CN1798837A - Thioredoxin modification - Google Patents
Thioredoxin modification Download PDFInfo
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- CN1798837A CN1798837A CNA2004800150605A CN200480015060A CN1798837A CN 1798837 A CN1798837 A CN 1798837A CN A2004800150605 A CNA2004800150605 A CN A2004800150605A CN 200480015060 A CN200480015060 A CN 200480015060A CN 1798837 A CN1798837 A CN 1798837A
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Abstract
A human thioredoxin modification which comprises one of the following polypeptides: (a) a polypeptide having an amino acid sequence derived from the amino acid sequence of SEQ ID NO:2 by alteration or chemical modification of the cysteine residue at the 35-position; and (b) a polypeptide having an amino acid sequence derived from the amino acid sequence of SEQ ID NO:2 by substitution of the cysteine residue at the 35-position by another amino acid residue, and substitution, deletion, insertion or addition of one or more amino acids at the 32- and 35-positions (preferably at site(s) excluding from the 32- to the 35-positions) in the amino acid sequence of SEQ ID NO:2 and having an activity of inducing apoptosis.
Description
Technical field
The present invention relates to regulate and control by intravital endogenous and/or the unusual cell growth that exogenous factor causes and/or Trx (being called " the TRX ") modified protein of cell function, basically minimized peptide, the albumen that coding is modified and the DNA of minimized peptide basically, the recombinant expression vector that comprises this DNA, DNA with this DNA conversion, the TRX albumen of produce modifying and the method for minimized peptide basically, and comprise the TRX albumen of modification and/or the medicine or the pharmaceutical composition of minimized peptide basically.
Background technology
Trx is the little multifunctional protein of 12kD, at its avtive spot sequence-Cys-Gly-Pro-Cys (" the redox regulation and control of cell-stimulating ", Ann.Rev.Immunol.1997; Disulfide linkage/the dimercapto that has redox active 15:351-369).Trx in intestinal bacteria (Escherichia coli) as the enzyme of important synthetic deoxyribonucleotide, the hydrogen donor of ribonucleotide reductase and after separating, it separates from various protokaryons and biology eucaryon and identifies.The T chronic myeloid leukemia derivative factor (ADF) of growing up at first is accredited as the IL-2 receptor-inducible factor of the T lymphocyte generation of infecting HTLV-1 by the inventor, and is the Trx.Trx is used for for example activated protein-1 and nuclear Factor-Kappa B (" the AP-1 transcriptional activity is subjected to contacting directly and regulate and control between Trx and the Ref-1 " 1997 of redox transcription factor to removing free radical and regulation and control in the cell; 94:3633-3638) play an important role.In addition, the Trx controls the signal conduction of p38 mitogen activated protein kinases (MAPK) and apoptotic signal regulation and control kinases (ASK-1).
Simultaneously, we have reported that Trx is released to the extracellular and as cytokine or chemokine, but its mechanism of action is not also known.The outer TRX of cells involved is not produced than wild-type TRX much higher cell endocytic active report to the cell neutralization by modifying the TRX avtive spot by endocytosis as yet.
The present invention analyzes interior regulation activity of cell and the cell endocytic activity of TRX, and plans to provide based on the TRX of active modification and the method for producing the TRX that modifies.
The accompanying drawing summary
Fig. 1 shows the result of the flow cytometry of embodiment 2.
Fig. 2 shows the result of the flow cytometry of embodiment 2.
Fig. 3 shows the result of the Western trace of embodiment 2.
Fig. 4 shows the result that the apoptosis of embodiment 3 is analyzed.
Fig. 5 shows the measurement of use scintillometer
3The result of H-thymus pyrimidine absorbed dose.
Fig. 6 shows the result of reinforced effects among the embodiment 4-1 pair carcinostatic agent effect.In Fig. 6, CDDP (-) and CDDP (+) represent that respectively 3 μ g/mL cis-platinums do not exist or exist.TRX-WT, TRX-CS and TRX-C35S represent the wild-type Trx respectively, the Trx that Trx that two position C32S and C35S modify and C35S modify.In CDDP (-), cell and 4% (TRX-WT) and 3% (TRX-CS) dead among the TRX-C35S increase about 4 times.At CDDP (+) (3 μ g/mL), cell (26%) dead among the TRX-C35S relatively increases about 5 or 2 times with 5% (TRX-WT) or 13% (TRX-CS).
Fig. 7 shows the result of reinforced effects among the embodiment 4-2 pair carcinostatic agent effect.In Fig. 7, CDDP, rec (-) and rec (+) represent cis-platinum respectively, reorganization Trx (C35S-TRX) does not exist or exists.The right upper quadrant representative is two positive, and it is the zone (zone with anticancer effect) of expression necrocytosis.For example, after handling 1 hour with C35S-TRX, at the following pair positive cell of situation of the CDDP (3 μ g/mL) that has 10 μ g/mL C35S-TRX from 2 times of 10% raisings to 22%.In CDDP (6 μ g/mL), two positive cells improve 4 times to 76% from 18%.In addition, after handling 1 hour with C35S-TRX, at the following pair positive cell of situation of the CDDP (3 μ g/mL) that has 10 μ g/mLC35S-TRX from 4.7 times of 7% raisings to 33%.At CDDP (6 μ g/mL), two positive cells improve 3 times to 70% from 23%.
Summary of the invention
Regulation activity and cell endocytic activity in the cell of inventor's detailed analysis TRX, and the recombinate endocytosis activity of wild-type TRX of the outer people of last successful identification of cell.In addition, they have successfully prepared the TRX based on active modification.In other words, theme of the present invention is as follows:
1. the Trx that comprises people's modification of one of following polypeptide:
(a) has in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the polypeptide of the aminoacid sequence of other aminoacid replacement;
(b) has in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the polypeptide of the aminoacid sequence of chemically modified;
(c) have in the SEQ ID NO:2 aminoacid sequence except that 32 and 35, there are one or more replacements in position in the preferred SEQ ID NO:2 aminoacid sequence except that 32 to 35, disappearance, the amino acid whose aminoacid sequence that inserts or add, and have the polypeptide of apoptotic activity; With
(d) by be coded in halfcystine in the SEQ ID NO:2 aminoacid sequence by the also proteic DNA of people's oxygen of the modification of other aminoacid replacement or can with the polypeptide of the dna encoding of its complementary strand hybridize under stringent condition.
2. the Trx of modifying according to the 1st people, wherein said other amino-acid residue is a Serine.
3. constitute by following any DNA and the gene of the Trx that the people with apoptotic activity of encoding modifies:
(1) polynucleotide of coded polypeptide, or the complementary strand of these polynucleotide, this polypeptide has in amino acid SEQ ID NO:2 aminoacid sequence 35 cysteine residues by other aminoacid replacement, and in this external SEQ ID NO:2 aminoacid sequence except that 32 and 35, preferably there are one or more replacements in the position except that 32 to 35, disappearance, the amino acid whose aminoacid sequence that inserts or increase, and have activity inducing apoptosis; With
(2) cysteine residues that is coded in 35 of SEQ ID NO:2 aminoacid sequences is modified the DNA of Trx or the DNA that can have the active polypeptide of apoptosis with complementary strand hybridize under stringent condition and the coding of above-mentioned DNA by the people of other aminoacid replacement.
4. but introduce the recombinant expression vector of the 3rd gene with phraseology.
5. the transformant that transforms with the 4th expression vector.
6. be used to produce the method for the polypeptide with activity inducing apoptosis, described method comprises cultivates the 5th transformant.
7. a polypeptide that is used for the cell endocytic of biologically active substance comprises-Cys-Gly-Pro-A ,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
8. comprise by-Cys-Gly-Pro-A ,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(any amino acid of A representative except that Cys) represents is being used for the cell endocytic of biologically active substance.
9. producing can be by the method for the biologically active substance mixture of endocytosis in the cell, this method comprises biologically active substance is attached to and comprises by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
10. can be by the biologically active substance mixture of endocytosis in the cell, wherein biologically active substance is attached to and comprises by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
11. the 10th mixture, wherein comprise by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents is to have 35 cysteine residues of SEQ ID NO:2 aminoacid sequence by the polypeptide of the aminoacid sequence of other aminoacid replacement.
12. the 10th or 11 mixture, wherein above-mentioned biologically active substance is albumen or polypeptide.
Can be 13. produce by the method for the polypeptide complex of endocytosis in the cell, this method comprises cultivates the transformant that conversion has recombinant vectors, this recombinant vectors has the polypeptide of coding mixture, biologically active polypeptides has been attached to and has comprised by-Cys-Gly-Pro-A in the mixture,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
14. comprise the Trx carcinostatic agent of the 1st or 2 modification.
15. comprise the anticancer toughener of Trx of the 1st or 2 modification.
16. comprise the 1st or 2 the Trx of modification and the carcinostatic agent composition of other carcinostatic agent.
17. treatment method for cancer, this method comprise the Trx of administration the 1st or 2 s' modification, if desired, are administered into cancered patient with other carcinostatic agent.
18. comprise the 10th to 12 any one mixture and if desired, comprise the medicine acceptable carrier, the medicine of vehicle or thinner or pharmaceutical composition.
The present invention will be described in more detail below.
Trx (hTRX) represents to comprise 105 amino acid whose polypeptide of SEQ ID NO:2 representative here.
HTRX of the present invention belong to the Trx superfamily and as example be that those have-Cys-Gly-Pro-Cys-in its active centre ,-Cys-Pro-Tyr-Cys-,-Cys-Pro-His-Cys-, or-polypeptide of Cys-Pro-Pro-Cys-.Wherein, the active centre have-Trx of Cys-Gly-Pro-Cys-sequence is preferred.
In first embodiment of the present invention, have been found that induced expression apoptosis activity and cell growth inhibiting activity by 35 halfcystine being converted into TRX that other amino acid preparation modifies, the TRX of modification is effectively as carcinostatic agent.
Second embodiment of the present invention is based on following discovery, promptly by or cysteine residues is converted into other amino acid makes the cell endocytic of the TRX that modifies sharply increase with the TRX that modifies or chemically modified cysteine residues (specifically being sulfhydryl residue) is modified with preparation, and show that for the first time the entity that promotes the cell endocytic effect is based on by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-Cys-Pro-Pro-A-(A representative except that Cys any amino acid or the halfcystine of chemically modified) aminoacid sequence represented.
The Cys that the above-mentioned avtive spot C end of TRX is 35 can be with 19 amino acid (Gly, Ala, Met, Ser, The, Lys, Arg, His, Val, Leu, Ile, Phe, Tyr, Trp, Pro, Gly, Asp, Gln, Asn) arbitrary replacement, and preferably replacing with Ser.
The halfcystine of chemically modified comprises that (R is any organic group by SR for sulfydryl (SH) group of those halfcystines; it comprises; for example; straight or branched alkyl with 1-18 carbon atom; straight or branched thiazolinyl with 2-18 carbon atom; straight or branched alkynyl with 2-18 carbon atom; straight or branched alkoxyalkyl with 2-18 carbon atom has the straight or branched hydroxyalkyl of 1-18 carbon atom, has the acyl group of 1-18 carbon atom; aryl with 6-18 carbon atom; have the aralkyl of 7-18 carbon atom etc., these groups can be with substituting group halogen atom for example, hydroxyl; nitro; amino, alkyl monosubstituted amino, dialkyl amido; methoxyl group, carboxyl or ester group replace) chemically modified that replaces of the group of representative.The TRX of chemically modified can use known method easily synthetic like this.
The gene of the Trx that Trx that the present invention preferably modifies and coding are modified is shown in SEQ ID NO:11.
Have been found that Trx induced expression apoptosis activity and cell growth inhibiting activity that the present invention modifies, and effectively as carcinostatic agent.
In addition, the Trx of the present invention's modification can make up by the carcinostatic agent with other and strengthen anticancer effect.Particularly, the Trx of modification strengthens the effect of carcinostatic agent, and reduces side effect by inducing anticancer effect with concentration and carcinostatic agent combination less than effective anticancer.
Effect is comprised Zorubicin by aforesaid combination enhanced carcinostatic agent, methotrexate, taxol, 5-fluor-uracil, vinealeucoblastine(VLB), vincristine(VCR), mitomycin, cis-platinum, daunomycin, etoposide, taxotere etc.
Trx that the present invention modifies and carcinostatic agent administration simultaneously or can separate administration.
Embodiment preferred of the present invention relates to the treatment method for cancer, wherein the Trx and the carcinostatic agent of administration modification.
The cancer that can be treated is not concrete the qualification, comprises cancer of the stomach, colorectal carcinoma, the rectum cancer, liver cancer, gall-bladder/cholangiocarcinoma, carcinoma of the pancreas, lung cancer, mammary cancer, bladder cancer, cervical cancer etc.
The treatment significant quantity of carcinostatic agent that comprises the Trx of modification of the present invention is each adult cancer patients every day about 0.01 to 100mg, and the treatment significant quantity is about 0.001 to 10mg when as anticancer toughener.
In embodiment preferred of the present invention, the Trx that the present invention modifies can prepare by known genetic engineering technique based on the Trx of SEQ ID NO:2.Except that 32 and 35, preferably the position except that 32 to 35 has one or more replacements to the albumen of described modification in SEQ ID NO:2 aminoacid sequence, disappearance, and the amino acid that adds or insert, and have apoptotic activity.Such sudden change (replace, disappearance is added and inserted) comprises the sudden change (as, allelotrope) of artificial sudden change and natural generation.The method that produces artificial mutation can include, but not limited to rite-directed mutagenesis (Nucleic Acids Res.10,6487-6500,1982) etc.Only otherwise lose apoptotic activity, the amino acid whose number of sudden change is unrestricted, but preferably at 20 amino acid, more preferably at 15 amino acid, more preferably at 10 amino acid with most preferably again in 5 amino acid scopes.
In another embodiment preferred of the present invention, the DNA that comprises the Trx that coding is modified, this albumen in SEQ ID NO:2 aminoacid sequence 35 cysteine residues by other aminoacid replacement, or can have the DNA of polypeptide of apoptotic activity with its complementary strand hybridize under stringent condition and coding, and the albumen of dna encoding has apoptotic activity.
" stringent condition " refers to only specific hybrid take place and the condition that do not have non-specific hybridization here.Such condition normally is equal to approximately the condition of " 1 * SSC and 0.1% SDS are at 37 ℃ ", preferably approximately " 0.5 * SSC and 0.1% SDS are at 42 ℃ " and more preferably " 0.2 * SSC and 0.1% SDS are at 65 ℃ ".The DNA of the polypeptide of the present invention that DNA of the present invention usually suddenlys change with 35 quilts of coding (as, the DNA that SEQ ID NO:11 describes) has high homology.High homology refers to that homology is 75% or more, preferred 90% or more, more preferably 95% or more and particularly 99% or more.
Albumen of the present invention can obtain by the described gene of the present invention in back is incorporated in the expression vector and expresses in proper host cell.As carrier, might select and use those known carriers suitably, for example pTrc-HisA etc.Host cell comprises eukaryotic cell such as mammalian cell and yeast cell, and prokaryotic cell prokaryocyte such as intestinal bacteria (Escherichiacoli), subtilis (Bacillus subtilis), the cell of algae and fungi can use wherein member arbitrarily.
Need not to be limited by theory, believe the TRX induced expression apoptosis activity or the cell growth inhibiting activity of modification, because as 35 Cys after for example Ser replaces by other amino acid, the TRX of modification works as the antagonist of TRX.
In addition, it is believed that 35 Cys by other amino acid for example the TRX of the modification that replaces of Ser be attached to cell surface with 32 Cys owing to do not have Cys and be not joined to cell surface at 35 at 35, the TRX that the result modifies by endocytosis rapidly in cell.This mechanism also obtains the support of the following fact, i.e. TRX (35 Cys of all Ser32 modifications; SEQ IDNO:14), Ser
32Ser
35TRX (SEQ ID NO:13) and wild-type TRX (32 and 35 Cys of modifying; SEQ ID NO:2) seldom by endocytosis in cell.
In other words, Cys-Gly-Pro-A clearly,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-aminoacid sequence of Cys-Pro-Pro-A-(A represents any amino acid beyond the Cys) representative, especially Cys-Gly-Pro-A (A is the same) is playing a part the biologically active substance endocytosis in the cell guiding peptide, and might be attached to biologically active substance by tetrapeptide and will be difficult to endocytosis to intracellular biologically active substance endocytosis.
Here " TRX of modification " is used to represent to comprise the term of variant and modifier.
Biologically active substance comprises the medical compounds that is acted on by endocytosis, oligopeptides, polypeptide, monose, disaccharides or polysaccharide, fat etc., preferred exemplary medical compounds, oligopeptides and polypeptide (comprising glycoprotein).Might be attached to guiding N of peptide or C-terminal by the suitable sequence that comprises above-mentioned tetrapeptide and strengthen selectivity target cell.The medical compounds of example such as biologically active substance, carcinostatic agent, antiviral agent etc.The biologically active peptides of example such as enzyme, antibody, hormone etc.
Guiding peptide of the present invention can be attached to biologically active substance by spacer peptide if desired.For example, when biologically active substance is a polypeptide, above-mentioned guiding peptide is attached to biologically active polypeptides by spacer peptide and forms mixture, and might be incorporated into by the DNA of the mixture of will encoding in the suitable recombinant vectors, transform for example intestinal bacteria of host bacteria, zooblast is CHO for example, or yeast, and cultivates transformant and prepare mixture.
Resectable short-chain peptide might interior biologically active substance, especially biological activity oligopeptides or the polypeptide introduced of cell as the interval in the cell by introducing.Resectable peptide like this comprises-AYVHDAPVK-, it is the sequence that interleukin-11 β (pro-IL-1 β) cleavage site is cut by L-Cysteine HCL Anhydrous (caspase)-1 specificity before the people, with-GDEVDGVK-, its be the people poly--ADP-ribose polymerase (PARP) cleavage site is by the sequence of cysteine proteinase-3 and cutting such as-7 grades.
Optimum implementation of the present invention
The present invention is described with more details with reference to following examples, but these examples are only represented the purpose of example, rather than scope of the present invention is limited.
[embodiment 1]
The production of the TRX of reorganization wild-type TRX and modification
1. intestinal bacteria (E.coli) expression system construction
The Nucleotide of the TRX gene correspondence position by replacing SEQ ID NO:1 makes 32 and/or 35 cysteine residues of TRX polypeptide be replaced by serine residue.At 32 genetic mutation (TRX-C32S that replaced cysteine residues with serine residue; SEQ ID NO:14) plasmid DNA by people TRX gene (SEQ ID NO:1) is inserted carrier pcDNA3.1 is as template, use primer 5 '-GGA TCC GTG AAG CAG ATC GAG AGC AAG-3 ' (SEQ ID NO:3) and 5 '-CTT GAT CAT TTT GCA AGG CCC AGA CCA-3 ' (SEQ ID NO:5) and primer 5 '-PCR of GTC GAC TTA GAC TAA TTC ATTAAT GGT GGC-3 ' (SEQ ID NO:4) and 5 '-TGG TCT GGG CCT TGCAAA ATG ATC AAG-3 ' (SEQ ID NO:6) and obtaining.Subsequently, two dna fragmentation balanced mix of amplification, and interpolation primer 5 '-GGA TCC GTG AAG CAGATC GAG AGC AAG-3 ' (SEQ ID NO:3) and 5 '-GTC GAC TTA GAC TAATTC ATT AAT GGT GGC-3 ' (SEQ ID NO:4), then by pcr amplification total length TRX-C32S.PCR is with 95 ℃ of cycling conditions 1 minute, and 56 ℃ of annealing in 1 minute and 72 ℃ extended and carry out in 2 minutes.Similar, replace the genetic mutation (TRX-C35S of cysteine residues with serine residue in the TRX35 position; SEQ ID NO:12) or at 32 and 35 of the TRX genetic mutation (TRX-C32S/C35S that replace cysteine residues with serine residue; SEQID NO:13) uses primer 5 '-CTT GAT CAT TTT GGA AGG CCC ACACCA-3 ', (SEQ ID NO:7) and 5 '-TGG TGT GGG CCT TCC AAA ATGATC AAG-3 ', (SEQ ID NO:8) or 5 '-TGG TCT GGG CCT TCC AAA ATGATC AAG-3 ', (SEQ ID NO:9) and 5 '-CTT GAT CAT TTT GGA AGG CCCAGA CCA-3 ', the primer that (SEQ ID NO:10) replaces being used to preparing the TRX-C32S gene prepares by PCR.Wild-type TRX (TRX-WT) by personnel selection TRX cDNA (SEQ IDNO:1) be inserted among the carrier pcDNA3.1 plasmid DNA as template use primer 5 '-GGA TCC GTG AAG CAG ATC GAG AGC AAG-3 ' (SEQ ID NO:3) and 5 '-GTC GAC TTA GAC TAA TTC ATT AAT GGT GGC-3 ' (SEQ IDNO:4) obtains by pcr amplification total length TRX-WT.By the TRX-WT of pcr amplification, TRX-C32S, the dna fragmentation of TRX-C35S or TRX-C32S/C35S are connected to TOPO cloning vector (available from Invitrogen) and are incorporated into subsequently in the E.coli host cell.From the clone who transforms, collect plasmid DNA, identify the sequence of the DNA that inserts by dna sequencing.Subsequently, plasmid DNA is cut with restriction enzyme BamHI and SalI, and the fragment that obtains is connected to the expression of recombinant proteins carrier pQE80L (available from Qiagen) that adds histidine mark, Transformed E .coli host cell XL-1Blue then.
2. the cultivation of the E.coli cell of the TRX of generation reorganization TRX wild-type or modification
Conversion has the E.coli cell of plasmid DNA pQE80L to be inoculated in the 3Lterrific broth liquid nutrient medium (BRL) (containing 100 μ g/mL penbritins) after pre-the cultivation, and cultivated 4 hours, inserted the TRX gene of TRX wild type gene or modification in this plasmid.Subsequently, add IPTG to final concentration 1mM, culture was continued to cultivate 2 to 4 hours.
3. the purifying of the TRX of reorganization TRX wild-type and modification
The cell of collecting is suspended from the lysis buffer (proteinase inhibitor, 0.8mM imidazoles, 2 mercapto ethanol) that contains the 2mM N,O-Diacetylmuramidase, uses the ultrasonic apparatus fragmentation.15,000rpm is after centrifugal 30 minutes, collects supernatant, and goes up sample to (Qiagen provides) (the Ni post was replaced PBS in advance) on the PBS equilibrated Ni agarose column.Behind last sample, post is with the PBS washing that contains the 20mM imidazoles, and with the PBS wash-out that contains the 80mM imidazoles.The sample solution of wash-out uses the PD-10 post to replace with PBS solution.
[embodiment 2]
The TRX endocytosis of reorganization TRX wild-type or modification is in cultured cells
1. with cell bonded ability
Final concentration is the fluorescently-labeled TRX-WT of 1 μ g/ml, and TRX-C35S or TRX-C32S/C35S are added to 5 * 10
5The human T-cell that HTLV-1 infects is in the cell of ATL2, hatches 30 minutes at 4 ℃, uses damping fluid (0.1% contains the phosphate buffered saline buffer of sodium azide) washing to be used for Flow Cytometry then.Carry out the analysis (Fig. 1) of Flow Cytometry.As a result, identify and have only TRX-C35S to can be incorporated into cell.Also identify this combination and suppressed (Fig. 2) by the TRX-WT of excessive coexistence.
2. cell endocytic
Histidine-tagged recombinant protein, TRX-WT, TRX-C35S or TRX-32S/C35S add in the ATL2 cell with final concentration 10 μ g/mL, and 4 ℃ or 37 ℃ of incubations one hour.Subsequently, collecting cell, with the phosphate buffered saline buffer washing, 1 * 107 cell is suspended in the hypotonic damping fluid then, and breaks by nitrogen cell homogenates device (available from Pearl).1, after 000g is centrifugal, collect supernatant, and pass through 10 the cytosol fraction of the centrifugal collection supernatant of 000g.The cytosol fraction uses SDS-polyacrylamide gel electrophoresis and anti--TRX monoclonal antibody to analyze (Fig. 3) by the Western blotting.As a result, evaluation only detects TRX-C35S in the cytosol fraction.
[embodiment 3]
The biological activity of the TRX of TRX wild-type or modification
1. activity inducing apoptosis
Wherein introduced TRX-WT, the human T-cell of TRX-C35S or TRX-C32S/C35S gene is Jurkat, cultivates 72 hours to analyze inductive apoptosis (Fig. 4) in serum-free RPMI substratum.As a result, compare with the Jurkat cell of introducing TRX-WT or TRX-C32S/C35S, the apoptosis with Jurkat cell of high expression level TRX-C35S in the cell is further promoted.
2. cytostatic activity
5 * 10
5The human peripheral blood mononuclear cell is suspended in the RPMI substratum that 1ml contains 10% foetal calf serum, adds the pHA of final concentration 28ng/ml and the TRX-WT of 10 μ g/mL then, TRX-C32S/C35S or TRX-C35S, and cultivate.After 96 hours, add the 3H-thymidine and measure its absorbed dose (Fig. 5) by scintillometer.As a result, identify with the cell that does not have TRX and compare to have that ability of cell proliferation is enhanced in the reorganization TRX-WT proteic cell, and cell proliferation is suppressed in having the proteic cell of reorganization TRX-C35S.
[embodiment 4]
1. the reinforced effects 1 of carcinostatic agent performance
2. the reinforced effects 2 of carcinostatic agent performance
Recombinant C 35S-TRX albumen adds into concentration 10 μ g/mL that the human T-cell is in the culture of Jurkat, and incubation added cis-platinum in one hour or three hours then to final concentration 3 μ g/mL or 6 μ g/mL.Annexin V-FITC and propidium diiodide are incorporated into cell after cultivating 24 hours, by the quantity of apoptotic pair of positive cell of flow cytometry analysis showed.The result, in adding proteic group of recombinant C 35S-TRX in advance, the quantity of the two positive cells of annexin V-FITC and propidium diiodide is compared increase with the group of not adding recombinant protein, and increases about 2 times and increase about 4 times in the group with 6 μ g/mL cis-platinums in the group with 3 μ g/mL cis-platinums.Higher than the effect of 1 hour group of pre-treatment with 3 hours group of C35S-TRX albumen pre-treatment.
As mentioned above, described the preferred embodiment of the invention, those skilled in the art should understand that and to carry out various changes and modifications and do not depart from essence of the present invention.Therefore, scope of the present invention should only be determined by claim.
According to the present invention, might stablize and produce TRX albumen with the active modification of high cell growth inhibiting and exploitation utilization on a large scale and derive from the carrier that to be swallowed the peptide sequence of the TRX that modifies in the cell in fast.With the TRX albumen of modifying or derive from the biologically active substance (protein, lipid, nucleic acid, organic compound, mineral compound) that the peptide of the TRX of modification merges and to be used to various fields treatment of diseases and/or prevention.
Sequence table
<110〉Independent Administrative Leged Industrial Technology Complex Inst
(NATIONAL?INSTITUTE?OF?ADVANCED?INDUSTRIAL?SCIENCE?AND?TECHNOLOGY)
<120〉Trx of Xiu Shiing (Thioredoxin derivatives)
<130>SCT053879-47
<160>14
<170>Patentln?version?3.1
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<212>DNA
<213>Homo?sapiens
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<222>(1)..(318)
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atg?gtg?aag?cag?atc?gag?agc?aag?act?gct?ttt?cag?gaa?gcc?ttg?gac 48
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
gct?gca?ggt?gat?aaa?ctt?gta?gta?gtt?gac?ttc?tca?gcc?acg?tgg?tgt 96
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Cys
20 25 30
ggg?cct?tgc?aaa?atg?atc?aag?cct?ttc?ttt?cat?tcc?ctc?tct?gaa?aag 144
Gly?Pro?Cys?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
tat?tcc?aac?gtg?ata?ttc?ctt?gaa?gta?gat?gtg?gat?gac?tgt?cag?gat 192
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
gtt?gct?tca?gag?tgt?gaa?gtc?aaa?tgc?atg?cca?aca?ttc?cag?ttt?ttt 240
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
aag?aag?gga?caa?aag?gtg?ggt?gaa?ttt?tct?gga?gcc?aat?aag?gaa?aag 288
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
ctt?gaa?gcc?acc?att?aat?gaa?tta?gtc?taa 318
Leu?Glu?Ala?Thr?Ile?Asn?Glu?Leu?Val
100 105
<210>2
<211>105
<212>PRT
<213>Homo?sapiens
<400>2
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Cys
20 25 30
Gly?Pro?Cys?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
Leu?Glu?Ala?Thr?Ile?Asn?Glu?Leu?Val
100 105
<210>3
<211>27
<212>DNA
<213>Homo?sapiens
<400>3
ggatccgtga?agcagatcga?gagcaag 27
<210>4
<211>30
<212>DNA
<213>Homo?sapiens
<400>4
gtcgacttag?actaattcat?taatggtggc 30
<210>5
<211>27
<212>DNA
<213>Homo?sapiens
<400>5
cttgatcatt?ttgcaaggcc?cagacca 27
<210>6
<211>27
<212>DNA
<213>Homo?sapiens
<400>6
tggtctgggc?cttgcaaaat?gatcaag 27
<210>7
<211>27
<212>DNA
<213>Homo?sapiens
<400>7
cttgatcatt?ttggaaggcc?cacacca 27
<210>8
<211>27
<212>DNA
<213>Homo?sapiens
<400>8
tggtgtgggc?cttccaaaat?gatcaag 27
<210>9
<211>27
<212>DNA
<213>Homo?sapiens
<400>9
tggtctgggc?cttccaaaat?gatcaag 27
<210>10
<211>27
<212>DNA
<213>Homo?sapiens
<400>10
cttgatcatt?ttggaaggcc?cagacca 27
<210>11
<211>318
<212>DNA
<213>Homo?sapiens
<220>
<221>misc_feature
<222>(103)..(105)
<223>nnn?stands?for?any?base?for?coding?Lys,Asn,Arg,Ser,Thr,Ile,
Met,Glu,Asp,Gly,Ala,Val,Gln,His,Pro,Leu,Tyr,Trp,or?Ph
e.
<220>
<221>CDS
<222>(1)..(318)
<223>The’Xaa’at?location?35?stands?for?Lys,Asn,Arg,Ser,Thr,Ile,
Met,Glu,Asp,Gly,Ala,Val,Gln,His,Pro,Leu,Tyr,Trp,or?Ph
e.
<400>11
atg?gtg?aag?cag?atc?gag?agc?aag?act?gct?ttt?cag?gaa?gcc?ttg?gac 48
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
gct?gca?ggt?gat?aaa?ctt?gta?gta?gtt?gac?ttc?tca?gcc?acg?tgg?tgt 96
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Cys
20 25 30
ggg?cct?nnn?aaa?atg?atc?aag?cct?ttc?ttt?cat?tcc?ctc?tct?gaa?aag 144
Gly?Pro?Xaa?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
tat?tcc?aac?gtg?ata?ttc?ctt?gaa?gta?gat?gtg?gat?gac?tgt?cag?gat 192
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
gtt?gct?tca?gag?tgt?gaa?gtc?aaa?tgc?atg?cca?aca?ttc?cag?ttt?ttt 240
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
aag?aag?gga?caa?aag?gtg?ggt?gaa?ttt?tct?gga?gcc?aat?aag?gaa?aag 288
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
ctt?gaa?gcc?acc?att?aat?gaa?tta?gtc?taa 318
Leu?Glu?Ala?Thr?Ile?Asn?Glu?Leu?Val
100 105
<210>12
<211>318
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(1)..(318)
<223>
<400>12
atg?gtg?aag?cag?atc?gag?agc?aag?act?gct?ttt?cag?gaa?gcc?ttg?gac 48
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
gct?gca?ggt?gat?aaa?ctt?gta?gta?gtt?gac?ttc?tca?gcc?acg?tgg?tgt 96
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Cys
20 25 30
ggg?cct?tcc?aaa?atg?atc?aag?cct?ttc?ttt?cat?tcc?ctc?tct?gaa?aag 144
Gly?Pro?Ser?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
tat?tcc?aac?gtg?ata?ttc?ctt?gaa?gta?gat?gtg?gat?gac?tgt?cag?gat 192
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
gtt?gct?tca?gag?tgt?gaa?gtc?aaa?tgc?atg?cca?aca?ttc?cag?ttt?ttt 240
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
aag?aag?gga?caa?aag?gtg?ggt?gaa?ttt?tct?gga?gcc?aat?aag?gaa?aag 288
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
ctt?gaa?gcc?acc?att?aat?gaa?tta?gtc?taa 318
Leu?Glu?Ala?Thr?Ile?Asn?Glu?Leu?Val
100 105
<210>13
<211>318
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(1)..(318)
<223>
<400>13
atg?gtg?aag?cag?atc?gag?agc?aag?act?gct?ttt?cag?gaa?gcc?ttg?gac 48
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
gct?gca?ggt?gat?aaa?ctt?gta?gta?gtt?gac?ttc?tca?gcc?acg?tgg?tct 96
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Ser
20 25 30
ggg?cct?tcc?aaa?atg?atc?aag?cct?ttc?ttt?cat?tcc?ctc?tct?gaa?aag 144
Gly?Pro?Ser?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
tat?tcc?aac?gtg?ata?ttc?ctt?gaa?gta?gat?gtg?gat?gac?tgt?cag?gat 192
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
gtt?gct?tca?gag?tgt?gaa?gtc?aaa?tgc?atg?cca?aca?ttc?cag?ttt?ttt 240
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
aag?aag?gga?caa?aag?gtg?ggt?gaa?ttt?tct?gga?gcc?aat?aag?gaa?aag 288
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
ctt?gaa?gcc?acc?att?aat?gaa?tta?gtc?taa 318
Leu?Glu?Ala?Thr?Ile?Ash?Glu?Leu?Val
100 105
<210>14
<211>318
<212>DNA
<213>Homo?Sapiens
<220>
<221>CDS
<222>(1)..(318)
<223>
<400>14
atg?gtg?aag?cag?atc?gag?agc?aag?act?gct?ttt?cag?gaa?gcc?ttg?gac 48
Met?Val?Lys?Gln?Ile?Glu?Ser?Lys?Thr?Ala?Phe?Gln?Glu?Ala?Leu?Asp
1 5 10 15
gct?gca?ggt?gat?aaa?ctt?gta?gta?gtt?gac?ttc?tca?gcc?acg?tgg?tct 96
Ala?Ala?Gly?Asp?Lys?Leu?Val?Val?Val?Asp?Phe?Ser?Ala?Thr?Trp?Ser
20 25 30
ggg?cct?tgc?aaa?atg?atc?aag?cct?ttc?ttt?cat?tcc?ctc?tct?gaa?aag 144
Gly?Pro?Cys?Lys?Met?Ile?Lys?Pro?Phe?Phe?His?Ser?Leu?Ser?Glu?Lys
35 40 45
tat?tcc?aac?gtg?ata?ttc?ctt?gaa?gta?gat?gtg?gat?gac?tgt?cag?gat 192
Tyr?Ser?Asn?Val?Ile?Phe?Leu?Glu?Val?Asp?Val?Asp?Asp?Cys?Gln?Asp
50 55 60
gtt?gct?tca?gag?tgt?gaa?gtc?aaa?tgc?atg?cca?aca?ttc?cag?ttt?ttt 240
Val?Ala?Ser?Glu?Cys?Glu?Val?Lys?Cys?Met?Pro?Thr?Phe?Gln?Phe?Phe
65 70 75 80
aag?aag?gga?caa?aag?gtg?ggt?gaa?ttt?tct?gga?gcc?aat?aag?gaa?aag 288
Lys?Lys?Gly?Gln?Lys?Val?Gly?Glu?Phe?Ser?Gly?Ala?Asn?Lys?Glu?Lys
85 90 95
ctt?gaa?gcc?acc?att?aat?gaa?tta?gtc?taa 318
Leu?Glu?Ala?Thr?Ile?Asn?Glu?Leu?Val
100 105
Claims (18)
1. the Trx who contains the modification of one of following polypeptide:
A) has in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the polypeptide of the aminoacid sequence of other aminoacid replacement;
B) has in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the polypeptide of the aminoacid sequence of chemically modified;
C) have in the SEQ ID NO:2 aminoacid sequence except that 32 and 35, preferably there are one or more replacements in the position except that 32 to 35, disappearance, the amino acid whose aminoacid sequence that inserts or add, and the polypeptide with activity inducing apoptosis; With
D) by be coded in cysteine residues in the SEQ ID NO:2 aminoacid sequence by the Trx's of the modification of other aminoacid replacement DNA or can with the polypeptide of the dna encoding of its complementary strand hybridize under stringent condition.
2. according to the Trx of the modification of claim 1, wherein said other amino-acid residue is a Serine.
3. contain Trx's the gene that following any DNA and coding have the modification of apoptotic activity:
(1) polynucleotide of coded polypeptide, or the complementary strand of these polynucleotide, this polypeptide has in SEQ ID NO:2 aminoacid sequence 35 cysteine residues by other aminoacid replacement, in this external SEQ ID NO:2 aminoacid sequence except that 32 and 35, preferably there are one or more replacements in the position except that 32 to 35, disappearance, the amino acid whose aminoacid sequence that inserts or increase, and have activity inducing apoptosis; With
(2) be coded in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the Trx's of the modification of other aminoacid replacement DNA, or complementary strand hybridize under stringent condition that can DNA above-mentioned with it and the DNA that coding has the active polypeptide of apoptosis.
4. but introduce the recombinant expression vector of the gene of claim 3 with phraseology.
5. use the transformant of the expression vector conversion of claim 4.
6. be used to produce the method for the polypeptide with activity inducing apoptosis, described method comprises the transformant of cultivating claim 5.
7. polypeptide that is used for the cell endocytic of biologically active substance, it comprises by-Cys-Gly-Pro-A ,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-aminoacid sequence that Cys-Pro-Pro-A-(any amino acid of A representative except that Cys) represents.
8. comprise by-Cys-Gly-Pro-A ,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(any amino acid of A representative except that Cys) represents is used for the cell endocytic of biologically active substance.
9. producing can be by the method for the biologically active substance mixture of endocytosis in the cell, this method comprises biologically active substance is attached to and comprises by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
10. can be by the biologically active substance mixture of endocytosis in the cell, wherein biologically active substance is incorporated into and comprises by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
11. the mixture of claim 10, wherein comprise by-Cys-Gly-Pro-A,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents is to have in the SEQ ID NO:2 aminoacid sequence 35 cysteine residues by the polypeptide of the aminoacid sequence of other aminoacid replacement.
12. the mixture of claim 10 or 11, wherein above-mentioned biologically active substance is albumen or polypeptide.
Can be 13. produce by the method for the polypeptide complex of endocytosis in the cell, this method comprises cultivates the transformant that conversion has recombinant vectors, this recombinant vectors has the polypeptide of coding mixture, biologically active polypeptides has been incorporated into and has comprised by-Cys-Gly-Pro-A in the mixture,-Cys-Pro-Tyr-A-,-Cys-Pro-His-A-or-polypeptide of the aminoacid sequence that Cys-Pro-Pro-A-(A representative except that Cys any amino acid) represents.
14. comprise the carcinostatic agent of Trx of the modification of claim 1 or 2.
15. comprise the anticancer toughener of Trx of the modification of claim 1 or 2.
16. comprise the Trx of modification of claim 1 or 2 and the carcinostatic agent composition of other carcinostatic agent.
17. the treatment method for cancer, this method comprises the Trx of the modification of administration claim 1 or 2, if desired, is administered into cancered patient with other carcinostatic agent.
18. medicine or pharmaceutical composition comprise any one mixture of claim 10 to 12, and if desired, comprise the medicine acceptable carrier, vehicle or thinner.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP093342/2003 | 2003-03-31 | ||
JP2003093342 | 2003-03-31 | ||
JP349109/2003 | 2003-10-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1798837A true CN1798837A (en) | 2006-07-05 |
Family
ID=36819196
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNA2004800150605A Pending CN1798837A (en) | 2003-03-31 | 2004-03-30 | Thioredoxin modification |
Country Status (1)
Country | Link |
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CN (1) | CN1798837A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101838653B (en) * | 2010-01-23 | 2013-06-05 | 河北师范大学 | Rice secretary type thioredoxin gene and application thereof |
CN107011413A (en) * | 2017-05-31 | 2017-08-04 | 浙江省农业科学院 | Tripeptides CGP with function of blood sugar reduction and application thereof |
CN107245104A (en) * | 2017-06-13 | 2017-10-13 | 厦门大学 | A kind of raising compound or material enter method and the application of cell efficiency |
CN110257347A (en) * | 2019-06-25 | 2019-09-20 | 成都英普博集生物科技有限公司 | Thioredoxin mutant, preparation method and its application in recombination fusion protein production |
-
2004
- 2004-03-30 CN CNA2004800150605A patent/CN1798837A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101838653B (en) * | 2010-01-23 | 2013-06-05 | 河北师范大学 | Rice secretary type thioredoxin gene and application thereof |
CN107011413A (en) * | 2017-05-31 | 2017-08-04 | 浙江省农业科学院 | Tripeptides CGP with function of blood sugar reduction and application thereof |
CN107011413B (en) * | 2017-05-31 | 2020-06-16 | 浙江省农业科学院 | Tripeptide CGP with blood sugar reducing function and application thereof |
CN107245104A (en) * | 2017-06-13 | 2017-10-13 | 厦门大学 | A kind of raising compound or material enter method and the application of cell efficiency |
CN110257347A (en) * | 2019-06-25 | 2019-09-20 | 成都英普博集生物科技有限公司 | Thioredoxin mutant, preparation method and its application in recombination fusion protein production |
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Open date: 20060705 |