CN1294987C - Protein having antitumor function, and high performance expression in vitro - Google Patents
Protein having antitumor function, and high performance expression in vitro Download PDFInfo
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- CN1294987C CN1294987C CNB031098614A CN03109861A CN1294987C CN 1294987 C CN1294987 C CN 1294987C CN B031098614 A CNB031098614 A CN B031098614A CN 03109861 A CN03109861 A CN 03109861A CN 1294987 C CN1294987 C CN 1294987C
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- C07K14/4716—Muscle proteins, e.g. myosin, actin
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Abstract
The present invention discloses an application of cardiac troponin I to preparing a medicine for inhibiting tumors. Research indicates that the cardiac troponin I has the function of inhibiting tumors. The present invention has the technical scheme that the cardiac troponin I is applied to preparing a medicine for inhibiting tumors. The effective dose of the cardiac troponin I is 1 microgram to 1 milligram per kilogram of body weight every day in the medicine for inhibiting tumors. One or more pharmacologically acceptable carriers can be also added to the medicine for inhibiting tumors during preparation. The present invention also discloses a cardiac troponin I coding gene effectively expressed in a colibacillus and pichia pastoris expression system. The medicine of the present invention does not directly act on the tumor cells, has broad spectrum function of inhibiting tumors, does not generate medicine tolerance, and has profound practical significance.
Description
Technical field
The present invention relates to proteinic new purposes and expression thereof, particularly relate to the new purposes and the vivoexpression method thereof of cardiac muscle troponin I.
Background technology
Malignant tumor is the most serious class disease of harm humans health.The common method of treatment malignant tumor has at present: surgical operation therapy, chemical medicinal treatment, radiotherapy, biotherapy and Chinese medical and herbal therapy.Wherein commonly used with surgical operation therapy, chemical medicinal treatment, radiotherapy.Surgical operation therapy is as the first-selection and the major programme of most of oncotherapys, makes that countless tumor patients rehabilitate, life-saving or improved quality of life.But aspect oncotherapy, surgical operation therapy only is applicable to the patient of certain condition and scope, and because tumor can recur and shift, excision can both not reach the purpose of radical cure.Extremely important and requisite effect is also being brought into play in chemical medicinal treatment and the radiocurable tumor treatment aspect that is used in, and also in continuous development.But at present chemotherapeutics therapy of using and radiotherapy all have infringement to all active proliferative cells in the health, as hemopoietic system, gastrointestinal tract etc., so symptoms such as the patient of chemicotherapy often has hemogram reduction, gastrointestinal hemorrhage, feels sick, vomitings.And owing to being subjected to the restriction that many-sided reasons such as drug resistance appear in patient's body and spiritual aspect holding capacity and tumor cell, chemicotherapy can not kill all tumor cells, for the recurrence of tumor and the deterioration of transfer and disease have stayed hidden danger.Biotherapy and Chinese medical and herbal therapy also are the antineoplastic reasonable method, but require further study to improve curative effect.Therefore, the exploitation have obvious therapeutic effect, safety antitumor drug be the extremely urgent task that the world of medicine faces.
Studies show that the unrestricted invasive growth of malignant entity tumor and transfer thereof depend on the continuous generation of neovascularity, can suppress growth of tumor significantly so suppress angiogenesis.Suppress angiogenesis, the blood supply of blocking-up tumor body is the New Policy that is different from conventional antineoplaston, is also referred to as " tumor starvation ", has become the focus of antineoplaston research.Under the normal physiological state, ovulating except female reproductive system, corpus luteum generates, and processes such as pregnancy have outside the of short duration new vessels generative process, and adult vascular system hypertrophy is in relative static conditions.But in tumor tissues, for adapting to the quick growth of tumor tissue, angiogenesis is very active, in time to be tumor tissue growth supply blood.Blood capillary in tumor tissues source has two kinds: a kind of is the VEGF that tumor cell etc. produces, and induces the tumor body to generate blood capillary; Another kind is that the host blood vessel that remains in the tumor body gradually becomes tumor vessel, both the tumorization of host blood vessel.Different with normal blood vessels, tumor vascular vascular endothelial cell is imperfect, and basement membrane is sparse and be easy to reveal.Behind the tumor-blood-vessel growth no longer further differentiation reconstruct into tremulous pulse and vein, no smooth muscle and teleneuron belong to passive blood vessel, its blood flow body that places one's entire reliance upon circulates.Newborn blood capillary is a first stop tumor-infiltrated and that shift, and tumor cell enters blood circulation to metastasis by the tumor wall of micrangium.
Entity tumor growth needs a large amount of blood supplies.Many experimentatioies confirm to suppress neovascularity generation in the animal body, can be cut to the blood supply that tumor growth provides nutrient effectively, make the atrophy of tumor body so that disappear.The application of anti-neovascularization medicaments TNP-470 and anti-bFGF, VEGF monoclonal antibody, the competitive blocking VEGF signal conduction of saltant vegf receptor FlK-1, α V β 3 overexpression inducing endothelial cell apoptosis and the formation of the special inhibition tumor neogenetic blood vessels of Angiostatin etc., all can suppress tumor growth, be a kind of novel, promising antitumor drug, can remedy the limitation of current clinical cancer therapy means and replace the part treatment means.Because this class medicine is to reach by the formation that suppresses new vessels to suppress tumor growth, and the growth of people's normal tissue cell does not rely on the generation of new vessels, so normal structure is not almost had toxic and side effects.This class medicine does not directly act on tumor cell, so the inhibition tumor has broad spectrum activity and does not develop immunity to drugs, be current effective, safe, promising antitumor drug of generally acknowledging in the world, be mainly used in original position and shift the treatment of solid tumor and the operative treatment of auxiliary tumor, also can be used for preventing the recurrence and the transfer of tumor.Therefore, develop a kind of inhibiting antitumor drug of blood vessel hyperplasia that has, aspect the preventing and treating of malignant tumor, have the important clinical meaning.
Cardiac muscle troponin I is one of three subunits of cardiac troponin, is often used as the mark of myocardial infarction diagnosis at present, has the SEQ ID № in the sequence table: 1 amino acid residue sequence, form by 210 amino acid residues.
Escherichia coli and pichia yeast expression system are to be used to one of the most successful expression system of expressing foreign protein, and development in recent years is very fast, and existing at present hundreds of kind albumen is expressed in this expression system.Pichia sp. is a kind of methanol auxotype yeast, its growth rapidly, condition of culture is simple, can the high density continuous culture, genetic manipulation is similar to saccharomyces cerevisiae, technology is quite ripe, is the high expressed strain of using always in the commercial production.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide cardiac muscle troponin I.
The inventor studies show that, cardiac muscle troponin I has the effect that suppresses tumor proliferative.Therefore technical scheme of the present invention is:
The application of cardiac muscle troponin I in the medicine of preparation inhibition tumor.
When needing, be in the medicine of active component with the cardiac muscle troponin I, can also adding one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.
In the medicine of the present invention, the effective dose of cardiac muscle troponin I is one 1 milligrams of 1 micrograms/kg body weight/sky, can make various ways such as injection, lyophilized injectable powder.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
Another object of the present invention provides the encoding gene of the cardiac muscle troponin I that efficiently expresses in Pichia sp. and escherichia expression system.
Cardiac muscle troponin I encoding gene provided by the invention has two kinds, and first kind is the high cardiac muscle troponin I encoding gene that efficiently expresses in pichia yeast expression system, is the SEQ ID № in the sequence table: 2.
The DNA sequence of sequence 2 is made up of 633 nucleotide in the sequence table, the protein of sequence 1 in the code sequence tabulation.
The expression vector and the cell line that contain said gene all belong to protection scope of the present invention, as the pPIC9K-AAF carrier, and (physical map is as shown in Figure 1) and GS115 Pichia sp. cell line.
Second kind of cardiac muscle troponin I encoding gene provided by the invention is the cardiac muscle troponin I encoding gene that efficiently expresses in escherichia expression system, is the SEQ ID № in the sequence table: 3.
The DNA sequence of sequence 3 is made up of 633 nucleotide in the sequence table, the protein of sequence 1 in the code sequence tabulation.
The expression vector and the cell line that contain said gene all belong to protection scope of the present invention, as the pET-AAF carrier, and (physical map is as shown in Figure 3) and BL21-DE3 Bacillus coli cells system.
The present invention has creatively disclosed the anti-tumor activity of cardiac muscle troponin I.The medicine that with the cardiac muscle troponin I is active component does not directly act on tumor cell, suppresses tumor and has broad spectrum activity and do not develop immunity to drugs, and has far-reaching practice significance.
Description of drawings
Fig. 1 is the physical map of expression plasmid pPIC9K-AAF
Fig. 2 is the restriction enzyme digestion and electrophoresis collection of illustrative plates of plasmid pPIC9K-AAF
Fig. 3 is the GS115 Pichia sp. bacteria culture fluid electrophoresis pattern behind the methanol induction
Fig. 4 is the physical map of expression plasmid pET-AAF
Fig. 5 is the restriction enzyme digestion and electrophoresis collection of illustrative plates of expression plasmid pET-AAF
Fig. 6 is the BL21-DE3 escherichia coli culture fluid electrophoresis pattern of IPTG after inducing
The specific embodiment
The Pichia yeast vivoexpression of embodiment 1, cardiac muscle troponin I
(1) the cardiac muscle troponin I gene is synthetic
The full length sequence of sequence 2 in the artificial synthesized sequence table, and its 5 ' end add the NdeI restriction enzyme site, 3 ' end add the NotI restriction enzyme site, obtain sequence A AF1.
(2) cardiac muscle troponin I vivoexpression
A, the complete sequence of synthetic is packed in the pGEM-T-EASY plasmid, make up plasmid pGEM-AAF1.
The structure of b, expression plasmid
Employing is available from the efficient expression plasmid pPIC9K of American I nvitrogen company, and with NdeI and NotI enzyme action pGEM-AAF1 plasmid and pPIC9K plasmid respectively, gel electrophoresis is collected target fragment, with the connection of spending the night of 16 ℃ of T4 ligases.Through the conversion of conventional method, choose bacterium, amplification obtains expression plasmid pPIC9K-AAF, and its structure collection of illustrative plates is as shown in Figure 1.The enzyme action qualification result proves that the structure of expression plasmid is correct as shown in Figure 2.
C, with plasmid pPIC9K-AAF SacI linearisation, adopt Zhejiang Xin Zhi company electricity gene introducing apparatus, linearizing pPIC9K-AAF is imported in the GS115 Pichia yeast (available from Invitrogen company), through cultivating, select, processes such as screening obtain the monoclonal bacterium of anti-G418mg/ml.
D, select the monoclonal bacterium, be inoculated in the 5ml culture medium, 30 ℃ are spent the night, and change over to then in the 250ml culture medium, continue to be cultured to OD
600=10-20 collects thalline, is diluted to OD with no carbon source culture medium
600=50, continue to cultivate 24 hours, adding methanol to final concentration is 1% abduction delivering, adds methanol once every 24 hours, to 96 hours.The centrifuging and taking supernatant carries out gel electrophoresis analysis and functional examination.Get the 10ml supernatant, use the 12%SDS-PAGE electrophoresis, and dye through Coomassie brilliant blue.As shown in Figure 3, the result shows the position at 32kD, and inductive protein expression is arranged, and according to the BSA contrast, calculating expression is 120mg/L.
(3) functional examination of cardiac muscle troponin I of the present invention
Get 24 kunming mices, be divided into and go up behind the cleer and peaceful methanol induction two groups of supernatants before the methanol induction, cervical region subcutaneous vaccination S180 ascites cells tumor strain respectively, go up cleer and peaceful methanol induction before the subcutaneous injection methanol induction respectively after three days after supernatant 0.5ml/ only, every injection in 12 hours once, injected continuously 7 days.Observation also cuts tumor tissue, weighs, and supernatant group tumor body weight is respectively 0.82+0.22 gram and 0.11+0.05 gram behind the preceding cleer and peaceful methanol induction of methanol induction as a result, and tumour inhibiting rate is 86.6%.
The escherichia coli vivoexpression of embodiment 2, cardiac muscle troponin I
(1) artificial gene is synthetic: synthetic gene sequence 3 total length 1-210 bases add that at 5 ' end SnabI restriction enzyme site and 3 ' end add the NotI restriction enzyme site, obtain sequence A FF2 respectively.
(2) cardiac muscle troponin I vivoexpression
A, the complete sequence of synthetic is packed in the pGEM-T-EASY plasmid, make up the pGEM-AAF2 cloning vehicle.
The structure of b, expression plasmid
Employing is available from the efficient expression plasmid pET21b of U.S. Novagen company, and with SnabI and NotI enzyme action pGEM-AAF2 plasmid and pET21b plasmid respectively, gel electrophoresis is collected target fragment, with the connection of spending the night of 16 ℃ of T4 ligases.Through the conversion of conventional method, choose bacterium, amplification obtains expression plasmid pET-AAF, and its structure collection of illustrative plates is as shown in Figure 4.The enzyme action qualification result as shown in Figure 5, proves that the structure of expression plasmid is correct.
C, plasmid pET-AAF is imported in the BL21-DE3 escherichia coli with conversion method,, select processes such as screening, acquisition monoclonal bacterium through cultivating.
D, select the monoclonal bacterium, be inoculated in the 5ml culture medium, 37 ℃ are spent the night, and change over to then in the 250ml culture medium, continue to be cultured to OD
600=0.8, add IPTG to final concentration be 1%37 ℃ of abduction deliverings 4 hours.The centrifuging and taking precipitation is used the lysate suspendible, ultrasonication, and centrifugal 30 minutes of 10000g abandons supernatant, and washing of precipitate twice is at last with the dissolving of 8M carbamide, through 50mM phosphoric acid buffer saline solution dialyzed overnight.Get 10ul, use the 12%SDS-PAGE electrophoresis, and dye through Coomassie brilliant blue.As shown in Figure 6, the result shows the position at 29KD, and inductive protein expression is arranged, and according to the BSA contrast, calculating expression is 85mg/L.
(3) functional examination of cardiac muscle troponin I of the present invention
Get 24 kunming mices, be divided into phosphate buffer and express purification liquid group, cervical region subcutaneous vaccination S180 ascites cells tumor strain respectively, difference subcutaneous injection phosphate buffer and expression purification liquid 1.0mg albumen/kg after three days, every injection in 12 hours once, injected continuously 7 days.Observation also cuts tumor tissue, weighs, and phosphate buffer and expression purification liquid group tumor body weight are respectively 1.02+0.28 gram and 0.15+0.06 gram as a result, and tumour inhibiting rate is 85.3%.
The body internal stability experiment of the escherichia coli vivoexpression purification liquid of embodiment 3, cardiac muscle troponin I:
Get 24 kunming mices, be divided into phosphate buffer and express purification liquid group, respectively subcutaneous injection phosphate buffer and expression purification liquid 2.0mg albumen/kg, injected 8 hours, eyeground vein is got blood, and separation of serum adopts conventional euzymelinked immunosorbent assay (ELISA) to measure serum cardiac muscle troponin I concentration.Phosphate buffer and express purification liquid group serum cardiac muscle troponin I concentration and be respectively 0.52+0.08ng/ml, 3.55+0.12ng/ml as a result shows in vivo stable higher of cardiac muscle troponin I.
Sequence table
<160>3
<210>1
<211>210
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Met?Ala?Asp?Gly?Ser?Ser?Asp?Ala?Ala?Arg?Glu?Pro?Arg?Pro?Ala
1 5 10 15
Pro?Ala?Pro?Ile?Arg?Arg?Arg?Ser?Ser?Asn?Tyr?Arg?Ala?Tyr?Ala
20 25 30
Thr?Glu?Pro?His?Ala?Lys?Lys?Lys?Ser?Lys?Ile?Ser?Ala?Ser?Arg
35 40 45
Lys?Leu?Gln?Leu?Lys?Thr?Leu?Leu?Leu?Gln?Ile?Ala?Lys?Gln?Glu
50 55 60
Leu?Glu?Arg?Glu?Ala?Glu?Glu?Arg?Arg?Gly?Glu?Lys?Gly?Arg?Ala
65 70 75
Leu?Ser?Thr?Arg?Cys?Gln?Pro?Leu?Glu?Leu?Ala?Gly?Leu?Gly?Phe
80 85 90
Ala?Glu?Leu?Gln?Asp?Leu?Cys?Arg?Gln?Leu?His?Ala?Arg?Val?Asp
95 100 105
Lys?Val?Asp?Glu?Glu?Arg?Tyr?Asp?Ile?Glu?Ala?Lys?Val?Thr?Lys
110 115 120
Ash?Ile?Thr?Glu?Ile?Ala?Asp?Leu?Thr?Gln?Lys?Ile?Phe?Asp?Leu
125 130 135
Arg?Gly?Lys?Phe?Lys?Arg?Pro?Thr?Leu?Arg?Arg?Val?Arg?Ile?Ser
140 145 150
Ala?Asp?Ala?Met?Met?Gln?Ala?Leu?Leu?Gly?Ala?Arg?Ala?Lys?Glu
155 160 165
Ser?Leu?Asp?Leu?Arg?Ala?His?Leu?Lys?Gln?ValLys?Lys?Glu?Asp
170 175 180
Thr?Glu?Lys?Glu?Asn?Arg?Glu?Val?Gly?Asp?Trp?Arg?Lys?Ash?Ile
185 190 195
Asp?Ala?Leu?Ser?Gly?Met?Glu?Gly?Arg?Lys?Lys?Lys?Phe?Glu?Ser
200 205 210
<210>2
<211>633
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
atggctgacg?gttcctccga?cgctgctaga?gagccaagac?cagctccagc?tccaatcaga 60
agaagatcct?ccaactacag?agcttacgct?accgagccac?acgctaagaa?gaagtccaag 120
atctccgctt?ccagaaagtt?gcaattgaag?accttgttgt?tgcaaatcgc?taagcaagag 180
ttggagagag?aggctgagga?gagaagaggt?gagaagggta?gagctttgtc?caccagatgt 240
caaccattgg?agttggctgg?tttgggtttc?gctgagttgc?aagacttgtg?tagacaattg 300
cacgctagag?tcgacaaggt?cgacgaggag?agatacgaca?tcgaggctaa?ggtcaccaag 360
aacatcaccg?agatcgctga?cttgacccaa?aagatcttcg?acttgagagg?taagttcaag 420
agaccaacct?tgagaagagt?cagaatctcc?gctgacgcta?tgatgcaagc?tttgttgggt 480
gctagagcta?aggagtcctt?ggacttgaga?gctcacttga?agcaagtcaa?gaaggaggac 540
accgagaagg?agaacagaga?ggtcggtgac?tggagaaaga?acatcgacgc?tttgtccggt 600
atggagggta?gaaagaagaa?gttcgagtcc?taa 633
<210>3
<211>633
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atggctgacg?gttcttctga?cgctgctcgt?gaaccgcgtc?cggctccggc?tccgatccgt 60
cgtcgttctt?ctaactaccg?tgcttacgct?actgaaccgc?acgctaaaaa?aaaatctaaa 120
atctctgctt?ctcgtaaact?gcagctgaaa?actctgctgc?tgcagatcgc?taaacaggaa 180
ctggaacgtg?aagctgaaga?acgtcgtggt?gaaaaaggtc?gtgctctgtc?tactcgttgc 240
cagccgctgg?aactggctgg?tctgggtttc?gctgaactgc?aggacctgtg?ccgtcagctg 300
cacgctcgtg?ttgacaaagt?tgacgaagaa?cgttacgaca?tcgaagctaa?agttactaaa 360
aacatcactg?aaatcgctga?cctgactcag?aaaatcttcg?acctgcgtgg?taaattcaaa 420
cgtccgactc?tgcgtcgtgt?tcgtatctct?gctgacgcta?tgatgcaggc?tctgctgggt 480
gctcgtgcta?aagaatctct?ggacctgcgt?gctcacctga?aacaggttaa?aaaagaagac 540
actgaaaaag?aaaaccgtga?agttggtgac?tggcgtaaaa?acatcgacgc?tctgtctggt 600
atggaaggtc?gtaaaaaaaa?attcgaatct?taa 633
Claims (3)
1, the application of cardiac muscle troponin I in the medicine of preparation inhibition tumor; Wherein, described cardiac muscle troponin I is the SEQ ID No:1 in the sequence table.
2, application according to claim 1 is characterized in that: in the medicine of described inhibition tumor, the effective dose of cardiac muscle troponin I is 1 microgram-1 milligram/kg body weight/sky.
3, application according to claim 1 and 2 is characterized in that: also add one or more pharmaceutically acceptable carriers in the medicine of preparation inhibition tumor.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031098614A CN1294987C (en) | 2003-04-15 | 2003-04-15 | Protein having antitumor function, and high performance expression in vitro |
| PCT/CN2004/000339 WO2004091650A1 (en) | 2003-04-15 | 2004-04-12 | Application of cardiac troponin i in preparing antitumour medicaments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031098614A CN1294987C (en) | 2003-04-15 | 2003-04-15 | Protein having antitumor function, and high performance expression in vitro |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1537633A CN1537633A (en) | 2004-10-20 |
| CN1294987C true CN1294987C (en) | 2007-01-17 |
Family
ID=33163870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031098614A Expired - Fee Related CN1294987C (en) | 2003-04-15 | 2003-04-15 | Protein having antitumor function, and high performance expression in vitro |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1294987C (en) |
| WO (1) | WO2004091650A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245102A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Anti-tumor recombinant protein GPTI and coding gene and application thereof |
| CN107245103A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Anti-tumor recombinant protein IFTI and coding gene and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312173C (en) * | 2004-12-23 | 2007-04-25 | 复旦大学 | Section of synthesized peptide S28-42 of troponin I of human cardiac muscle and application |
| CN102146372B (en) * | 2011-01-24 | 2012-09-05 | 四川农业大学 | Method for cloning complete sequence of goat TNNC2 gene coding region |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837680A (en) * | 1996-02-16 | 1998-11-17 | Children's Medical Center Corporation | Pharmaceutical compositions comprising troponin subunits, fragments and analogs thereof and methods of their use to inhibit angiogenesis |
-
2003
- 2003-04-15 CN CNB031098614A patent/CN1294987C/en not_active Expired - Fee Related
-
2004
- 2004-04-12 WO PCT/CN2004/000339 patent/WO2004091650A1/en unknown
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837680A (en) * | 1996-02-16 | 1998-11-17 | Children's Medical Center Corporation | Pharmaceutical compositions comprising troponin subunits, fragments and analogs thereof and methods of their use to inhibit angiogenesis |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245102A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Anti-tumor recombinant protein GPTI and coding gene and application thereof |
| CN107245103A (en) * | 2016-12-30 | 2017-10-13 | 广西壮族自治区药用植物园 | Anti-tumor recombinant protein IFTI and coding gene and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1537633A (en) | 2004-10-20 |
| WO2004091650A1 (en) | 2004-10-28 |
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