CN1847262A - One kind of protein and its use - Google Patents
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- CN1847262A CN1847262A CN 200510025054 CN200510025054A CN1847262A CN 1847262 A CN1847262 A CN 1847262A CN 200510025054 CN200510025054 CN 200510025054 CN 200510025054 A CN200510025054 A CN 200510025054A CN 1847262 A CN1847262 A CN 1847262A
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Abstract
The present invention discloses one kind of protein derived from snake venom and the protein has function similar to that of activating protein C and is used in preventing and treating pyemia. The present invention also discloses the preparation process of the protein, and the protein is prepared through chromatographic process with combined anion exchange and gel filtering to separate and purify the protein from snake venom. Compared with activating protein C, the present invention has the advantages of wide source, simple production process, low cost, etc.
Description
Technical field
The present invention relates to a kind ofly be used for prevention and treat pyemic protein, specifically a kind ofly be used for prevention and treat pyemic albumen with activated protein C is intimate.
Background technology
Sepsis is a common complication after severe trauma, burn, shock, the major operation, is to cause a human dead common disease kind, and it has high M ﹠ M.(Alerti C, Brun-Buission C, Burchardi H, etal.Epidemiology of sepsis and infection in ICU patients from an international multicentercohort study[J] .Intensive Care Med, 2002,28 (2): 108-121.) Sepsis is meant by the systemic inflammatory response syndrome that infects or have highly suspicious focus of infection to cause, its pathogenic bacteria comprises bacterium, fungi, parasite and virus etc., clinical symptom include but not limited to (1) body temperature＞38 ℃ or＜36 ℃; (2) heart rate＞90 time/minute; Breathe＞20 times/minute or PaCO
2＜32mm Hg; (4) the peripheral blood leucocyte counting＞12 * 10
9/ L,＜4 * 10
9/ L or ripe granulocyte＞10%; (5) organ dysfunction, HT or hypertension.(Wang Zhengguo. Sepsis research overview [J]. Chinese wound magazine, 2003,19 (1): 5-7.)
Since the eighties in 20th century, along with going deep into to the research of Sepsis pathogenesis, develop the various also rises immediately of medicine that Sepsis forms that are intended to suppress, and 80 multinomial clinical trials have been carried out, wherein several medicines likely have carried out the III clinical trial phase, but regrettably only have a medicine (activated protein C) to be developed success.(PoldermanKH and Girbes ARJ.Drug intervention trials in sepsis：divergent results[J].Lancet，2004，363：1721-1723.)。The exploitation of succeeding in developing to new treatment severe sepsis medicine of activated protein C (commodity are called Xigris) provides reference, particularly in natural product, seek, be called 1: PN: CN1847262 SEQID: 1 claimed protein (activated protein C like enzyme) at this with its intimate material.Because it is resistance to activated protein C that 64% patient is arranged in the phlebothrombosis patient approximately, so this part patient is invalid with the Xigris treatment more certainly after suffering from Sepsis, and activated protein C sample material just can remedy the deficiency of Xigris.In addition, the production process complexity of Xigris, production cost is higher, the expense of a course of treatment of each patient is about 6800 dollars, so high price limit its widespread use clinically.More than these reasons make the searching of 1: PN: CN1847262 SEQID: 1 claimed protein and exploitation that very important economy and social effect be arranged.(, Feng Jun, Zhao Wenjie. and activated protein C [J]. external medicine---synthetic drug, biochemical medicine, preparation medicine fascicle, 2002,23 (6): 348～351.)
There is abundant snake venom resource in China, and more than 500 kind of poisonous snake distributing all over the world just has kind more than 150 in China.Snake venom is a kind of mixture, and it contains numerous species and has different bioactive protein, and wherein a lot of protein act on the composition of human blood system.(Markland FS.Snake venoms and the hemostatic system[J] Toxicon, 1998,36 (12): 1749～1800.) still, do not find so far to utilize the snake venom one-component to prevent and treat pyemic report.
Successfully be developed as in treatment severe sepsis medicine and the snake venom based on activated protein C and had the multiple component that acts on blood system, be necessary to propose from snake venom, to seek 1: PN: CN1847262 SEQID: 1 claimed protein, treated the exploitation of Sepsis original new drug.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of be used for the prevention and treat pyemic protein, to overcome the deficiencies in the prior art.
Another technical problem that the present invention need solve provides the application of this albumen in the pyemic medicine of preparation treatment.
The invention discloses a kind of protein, the 1: PN: CN1847262 SEQID: 1 claimed protein of forming by 124 amino-acid residues, external it can significant prolongation human plasma activated partial thromboplastin time again can the special colour developing peptide substrates of hydrolytic activation C albumen, it can significantly reduce the mortality ratio of Sepsis experimental animal in vivo.Above-mentioned 1: PN: CN1847262 SEQID: 1 claimed protein can be also can be reorganization 1: PN: CN1847262 SEQID: 1 claimed protein with genetic engineering means preparation from natural product extraction.The molecular weight of above-mentioned 1: PN: CN1847262 SEQID: 1 claimed protein 15.3kD (± 5000kD).The primary structure of above-mentioned 1: PN: CN1847262 SEQID: 1 claimed protein aminoacid sequence is as shown in Figure 4 formed.Its aminoacid sequence such as SEQ ID NO:1:
Ser Leu Ile Gln Phe Glu Thr Leu Ile Met Lys Val Ala Lys Lys
5 10 15
Ser Gly Ile Phe Trp Tyr Ser Asn Tyr Gly Cys Tyr Cys Gly Trp
20 25 30
Gly Gly Gln Gly Arg Pro Gln Asp Ala Thr Asp Arg Cys Cys Phe
35 40 45
Val His Asp Cys Cys Tyr Gly Lys Val Thr Gly Cys Asp Pro Lys
50 55 60
Met Asp Val Tyr Ser Phe Ser Glu Glu Asn Gly Asp Ile Val Cys
65 70 75
Gly Gly Asp Asp Pro Cys Lys Lys Glu Ile Cys Glu Cys Asp Arg
80 85 90
Ala Ala Ala Ile Cys Phe Arg Asp Gln Leu Thr Asp Tyr Asn Asp
95 100 105
Lys Lys Tyr Trp Ala Phe Gly Ala Lys Asn Cys Pro Gln Glu Glu
110 115 120
Ser Glu Pro Cys
This prevention and treat pyemic protein source in agkistrodon halyx pallas venom (Agkistrodon halys venom), but be not limited to snake venom.
The invention also discloses this proteic preparation method, this method comprises the steps:
(1) to be dissolved in initial damping fluid be in the Tris-HCl damping fluid to snake venom, and 4 ℃ of dialysed overnight are taken out then, and centrifugal, centrifugal condition is 4 ℃, 10000g, and 30min gets supernatant;
(2) carry out anion-exchange chromatography with SOURCE 30Q as chromatography media and separate snake venom, the column volume of anion chromatography is 30ml, and flow velocity is 5ml/min; With 5 times of column volumes of initial damping fluid balance; Applied sample amount is 10ml; Earlier with the initial buffer solution elution of 3 times of column volumes absorbed portion not, re-use gradient elution, gradient liquid consists of initial damping fluid (A), and initial damping fluid+0.2mol/L NaCl damping fluid (B), B be from 0～100%, 20 times of CV of wash-out; Get the collection liquid in the peak 5;
(3) with molecular weight cut-off be 3000 cellulose acetate membrane ultrafiltration and concentration, get concentrated solution and do gel permeation chromatography that chromatography media is Superdex 75pre grade, the gel permeation chromatography column volume is 150ml; Flow velocity is 0.5ml/min; 1 times of column volume of PBS damping fluid balance; Applied sample amount is 2ml; 1.5 times of column volumes of PBS buffer solution elution; Get the collection liquid in the peak 2.
Among the preparation method of 1: PN: CN1847262 SEQID: 1 claimed protein of the present invention most preferably raw material use agkistrodon halyx pallas venom (Agkistrodonhalys), the SOURCE30Q that the anion-exchange chromatography filler uses Sweden Amersham Biosciences company to produce, the Superdex 75pregrade that the gel permeation chromatography filler uses Sweden Amersham Biosciences company to produce.
The be hydrolyzed mensuration of the special colour developing peptide of activated protein C ability of 6 agkistrodon halyx pallas venom component peaks that anion-exchange chromatography obtains, the result shows only has peak 5 components to have tangible hydrolysis L Pefachrome
The ability of PCa3297.
6 agkistrodon halyx pallas venom component peaks that anion-exchange chromatography is obtained mensuration of the special colour developing peptide of activated protein C ability that is hydrolyzed respectively, the result shows only has peak 5 components to have tangible hydrolysis L Pefachrome
The ability of PCa3297
The peak 5 that the anion exchange separation chromatographic separation arrives is behind gel permeation chromatography, the result obtains 2 component peaks, respectively these 2 component peaks are carried out prolonged human blood plasma activated partial thromboplastin time and hydrolysis colour developing peptide test mensuration, test-results shows that each the pipe collection liquid in the peak 2 can have the ability of stronger hydrolysis colour developing peptide again by significant prolongation human plasma activated partial thromboplastin time, and this shows that the component in the peak 2 is a 1: PN: CN1847262 SEQID: 1 claimed protein.
Carry out SDS-PAGE, wherein the concentration of separation gel is 12.5%, and the concentration that concentrates glue is 4%, and the 1: PN: CN1847262 SEQID: 1 claimed protein that obtains is carried out purity detecting and molecular weight determination.SDS-PAGE result is single band, sees accompanying drawing 3.Mobility (Rf) with the lower molecular weight standard protein is an X-coordinate, and the logarithmic value of lower molecular weight standard protein is that ordinate zou carries out regression Calculation, and the molecular weight that draws APCL is 15.3kD.
Sequencing shows that 1: PN: CN1847262 SEQID: 1 claimed protein is made of the sequence table of sequence set prejudice 124 amino acid.
1: PN: CN1847262 SEQID: 1 claimed protein is to the toxicity test of normal mouse, test-results shows: in 72h, all do not observe the death of mouse in any one dosage group, unusual variation takes place in outward appearance sign and the behavioral activity of also not observing mouse simultaneously, therefore can determine the LD of APCL to mouse
50Be lower than 400mg/kg bwt.
1: PN: CN1847262 SEQID: 1 claimed protein behind the injected in mice lipopolysaccharides 24h, begins to observe the death of mouse to the effect of Sepsis experiment mice, continues to observe to 72h.1: PN: CN1847262 SEQID: 1 claimed protein can significantly reduce the Sepsis mortality of mice, and low dose group reduces by 20%, and middle dosage group reduces by 65%, and high dose group reduces by 70%.
Venin-derived protein among the present invention, promptly the purposes of 1: PN: CN1847262 SEQID: 1 claimed protein is to be used for the preparation prevention and to treat pyemic medicine.
Proteinic preparation method venin-derived among the present invention compares with activated protein C, has convenient sources, produce simple, low cost and other advantages.
Description of drawings
Fig. 1 is the anion-exchange chromatography collection of illustrative plates.
Fig. 2 is the gel permeation chromatography collection of illustrative plates.
Fig. 3 is that the SDS-PAGE of 1: PN: CN1847262 SEQID: 1 claimed protein detects collection of illustrative plates.
Embodiment
Embodiment 1 anion-exchange chromatography separates agkistrodon halyx pallas venom
Take by weighing the 0.5g agkistrodon halyx pallas venom, be dissolved in 10ml 0.05mol/L, in the Tris-HCl damping fluid of pH7.6 (initial damping fluid), this damping fluid dialysed overnight (4 ℃), take out then, centrifugal (4 ℃, 10000g, 30min), abandon precipitation, collect supernatant, carry out anion-exchange chromatography, chromatography condition is as follows:
Chromatography media: SOURCE 30Q; Column volume: 30ml; Flow velocity: 5ml/min; Column equilibration: with 5 times of column volumes of initial damping fluid balance; Applied sample amount: 10ml; Wash-out: earlier with the initial buffer solution elution of 3 times of column volumes absorbed portion not, re-use gradient elution, gradient liquid consists of initial damping fluid (A), and initial damping fluid+0.2mol/L NaCl damping fluid (B), B be from 0～100%, 20 times of CV of wash-out; Collect: the 8.0ml/ pipe.
Adopt SOURCE 30Q anion-exchange chromatography medium that agkistrodon halyx pallas venom is separated, obtained 6 components, concrete outcome is seen accompanying drawing 1 and table 1.
The integral result of each component peaks of table 1 anion-exchange chromatography
| Retention volume (ml) | Peak face (mAU*ml) | Long-pending peak area per-cent (%) |
| 500.50 (peaks 5) 616.74 (peak 6), (33.19 peak 1) 135.97 (peak 2) 271.48 (peaks 3) 363.86 (peak 4) | 8261.61 2052.36 2139.33 7371.14 10790.87 4171.17 | 23.75 5.90 6.15 21.19 31.02 11.99 |
The mensuration of embodiment 2 prolonged human blood plasma activated partial thromboplastin times
Get thrombokinase (white bole-kephalin) suspension 0.1ml, add the normal people and mix fresh plasma 0.1ml, control group adds physiological saline 0.1ml, mixing, 5min is hatched in 37 ℃ of water-baths, adds 0.025mol/L calcium chloride solution 0.1ml, start stopwatch immediately, in the record clotting time, test group is except that using the agkistrodon halyx pallas venom parting liquid to replace the physiological saline, and other is identical with control group.[referring to, Wang Hongli. thrombus and hemostasis inspection technology, first version [M], Shanghai: Shanghai science tech publishing house, 1992,66-67.]
6 agkistrodon halyx pallas venom component peaks that anion-exchange chromatography is obtained are measured respectively, the result shows that peak 5 components have tangible prolongation activated partial thromboplastin time, wherein the time lengthening of the comparable contrast of a pipe collection liquid is 10 times, and other 5 component peaks only have the ability of more weak prolongation activated partial thromboplastin time.
The mensuration of the special colour developing peptide of embodiment 3 hydrolytic activation C albumen ability
Get 4 μ mol/L Pefachrome
PCa3297 (the special colour developing peptide of activated protein C, available from Sweden Pentapharm company) 0.2ml, add the 0.05mol/L Tris-imidazole buffer 1.65ml of pH8.4, add different agkistrodon halyx pallas venom parting liquid 0.2ml again, mixing, the variation (measuring 3min) of surveying absorption value at 405nm.Reference solution is for replacing the above-mentioned solution composition of snake venom solution with the 0.2ml aqueous solution.[referring to, Martinoli JL, Stocker K.Fast functionalprotein C assay using Protac, a novel protein C activator[J] .Thromb Res, 1986,43 (3): 253～264.]
6 agkistrodon halyx pallas venom component peaks that anion-exchange chromatography is obtained are measured respectively, and the result shows only has peak 5 components to have tangible hydrolysis L Pefachrome
The ability of PCa3297.
Embodiment 4 gel permeation chromatography purifying 1: PN: CN1847262 SEQID: 1 claimed proteins
Merging through the anion exchange separation chromatographic separation to peak 5 in each pipe collect liquid, be 3000 cellulose acetate membrane ultrafiltration and concentration with molecular weight cut-off, get concentrated solution and do gel permeation chromatography.Chromatography condition is as follows:
Chromatography media: Superdex 75pre grade; Column volume: 150ml; Flow velocity: 0.5ml/min; Column equilibration: PBS damping fluid (0.15mol/L NaCl, 0.2mol/L Na
2HPO
4-NaH
2PO
4, pH 7.0) and 1 times of column volume of balance; Applied sample amount: 2ml; Wash-out: 1.5 times of column volumes of PBS buffer solution elution; Collect: the 3ml/ pipe.
Tomographic results:
The peak 5 that the anion exchange separation chromatographic separation arrives is behind gel permeation chromatography, and the result obtains 2 component peaks, and concrete outcome is seen accompanying drawing 2 and table 2.
The integral result of each component peaks of table 2 gel permeation chromatography
| Retention volume (ml) | Peak face (mAU*ml) | Long-pending peak area per-cent (%) |
| (58.34 peak 1) 73.06 (peak 2) | 1374.8 6822.8 | 16.75 83.14 |
Respectively these 2 component peaks are carried out prolonged human blood plasma activated partial thromboplastin time and hydrolysis colour developing peptide test mensuration, test-results shows that each the pipe collection liquid in the peak 2 can have the ability of stronger hydrolysis colour developing peptide again by significant prolongation human plasma activated partial thromboplastin time, and this shows that the component in the peak 2 is a 1: PN: CN1847262 SEQID: 1 claimed protein.
The analyzing activated C albumen of embodiment 5 sodium dodecyl sulfate-polyacrylamide gel electrophoresiss (SDS-PAGE) sample enzyme
According to document [referring to, Li Jianwu, Yu Ruiyuan, Yuan Mingxiu etc. Biochemistry Experiment principle and method [M]. Beijing: BJ University Press, 1997,174～176.] carry out SDS-PAGE, wherein the concentration of separation gel is 12.5%, the concentration that concentrates glue is 4%, and the 1: PN: CN1847262 SEQID: 1 claimed protein that obtains is carried out purity detecting and molecular weight determination.
SDS-PAGE result is single band, sees accompanying drawing 3.Mobility (Rf) with the lower molecular weight standard protein is an X-coordinate, and the logarithmic value of lower molecular weight standard protein is that ordinate zou carries out regression Calculation, and the molecular weight that draws APCL is 15.3kD.Among the figure, M is the standard protein molecule; No. 1 is 1: PN: CN1847262 SEQID: 1 claimed protein.
The amino acid composition sequence of embodiment 6 1: PN: CN1847262 SEQID: 1 claimed proteins is measured
Sequencing shows that 1: PN: CN1847262 SEQID: 1 claimed protein is made of the sequence table of sequence set prejudice 124 amino acid.
Embodiment 7 1: PN: CN1847262 SEQID: 1 claimed proteins are to the effect of Sepsis laboratory animal
(1) lipopolysaccharides is to mouse LD
100Mensuration
Get 30 of 18～22g mouse, per 10 are divided into one group, male and female half and half, respectively with dosage tail vein injection lipopolysaccharides (the E.coli 055:B5 of 2mg/kg bwt (body weight), 10mg/kg bwt and 50mg/kg bwt, available from Sigma company) solution, injection speed is 0.5ml/min, observes the death condition of respectively organizing mouse in 72h.
After mouse is injected lipopolysaccharides 24h, begin to observe the death of mouse, continue to observe to 72h.The concrete death condition of respectively organizing mouse sees Table 3.Can find out that mouse is lower to the susceptibility of lipopolysaccharides from table, have only when the dosage of lipopolysaccharides reaches 50mg/kg bwt that its lethality rate just reaches 100%, the lipopolysaccharides that therefore definite this test is measured is to mouse LD
100Be 50mg/kg bwt, this dosage is equivalent to the lipopolysaccharides of every injected in mice 1.0mg approximately.
Table 3 LPS is to mouse LD
100Measurement result
| LPS dosage (mg/kg bwt) | Dead mouse number (only) | Mortality ratio (%) |
| 2 10 50 | 0 2 10 | 0 20 100 |
(2) 1: PN: CN1847262 SEQID: 1 claimed protein is to the toxicity test of normal mouse
Get 24 of 18～22g mouse, per 6 are divided into one group, and male and female half and half are respectively with the dosage tail vein injection APCL solution of 4mg/kg bwt, 40mg/kgbwt, 100mg/kg bwt and 400mg/kg bwt, injection speed is 0.5ml/min, observes the death condition of respectively organizing mouse in 72h.
Test-results shows: in 72h, all do not observe the death of mouse in any one dosage group, unusual variation takes place in outward appearance sign and the behavioral activity of also not observing mouse simultaneously, therefore can determine the LD of APCL to mouse
50Be lower than 400mg/kg bwt.
(3) 1: PN: CN1847262 SEQID: 1 claimed protein is to the effect of Sepsis experiment mice
Get 80 of 18～22g mouse, per 20 are divided into one group, and male and female half and half are divided into four groups.First group of dosage tail vein injection lipopolysaccharides solution with 50mg/kgbwt; Second group earlier with the 1: PN: CN1847262 SEQID: 1 claimed protein solution of the dosage tail vein injection 0.4mg/ml of 2mg/kg bwt, behind the 30min again with the lipopolysaccharides solution of the dosage tail vein injection 2mg/ml of 50mg/kg bwt; The 3rd group earlier with the 1: PN: CN1847262 SEQID: 1 claimed protein solution of the dosage tail vein injection 0.4mg/ml of 4mg/kg bwt, behind the 30min again with the lipopolysaccharides solution of the dosage tail vein injection 2mg/ml of 50mg/kg bwt; The 4th group earlier with the 1: PN: CN1847262 SEQID: 1 claimed protein solution of the dosage tail vein injection 0.4mg/ml of 8mg/kg bwt, behind the 30min again the lipopolysaccharides injection of solution speed with the dosage tail vein injection 2mg/ml of 50mg/kg bwt be 0.5ml/min, in 72h, observe the death condition of respectively organizing mouse, calculate mortality ratio.
Behind the injected in mice lipopolysaccharides 24h, begin to observe the death of mouse, continue to observe to 72h.Each death condition of organizing mouse sees Table 4.Can find out that 1: PN: CN1847262 SEQID: 1 claimed protein can significantly reduce the Sepsis mortality of mice from table, low dose group reduces by 20%, and middle dosage group reduces by 65%, and high dose group reduces by 70%.
Table 4 activated protein C sample enzyme reduces the test-results of Sepsis mouse death rate
| Group | APCL dosage (mg/kg bwt) | Dead mouse number (only) | Mortality ratio (%) |
| 1 | 0 | 20 | 100 |
| 2 | 2 | 16 | 80 |
| 3 | 4 | 7 | 35 |
| 4 | 8 | 6 | 30 |
Embodiment 8
In embodiment 7 times " with the effect of 1: PN: CN1847262 SEQID: 1 claimed protein ", inject lipopolysaccharides, inject 1: PN: CN1847262 SEQID: 1 claimed protein again and do replacement, also obtained similar test-results with elder generation to the Sepsis experiment mice.
Sequence table
＜110〉Shanghai Institute of Pharmaceutical Industry
＜120〉a kind of protein and uses thereof
<130>SPI058153
<160>1
<170>Patent In Version2.1
<210>1
<211>124
<212>PRT
＜213〉artificial sequence
<220>
<221>CHAIN
<400>1
Ser Leu Ile Gln Phe Glu Thr Leu Ile Met Lys Val Ala Lys Lys
5 10 15
Ser Gly Ile Phe Trp Tyr Ser Asn Tyr Gly Cys Tyr Cys Gly Trp
20 25 30
Gly Gly Gln Gly Arg Pro Gln Asp Ala Thr Asp Arg Cys Cys Phe
35 40 45
Val His Asp Cys Cys Tyr Gly Lys Val Thr Gly Cys Asp Pro Lys
50 55 60
Met Asp Val Tyr Ser Phe Ser Glu Glu Asn Gly Asp Ile Val Cys
65 70 75
Gly Gly Asp Asp Pro Cys Lys Lys Glu Ile Cys Glu Cys Asp Arg
80 85 90
Ala Ala Ala Ile Cys Phe Arg Asp Gln Leu Thr Asp Tyr Asn Asp
95 100 105
Lys Lys Tyr Trp Ala Phe Gly Ala Lys Asn Cys Pro Gln Glu Glu
110 115 120
Ser Glu Pro Cys
Claims (2)
1, a kind of protein is characterized in that, its aminoacid sequence is shown in SEQ ID NO:1.
2, albumen according to claim 1 is in preparation prevention with treat application in the pyemic medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025054 CN1847262A (en) | 2005-04-13 | 2005-04-13 | One kind of protein and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025054 CN1847262A (en) | 2005-04-13 | 2005-04-13 | One kind of protein and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1847262A true CN1847262A (en) | 2006-10-18 |
Family
ID=37077028
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510025054 Pending CN1847262A (en) | 2005-04-13 | 2005-04-13 | One kind of protein and its use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1847262A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692511A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Kit (Developing substrate method) for testing protein C (PC) |
-
2005
- 2005-04-13 CN CN 200510025054 patent/CN1847262A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692511A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Kit (Developing substrate method) for testing protein C (PC) |
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