territory of natural t-PA molecule, as F district, E district, K1 district, makes it only to contain K
2Functional domain and protease function territory.This deletion mutant t-PA is a strand, nonglycosylated molecule, at expression in escherichia coli.With respect to natural t-PA, its transformation period has in vivo prolonged 4～5 times, but has reduced by 5 times with fibrinous binding ability.Therefore certain limitation is arranged in clinical application.
Production method for the mutant rhTNK-tPA of tr-PA does not still have bibliographical information at present.
Summary of the invention
The purpose of this invention is to provide a kind of processing method of producing recombinant human histiotype plasminogen activator TNK mutant.
In order to achieve the above object, the present invention adopts the synthetic TNK-tPA gene that contains the said mutation site of full-length gene synthetic method, at Chinese hamster ovary cell (CHO-DHFR
-) the middle expression, the engineering cell strain through clone and screening acquisition high level expression TNK-tPA utilizes biological reactor for cell culture to continue the culturing engineering cell, the collecting cell culture supernatant, and through a series of column chromatographic isolation and purification technology, acquisition rhTNK-tPA goods.
The present invention may further comprise the steps successively:
(1) the natural t-PA gene of rite-directed mutagenesis obtains to contain the TNK-tPA mutant gene in mutational site;
(2) construction of expression vector pCdhfr;
(3) contain the dna artificial sequence synthetic of TNK-tPA gene with the XhoI/XbaI double digestion, recovery contains the dna fragmentation of the 1.7Kb of goal gene, be connected with carrier for expression of eukaryon pCdhfr with the XhoI/XbaI double digestion, transformed into escherichia coli E.coli JM109, be PCR preliminary screening clone with the universal primer on the carrier, get positive clone strain and cultivate, obtain recombinant expression plasmid pCdhfr-mt-PA;
(4) with TNK-tPA mutant expression plasmid pCdhfr-mt-PA transfection CHO (dhfr
-) cell, through the engineering cell strain of clone and screening acquisition high level expression TNK-tPA;
(5) utilize biological reactor for cell culture to continue the culturing engineering cell, the collecting cell culture supernatant obtains the rhTNK-tPA mutant through separation and purification.
The present invention compared with prior art has the following advantages: the expression level of goal gene 1. of the present invention in cell is up to 18000IU/10
6Cell/d.2. according to the strand ratio of the rhTNK-tPA of separation purifying technique of the present invention preparation up to more than 80%, purity is up to more than 95%.3. for industrially scalable, equipment required for the present invention is relatively simple, and cost is low, and processing condition are simple.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the restriction analysis figure of TNK-tPA eukaryon expression plasmid in the embodiments of the invention.
Embodiment
The structure of 1 engineering cell strain
1.1 material, method and result
11.1 material
1.1.1.1 bacterial classification
E.coli XL1-Blue (genotype hsdR17, supE44, recA1, endA1, gyrA46, thi, relA1, lac/F ' [proAB+, lac I q, lacZDM15 ∷ Tn10 (tetr)]).
1.1.1.2 clone
CHO (dhfr
-), Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell.
1.1.1.3 toolenzyme
Various restriction enzymes, T4 dna ligase, alkaline phosphatase, Taq archaeal dna polymerase are all available from Promega company.The Vent archaeal dna polymerase is available from Biolab company.Dna molecular amount standard DL2000 (2000,1000,750,500,250,100bp) and lamda-DNA/EcoRI (23130,9416,6557,4361,2322,2027,564,125bp) available from precious biotechnology Dalian company limited.
1.1.1.4 main agents
1.1.1.4.1 3mM HT (xanthoglobulin+thymus pyrimidine) concentrated solution
40.83 milligrams of xanthoglobulin (available from Sigma company), 72.7 milligrams of thymus pyrimidines (Sigma company) add 50 milliliters of pure water, dropwise add 1N sodium hydroxide,
1.1.1.4.2 IMDM nutrient solution
IMDM powder (available from Hyclone company) 17.7 gram NaHCO
32.0 gram, adding 950ml pure water is stirred to dissolving fully, transfers pH to 7.6 with 1N HCl, adds water to 1000 milliliters, and 4 ℃ of preservations add 10% dialysis foetal calf serum (available from Hyclone company) and two resisting before the use.
1.1.1.4.3 phosphate buffered saline buffer (PB)
Na
2HPO
4?????????????????7.1g
NaH
2PO
42H
2O????????????1.79g
Tween-80???????????????????0.1ml
NaN3?????????????????????0.2g
1.1.1.4.4 thrombin of beef solution
Get 10000U/ and prop up zymoplasm (available from Sigma company), be diluted to 330U/ml with PB, packing ,-20 ℃ of preservations are standby.
1.1.1.4.5 ox proplasmin solution (P liquid)
Get ox proplasmin (100U/ props up, available from Sigma company), add PB and be diluted to 0.1mg/ml, packing ,-20 ℃ of preservations are standby.
1.1.1.4.6 bovine fibrinogen solution (F liquid)
The Profibrinolysin of-20 ℃ of preservations is placed room temperature more than half an hour, take by weighing aequum and be dissolved in the PB damping fluid of reducing to 37 ℃ after boiling, make its final concentration reach 2mg/ml, 37 ℃ of insulations are to dissolving fully.
1.1.1.4.7 standard solution preparation
Get standard substance, be diluted to following concentration, 3000IU, 1500IU, 750IU, 375IU, 187.5IU, 93.25IU/ml with PB.
1.1.1.4.8 TNK-tPA dilution of sample
Get sample to be checked, be diluted to about 0.1 μ g/ml with PB.
1.1.2 key instrument equipment
High speed tabletop centrifuge TGL-16G goes up Hai Anting
High speed tabletop refrigerated centrifuge Sigma
High speed freezing centrifuge Beckman
Constant water bath box DT75-1 is homemade
CO
2Incubator Sanyo
Bechtop is homemade
The inversion opticmicroscope is homemade
Pcr amplification instrument PERKIN ELMER
Sorvall RC 5C plus high speed freezing centrifuge DU Pont
DF-C constant pressure and flow electrophoresis apparatus Beijing Orient instrument plant
Supersonic cell crusher Ningbo ultrasonic instrument factory
Enzyme connection instrument Model450 BIO-RAD
Spectronic601 ultraviolet spectrophotometer Milton
Electrophoretic blotting instrument BIO-RAD
11.3 method and result
11.3.1 the acquisition of goal gene
Utilize the natural t-PA gene of point mutation technology rite-directed mutagenesis, take the synthetic TNK-tPA gene that contains the mutational site of full-length gene synthetic method, that is: the ACG base with the 103rd Thr of natural type t-PA gene (wtPA) coding becomes AAC/Asn (T103N), can (K1 district) form an artificial glycosylation site in this site, the speed that the formation in this site causes t-PA to be eliminated in vivo descends, yet this site mutation causes descending with fibrinous binding ability, thereby reduces its specificity; By 117 AAC/Asn are sported CAA/Gln (N117Q), can eliminate above-mentioned 103 disadvantageous effects that glycosylation caused; In addition, 296 Lys of natural type t-PA, 297 His, 298 Arg and 299 Arg are all replaced with Ala[KHRR (296-299) AAAA], can strengthen the proteic ability of its binding fiber, and reduce its susceptibility PAI-1 (t-PA inhibitor).
According to the cDNA sequence of natural type t-PA, design said mutation site lays respectively at proteic 103,117, the 296-299 amino acids of t-PA, causes the amino acid in above-mentioned site different with natural type t-PA.Simultaneously, introduce Xho I and XbaI restriction endonuclease sites respectively in the upstream and downstream of gene, full length gene is 1.7kb, and is synthetic by precious biotechnology Dalian company limited.
The structure of 1 1.3.2 expression vector pCdhfr
The used expression vector pCdhfr of this expression system changes structure by pCI-neo and forms.The dhfr gene cDNA of pCdhfr derives from plasmid pSH2.After pSH2 digested with BglII, the Klenow enzyme was mended flat 3 ' recessed end, cut with the HindIII enzyme again, separated the fragment of 0.7kb in low melting-point agarose.Carrier pCI-neo cuts with the BstXI enzyme earlier, scabble 3 ' nose with the T4 archaeal dna polymerase then, cut with the HindIII enzyme again, adopt three segments to link to each other, the dhfr gene subclone of above-mentioned 0.7kb to the HindIII/BstXI site, is obtained carrying the coamplification expression vector pCdhfr of dhfr.
Because expression vector pCdhfr contains the dhfr gene, behind this plasmid transfection CHO (dhfr-) cell, under copy number under the effect of methotrexate (MTX), increase greatly, can drive efficiently expressing of foreign gene that this plasmid carries.This plasmid contains multiple clone site, can be used for the clone of foreign gene, and the upstream and downstream of multiple clone site contains CMV promotor and SV40 Poly (A) site respectively, and these sequences can make the foreign gene that inserts multiple clone site obtain effectively expressing.Also contain colibacillary replication origin and ampicillin resistance gene on this plasmid, they can make this plasmid increase in intestinal bacteria.
1.1.3.3 TNK-tPA construction of recombinant plasmid and evaluation
The dna artificial sequence synthetic that contains the TNK-tPA gene with the XhoI/XbaI double digestion, recovery contains the dna fragmentation of the 1.7Kb of goal gene, be connected with carrier for expression of eukaryon pCdhfr with the XhoI/XbaI double digestion, transformed into escherichia coli E.coli JM109, be PCR preliminary screening clone with the universal primer on the carrier, getting positive clone strain cultivates, rapid extraction plasmid in a small amount,, it inserts fragment restriction analysis, the result shows: the external source goal gene correctly is inserted in the expression vector, obtain recombinant expression plasmid pCdhfr-mt-PA, its restriction analysis as shown in Figure 1.
Simultaneously, the gene that inserts in order to confirm in the expression plasmid is the TNK-tPA gene, and its direction of insertion, dna sequence dna and design is in full accord, and insertion sequence is analyzed.The result shows, the sequence of the gene fragment of being inserted and design in full accord.
1.1.3.4 the transfection of CHO (dhfr-) cell and the screening of high expressing cell strain
Liposome method is adopted in the transfection of CHO (dhfr-) cell, and available from Life Technologies company, reference reagent box specification sheets is operated in transfection with test kit Lipofect Amine Plus in transfection.
With TNK-tPA mutant expression plasmid pCdhfr-mt-PA transfection CHO (dhfr-) cell, cultivate to contain two anti-selectivity nutrient solutions, changed one time nutrient solution every three days, come off up to most of necrocytosis and from culture plate, the cell clone of minority integrate foreign genes is under survival on the selectivity nutrient solution, and constantly division forms cell colony (clone).Formation from the transfection to the cell clone needs 2 time-of-weeks.In the cell clone process of growth, collect each hole culture supernatant, detect expression product.Choose the high clone of expression amount, with trysinization, divide to go in the Tissue Culture Plate again, use the selection nutrient solution that contains MTX instead and cultivate, in culturing process, most of necrocytosis has a few cell to form new clone again.Repeat aforesaid operations, MTX concentration is improved a level, in clone's screening process, MTX concentration is from 5 * 10
-8M progressively brings up to 5 * 10
-6M, correspondingly, the t-PA expression level also progressively improves, and is as shown in table 1.
TNK-tPA changes of expression level in the table 1 MTX pressurization screening process
| MTX concentration (M) | ??0 | ?5×10 -8 | ?1×10 -7 | ?2×10 -7 | ?5×10 -7 | ?1×10 -6 | ?2×10 -6 | ?5×10 -6 |
| Cell clone | ?4D1 | ??3F5 | ??6C3 | ??2G5 | ??5H1 | ??2A6 | ??3E9 | ??3C7 |
| TNK-tPA expression amount (IU/10 6cell/d) | ?1481 | ??4155 | ??8236 | ??10681 | ??12952 | ??17329 | ??17564 | ??18910 |
As can be seen from the above results: when medicine MTX concentration surpasses 1 * 10
-6During M, the TNK-tPA expression amount no longer obviously raises, thus we to select MTX concentration be 1 * 10
-6The cell strain 2A6 that screens during M is as engineering cell strain, called after CHO-G10.
The cultivation of 2 engineering cells in biological reactor for cell culture and the separation and purification of target protein
2.1 material, method and result
2.1.1 material
2.1.1.1 foetal calf serum
Dialysis-type foetal calf serum for Invitrogen life technologies company.
2.1.1.2 calf serum
Reinforced calf serum for Hyclone company.
2.1.1.3 cell culture medium
Employed IMDM and CHO-S-SFM II serum free medium are Invitrogen lifetechnologies company product.
2.1.1.4 purifying filler
Employed purifying filler POROS 20MC, Lysine Hyper D and Sephadex G-25 are respectively the products of PE company, Invitrogen life technologies company and Amersham Pharmacia company.
2.1.1.5 main agents
(1) pancreatin is a Hyclone company product
(2) imidazoles is a Tianjin Ke Miou scientific ﹠ technical corporation product.
(3) L-Arginine is an Invitrogen life technologies company product.
(4) ortho-phosphoric acid is the Guangzhou Chemical Reagent Factory product.
(5) sodium-chlor is the Guangzhou Chemical Reagent Factory product.
(6) Tween 20 is a SIGMA company product.
2 1.2 methods and result
2.1.2.1 cell recovery
Cell recovery method routinely: from cell bank, take out a freeze-stored cell, put rapidly in 37 ℃ of water-baths, the limit is shaken the limit gently and is melted, after treating that thawing finishes, the frozen pipe outer wall of sterilizing immediately, in clean bench, cell suspension transferred in the cell cultures square vase (T25) of IMDM substratum that about 10ml contains 10% foetal calf serum, in 37 ℃ 5%CO
2Cultivate in the incubator, changed fresh culture in second day, continue to cultivate after 1～2 day, observation of cell form and density under inverted microscope, the cell refractivity is good, adherent evenly, be paved with bottle at the bottom of 85% when above, can carry out passage and increase.
2.1.2.2 cell amplification
Comprise cell amplification and cell amplification two portions in biological reactor for cell culture in square vase.
At first, with the cell among the T25 with trysinization after, import in the T75 Tissue Culture Flask in 1: 1 ratio, cultivated 1～2 day, treat its stick bottle at the bottom of the time, import in the T175 Tissue Culture Flask in 1: 1 ratio again, increase in the T175 culturing bottle in 1: 4 ratio afterwards, be followed successively by 4,16,64 T175 culturing bottles.
Then, with the cell dissociation in these 64 T175 culturing bottles, and collect counting after, be inoculated in the biological reactor for cell culture, with the substratum amplification cultivation that contains 10% calf serum, controlled temperature is 37 ℃, and pH is 7.2, dissolved oxygen (DO) is 50%, and stirring velocity is 70～150 rev/mins.Judge the growing state of cell according to the variation of glucose consumption and other parameter.Usually inoculating cell began perfusion culture after 2～3 days, and initial groundwater increment is 2L/ days, increases groundwater increment gradually according to the glucose consumption situation then, and continous pouring can be replaced by serum-free culture after 3 days.
2.1.2.3 cell continous pouring in reactor is cultivated
Cell is finished amplification cultivation in reactor after, extrusion has blood serum medium, clean 3 times with P.B.S., change to serum free medium and carry out the continous pouring cultivation, groundwater increment is between 5～15L/ days, continue to cultivate about 30 days, honest and upright and thrifty 300L/ jar on the harvested cell/batch, the expression level of rhTNK-tPA is on average at 18000IU/10
6More than the cell/d.The cells and supernatant of being gathered in the crops changes purifying over to.
2.1.2.4 the separation and purification of rhTNK-tPA
2.1.2.4.1 filter, the serum-free culture supernatant that cell cultures is collected is removed cell debris through the positive press filtration of 1.2 μ m millipore filtrations.
2.1.2.4.2 Zn
++--POROS 20 MC chelating chromatography purifications
Filtering cell conditioned medium is adsorbed sample, after last sample finishes, at first use mobile phase A (30mMP.B.S, 0.01%Tween 20, pH7.6) are washed till under the sample peak, use Mobile phase B (30mM P.B then, 1.5M NaCl, 0.01%Tween 20, pH7.6) are washed till under the foreign protein peak, use moving phase C (30mMP.B at last, 0.5M NaCl, 0.01%Tween 20, pH7.6,0.3M imidazoles) be washed till under the target peak, collect target peak.
2.1.2.4.3 Lysine Hyper D affinity chromatography
Above-mentioned target peak is collected liquid on Lysine Hyper D affinity column, adsorb sample, begin wash-out after last sample finishes.At first use moving phase D (30mM P.B.S, 0.01%Tween 20, pH7.6) are washed till under the sample peak, use moving phase E (30mM P.B then, 1M NaCl, 0.01%Tween 20, pH7.6) are washed till under the foreign protein peak, use moving phase F (30mM P.B at last, 1M NaCl, 0.01%Tween 20, pH7.6,1ML-Arginine) wash-out target peak is collected this target peak.
2.1.2.4.4 Sephadex G-25 gel permeation chromatography
Above-mentioned target peak is collected liquid sample on the Sephadex G-25 gel column, use moving phase G (every 1000ml G liquid contains 60g L-Arginine, 15g ortho-phosphoric acid, 200mg Tween 20) wash-out then, collect the target protein peak, be the pure product of rhTNK-tPA albumen.
3, the evaluation of rhTNK-tPA
3.1 non-reduced SDS-PAGE measures rhTNK-tPA purity
Adopt the method for SDS discontinuous electro-phoresis to survey rhTNK-tPA purity.
Resolving gel concentration is 10%; Concentrated gum concentration is 5%; Sample preparation liquid is the Tris-HCl buffer that contains 0.5% tetrabromophenol sulfonphthalein, 1%SDS, 10% glycerine pH6.8.Sample and sample preparation liquid equivalent mixing, applied sample amount 10 μ g (then pressing sample on the maximum applied sample amount when protein content is less than 10 μ g in the maximum applied sample amount).Electrophoresis adopts constant current 30mA, the about 60Min of electrophoresis time.Electrophoresis finishes with the dyeing of Xylene Brilliant Cyanine G method, and its result has the feature electrophoresis band at the 68KD place, can obtain rhTNK-tPA purity according to the result of scanning image and be about 98%.
3.2 reduced form SDS-PAGE measures molecular weight and the single double-stranded ratio of rhTNK-tPA
Electrophoresis: except that sample preparation liquid adds the 5%DTT, all the other conditions are identical with non-reduced SDS-PAGE.
Molecular weight calculates: according to the logarithm and the relative mobility R of molecular weight
fBe linear relationship, can draw up linear equation: logM.V=k * R according to Marker
f+ a
The migration distance of measuring electrophoresis band with ruler just can obtain relative R
f, the substitution following formula can obtain molecular weight of albumen.
The molecular weight of single chain protein is about 68KD, and double-stranded proteic light chain is about 36KD, and heavy chain is about 42KD.Can obtain single, double chain ratio more than 8: 2 according to scanning image.
3.3 Western blot identifies rhTNK-tPA
Electrophoresis adopts the method for non-reduced SDS-PAGE, and fixedly film is pvdf membrane (steeps 0.5Min with methyl alcohol before using, use electrode buffer rinsing 0.5h again), and liquid 10%BAS blockades.Transfer current constant voltage 100V shifts 1.5h.One anti-be rabbit anti rec-t-PA, two anti-be goat anti-rabbit igg-HRP, colour developing liquid is DAB; Make the molecular weight sign with dying Marker in advance.
Its result has characteristic strip at the 68KD place.
3.4 rhTNK-tPA n terminal amino acid sequencing
RhTNK-tPA n terminal amino acid sequencing result shows that 15 amino-acid sequences of N-terminal are Ser-Tyr-Gln-Val-Ile-Cys-Arg-Asp-Glu-Lys-Thr-Gln-Met-Ile-Tyr, and are consistent with theoretical value.
3.5 rhTNK-tPA Determination of biological activity (bubble cracking process)
Agents useful for same: PB buffer:29g Na
2HPO
412H
2O and 3g NaH
2PO
42H
2The fixed molten 1000ml of O, pH7.4.The 2mg/ml fibrinogen solution, 0.1mg/ml Profibrinolysin solution, 33IU/ml thrombin solution.Standard substance (3000IU/ml, 1500IU/ml, 750IU/ml, 375IU/ml, 187.5IU/ml, 98.75IU/ml).
Sample preparation: be diluted to t-PA content with PB buffer and be about 750IU/ml.
Operation: with 0.12ml thrombin solution and 0.12ml sample (t-PA) mixing, ice-water bath is preserved (calling A liquid in the following text).The mixing in test tube with 1ml thrombin solution and 20 μ l Profibrinolysins, ice-water bath are preserved (calling the B pipe in the following text).Get A liquid 0.2ml and add in the B pipe, mixing is put into 37 ℃ of constant temperature, between beginning to clock fast.When rising to surface (all, indivedual bubbles are stained with on the wall and can not be remembered), bubble is terminal point, writing time.
According to the document record, this law Lg activity is a linear relationship with the Lg time, thereby can simulate linear equation.The data substitution of sample both can be drawn data.
The result shows that the rhTNK-tPA content of cells and supernatant is on average at 18000IU/10
6More than the cell/d, the rhTNK-tPA specific activity of pure product is 600,000IU/mg.
Animal test results before 3.6 rhTNK-tPA is clinical
Animal test results shows that rhTNK-tPA has tangible thrombolytic effect to dog acute coronary thrombus, rat aorta thrombus, rabbit lung thrombus, vitro human thrombus etc. before clinical; Not obvious to mouse general behavior state, central nervous system effect, the guinea pig ileum smooth muscle contraction there is not influence; Dog electrocardiogram(ECG, respiratory rate and depth of respiration there is not influence; Its macaque pharmacokinetic parameter and curve are close with reference product.The acute toxicity test of mouse shows that the MTD of female mice is greater than 3420.5mg/kg, and male mice is greater than 3102.3mg/kg; The long term toxicity test of rat and dog shows, the toxic reaction of its toxicity target organ liver is had reversibility; Local application's toxicity test shows that dog injection site vascular morphology is no abnormal, sees that in blood vessel surrounding soft tissue a small amount of remote hemorrhage and endochylema contain the scavenger cell of hemosiderin granule; Teratogenic test shows that female mouse of gestation and tire mouse are had slight toxic action, does not significantly cause birth defect but have.
Claims (3)
1. produce the processing method of recombinant human histiotype plasminogen activator TNK mutant, may further comprise the steps successively:
(1) the natural t-PA of rite-directed mutagenesis obtains to contain the TNK-tPA mutant gene in mutational site;
(2) construction of expression vector pCdhfr;
(3) contain the dna artificial sequence synthetic of TNK-tPA gene with the XhoI/XbaI double digestion, recovery contains the dna fragmentation of the 1.7Kb of goal gene, be connected with carrier for expression of eukaryon pCdhfr with the XhoI/XbaI double digestion, transformed into escherichia coli E.coli JM109, be PCR preliminary screening clone with the universal primer on the carrier, get positive clone strain and cultivate, obtain recombinant expression plasmid pCdhfr-mt-PA;
(4) with TNK-tPA mutant expression plasmid pCdhfr-mt-PA transfection CHO (dhfr
-) cell, through the engineering cell strain of clone and screening acquisition high level expression TNK-tPA;
(5) utilize biological reactor for cell culture to continue the culturing engineering cell, the collecting cell culture supernatant obtains the rhTNK-tPA mutant through separation and purification.
2. the processing method of production recombinant human histiotype plasminogen activator TNK mutant according to claim 1 is characterized in that: described expression vector pCdhfr changes structure by pCI-neo to form, and the dhfr gene cDNA of pCdhfr derives from plasmid pSH2; After pSH2 digested with BglII, the Klenow enzyme was mended flat 3 ' recessed end, cut with the HindIII enzyme again, separated the fragment of 0.7kb in low melting-point agarose; Carrier pCI-neo cuts with the BstXI enzyme earlier, scabble 3 ' nose with the T4 archaeal dna polymerase then, cut with the HindIII enzyme again, adopt three segments to link to each other, the dhfr gene subclone of above-mentioned 0.7kb to the HindIII/BstXI site, is obtained carrying the coamplification expression vector pCdhfr of dhfr.
3. the processing method of production recombinant human histiotype plasminogen activator TNK mutant according to claim 1 is characterized in that: in described step (5), the purification procedures of rhTNK-tPA mutant is followed successively by:
(1) filters;
(2) Zn
++--POROS 20 MC chelating chromatography purifications;
(3) Lysine Hyper D affinity chromatography;
(4) Sephadex G-25 gel permeation chromatography.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011015922A1 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | A highly efficient process of purification and production of recombinant tnk-tpa (tenecteplase) |
| CN108704122A (en) * | 2018-05-23 | 2018-10-26 | 广州铭康生物工程有限公司 | A kind of injection rhTNK-tPA lyophilized preparations and preparation method thereof |
| CN109628378A (en) * | 2019-01-25 | 2019-04-16 | 江苏丰华生物制药有限公司 | A method of utilizing peanut protein zymolyte culture Chinese hamster ovary celI |
| CN113337490A (en) * | 2021-06-18 | 2021-09-03 | 广州铭康生物工程有限公司 | Method for large-scale rapid separation and purification of rhTNK-tPAI/II type |
| CN115161308A (en) * | 2022-06-13 | 2022-10-11 | 江苏丰华生物制药有限公司 | Purification method of tenecteplase supernatant for injection |
-
2002
- 2002-07-19 CN CN 02134404 patent/CN1221567C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011015922A1 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | A highly efficient process of purification and production of recombinant tnk-tpa (tenecteplase) |
| CN108704122A (en) * | 2018-05-23 | 2018-10-26 | 广州铭康生物工程有限公司 | A kind of injection rhTNK-tPA lyophilized preparations and preparation method thereof |
| CN109628378A (en) * | 2019-01-25 | 2019-04-16 | 江苏丰华生物制药有限公司 | A method of utilizing peanut protein zymolyte culture Chinese hamster ovary celI |
| CN109628378B (en) * | 2019-01-25 | 2020-07-17 | 江苏丰华生物制药有限公司 | Method for culturing CHO (Chinese hamster ovary) cells by using peanut protein zymolyte |
| CN113337490A (en) * | 2021-06-18 | 2021-09-03 | 广州铭康生物工程有限公司 | Method for large-scale rapid separation and purification of rhTNK-tPAI/II type |
| CN115161308A (en) * | 2022-06-13 | 2022-10-11 | 江苏丰华生物制药有限公司 | Purification method of tenecteplase supernatant for injection |
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| Publication number | Publication date |
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| CN1221567C (en) | 2005-10-05 |
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Inventor after: Yang Qin Inventor after: Wang Junzhi Inventor after: Liu Longbin Inventor after: Wang Minrong Inventor after: Mu Guohui Inventor after: Wu Yinan Inventor after: Wang Sen Inventor before: Liu Longbin Inventor before: Yang Qin Inventor before: Wang Minrong Inventor before: Wang Junzhi |
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