JP2000046825A - Blood test container - Google Patents

Abstract

(57) [Problem] To provide a blood test container enclosing a blood coagulation promoter which can coagulate blood in a short time and has stable quality for a long period of time. SOLUTION: This blood test container comprises a bottomed tube whose one end is open and the other end is closed, and a stopper which seals the opening of the bottomed tube, comprising a protein folding factor, a peptide and / or a peptide. A blood test container in which a blood coagulation promoter is enclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood test container used in clinical test fields such as serum biochemical test and serum immunological test.

[0002]

2. Description of the Related Art Blood tests are generally performed for the purpose of preventing and diagnosing diseases, but most of the blood tests are serum tests such as serum biochemical tests, serum immunological tests, and blood cell tests. Serum required for those tests is usually separated from blood clots having different specific gravities by centrifugation, after coagulation of blood collected in a blood test container, using a pipette, or by decantation, and collected. I have.

Conventionally, blood test containers used for collecting blood have been made of glass or synthetic resins such as polystyrene, polymethyl methacrylate, polyethylene, and polyethylene terephthalate. However, blood collected from a subject requires a relatively long time to coagulate, and even a glass container which is considered to have the shortest blood coagulation time requires 40 to 60 minutes, and a synthetic resin container. It took more than 4 hours.

Conventionally, it usually takes two days or more after a blood sample is collected to obtain a test result, and the subject must return to the hospital to know the result. Also, blood coagulation time can be problematic, especially when urgent testing is required. Therefore, not only can important information be obtained at the time of consultation, but also effective treatment can be performed earlier, so that the waiting time for consultation is 30 to
It is desired that blood be collected and tested in about 60 minutes to obtain a result. Furthermore, in recent years, with the progress of inspection equipment, a quicker inspection has been further demanded.

In order to perform blood coagulation in a short time, blood coagulants have been studied. Blood clotting is initiated by the activation of blood coagulation factor XII, followed by a number of reaction steps and a complex pathway by which fibrinogen is eventually converted to fibrin.

[0006] Japanese Patent Application Laid-Open No. 5-157747 discloses an enzyme-based drug such as thrombin or snake venom enzyme, which promotes a reaction of converting fibrinogen into fibrin, which is the final stage of a blood coagulation reaction system, as a component that promotes blood coagulation. It is described to be used. However, when a synthetic resin container is used as a blood test container, the blood coagulation time is reduced to about 10 minutes, but during transportation, particularly during transportation or storage in summer, due to heat or moisture. There was a problem that thrombin was inactivated and the quality was unstable. Also,
Other blood coagulation factors have a large blood coagulation promoting effect,
There is a problem that the stability is poor and the quality is unstable when stored in a blood test container.

[0007]

SUMMARY OF THE INVENTION In view of the above situation, the present invention can coagulate blood in a short time, and
An object of the present invention is to provide a blood test container enclosing a blood coagulation accelerator having a stable quality for a long period of time.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a blood test container comprising a bottomed tube having one end opened and the other end closed, and a stopper for sealing the opening of the bottomed tube. A blood test container in which a factor and a peptide and / or a peptide-like blood coagulation promoter are enclosed.

Hereinafter, the present invention will be described in detail. The blood test container of the present invention contains a protein folding factor and a peptide and / or a peptide-like blood coagulation promoter. The present invention maintains and repairs the three-dimensional structure of a blood coagulation promoter which is easily inactivated by the influence of heat or the like by maintaining a protein folding factor together with the blood coagulation promoter, thereby maintaining the function of the blood coagulation promoter. It is.

The above-mentioned protein folding factor is not particularly limited as long as it has an action of maintaining the three-dimensional structure of the protein or an action of rapidly repairing a structural change, and includes molecular chaperone, peptidylprolyl-cis-trans-isomerase ( Hereinafter, also referred to as “PPIase”) and the like are suitably used. In the present invention, it is more effective to use a protein disulfide isomerase (hereinafter, also referred to as “PDI”) in combination. PDI is a general term for a group of enzymes involved in the formation of disulfide bonds contained in the three-dimensional structure of a protein.

[0011] The molecular chaperone is a kind of heat shock protein and is produced when cells are exposed to various environmental stresses such as temperature change. The molecular chaperone exists widely regardless of prokaryotes and eukaryotes, and GroE is particularly well known as a molecular chaperone produced from Escherichia coli.

It has been clarified that the above-mentioned GroE is nonspecifically involved in the formation of a three-dimensional structure of a protein in the presence of ATP regardless of the type of the protein. For example, GroEL, which is a component of the above GroE, has a characteristic structure composed of a total of 14 subunits in which a donut-shaped structure in which seven subunits are connected in a ring shape overlaps in two stages. It is known that GroEL captures a denatured protein in a concave portion of a donut structure, and efficiently folds into a protein having a correct tertiary structure with the consumption of nucleotides such as ATP and the binding of GroES which is a cofactor.

[0013] In addition to the above, examples of the molecular chaperone include Dnak of Escherichia coli and Ssal-4 of yeast.
p, Ssclp, Kar2pBip, Grp78; mammalian hsc70, HSP58, HSP10, HSP9
0; mold HSP60; plant-derived rubisco;
Escherichia coli SecB and the like.

The above-mentioned PPIase is an enzyme having an action of promoting the rotation of the peptide bond on the N-terminal side of the proline residue which is a constituent amino acid in the polypeptide chain. The N-terminal peptide bond of this proline residue cannot rotate freely, and spontaneous cis-trans isomerization hardly occurs. As a result, the conversion of the protein chain in which the structural change has occurred to the normal structure is limited by the cis-trans isomerization reaction of proline and the like, and spontaneous regeneration is inhibited. On the other hand, since the PPIase promotes the rotation of the peptide bond on the N-terminal side of the proline residue, it can promote the regeneration of the higher-order structure of the protein and can regenerate the denatured protein.

Examples of the PPIase include cyclophilin (protein binding to immunosuppressant cyclosporin A) and FKB.
P (protein binding to FK506, an immunosuppressant)
Of the types, any PPIase can be used.

The PPIase is not particularly limited, and examples of eukaryote-derived PPIase include those derived from pig.
9KPPIase, nanaA from Drosophila
Products, cyclophilin derived from higher plants such as tomato and corn, cyclophilin derived from Neurospora crassa, Nc
-FKBP, yeast-derived cyclophilin (Cycle
hilin), CRG protein, Sc-FKBP and the like. Also, for example, Cyclophilin-type PPIase (derived from calf rib gland) and FKBP-type PP available from Sigma
Iase (derived from human) and the like can be easily obtained.

Among the above PPIases, those derived from eubacteria are not particularly limited. E. coli-derived PPIase-α, PPIase-β, and the like (Protein Nucleic Acid Enzyme Vol. 36, No. 11 (199)
1)).

As the above-mentioned folding factors such as molecular chaperones and PPIases, those derived from eukaryotes, eubacteria or archaebacteria can be used. Preferably, they are derived from archaebacteria.

Among the archaeal-derived molecular chaperones,
A molecular chaperone belonging to HSP60 is obtained from Gross bacillus
It has been found to have a similar effect to E, but to a similar effect without the cofactor corresponding to GroES. Therefore, when attempting to obtain a large amount of molecular chaperones derived from archaebacteria, workability is excellent because the labor for preparing cofactors can be omitted.

On the other hand, PPIases derived from archaebacteria have been studied arbitrarily. As a result, PPIases have not only the N-terminal peptide bond cis-trans isomerization effect of the proline residue originally possessed by PPIase, but also the molecular aggregation of protein like chaperonin. It was found that they also have an inhibitory effect. Therefore, by using PPIase derived from archaebacteria, it is possible to more effectively perform the protein folding action.

The types of protein folding factors derived from archaebacteria are less than those of eubacteria such as Escherichia coli. Therefore, since archaea express protein folds with a minimum of types of fold factors, it is expected that the specificity of each fold factor for the protein is low and versatility is excellent. The protein folding effect is expected to be further improved by combining the respective folding factors of molecular chaperone, PPIase and PDI.

Among the archaea, it is more preferable to use archaea belonging to thermophilic archaea. Folding factors derived from thermophilic archae have excellent thermostability and are therefore advantageous when the blood coagulation promoter is exposed to higher temperatures. For example, among the thermophilic archaea, Methanococcus thermolystroficus (Methanococcus t)
Protein folding factors such as molecular chaperones and PPIase obtained from H. hemolithrophycus have excellent thermal stability and are hardly deactivated even when left at high temperatures such as 90 ° C and 100 ° C. Previously available molecular chaperones and PPIases were
It could not be used under high temperature conditions because it was almost inactivated at ℃. However, the above Methanococcus thermolystrophycus (Methanococcus)
s themolithrophycus) is obtained at medium temperatures such as 37-65 ° C,
It can be used under high temperature conditions such as 80 to 90 ° C.
In addition, Methanococcus voltae (Metanoco
Since Ccus voltae is a mesophilic bacterium, a protein folding factor derived from the bacterium has an effect of stabilizing the HBs antigen in a wide range of 4 to 40 ° C. Furthermore, a feature of these thermostable protein folding factors is that their activity is preserved for long periods.

The thermophilic archaebacteria are a general term for archaea that grow in a high temperature extreme environment such as a hot spring area or a seabed hot water bed, and are usually bacteria that grow in a temperature environment of 55 ° C. or higher.

The thermophilic archaebacteria is not particularly limited, and may be, for example, thermoplasma.
a) genus, Sulfolobus,
Genus Sulfurococcus,
The genus Desulforobus, the genus Acidinus, the genus Thermoproteus, the genus Desulfurococcus, the genus Thermofilum (genus Thermofilum), the genus Thermodiscus, Thermodiscus dip The genus Staphylothermus, the genus Thermococcus, the genus Pyrococcus (P
genus yrococcus, arceoglobus (Arc)
haeoglobus, Pyrobacrum (Pylob)
aculum), Sulfuria
sphaera), Aeropyr
um), Igneococcus
s) genus, Thermospherer
a) Genus, Hyperthermu (Hyperthermu)
s) Genus, Sulfophococcus
genus bococcus, Caldococcus
occus), Ferroglobu
s) Genus, Picophilus
And the genus Methanococcus.

The archaea used in the present invention are preferably archaea belonging to the genus Methanococcus. Methanococcus (Methanococcus)
ococcus) include Methanococcus thermolystrophycus (Methanococcus t).
hermolithotropicus; ATCC3
5097), Methanococcus voltae (Methan)
ococcus voltae; ATCC3327
3), Methanococcus deltae
ctus deltae; ATCC 35294), Methanococcus frischus (Methanococcus).
frisius; ATCC 43340), Methanococcus maripardis (Methanococcus)
maricipaludis; ATCC 43000), Methanococcus bannieri (Methanococcus)
vannielii; ATCC35089). Among them, Methanococcus thermolystroficus (Methanococcus thermolicus)
throphicus or Methanococcus voltae is more preferred.

In the present invention, the above-mentioned folding factors can be obtained by directly purifying those prepared from eukaryotes, eubacteria or archaebacteria. In addition, those expressed from DNA prepared from eukaryotes, eubacteria or archaebacteria can be used.

As a method for preparing the above-mentioned folding factor directly from a eukaryote, an eubacteria or an archaebacteria, a eukaryote, an eubacteria or an archaebacteria is cultured under a suitable medium and culture conditions, and the cultured cells are cultured. After crushing, a method obtained by a conventionally known purification method such as ion exchange chromatography and hydrophobic chromatography can be used.

In the present invention, it is necessary to clone a DNA base sequence encoding the above-described eukaryotic, eubacterial or archaeal folding factor and incorporating it into a fast-growing organism such as Escherichia coli or yeast. It is preferable because it can be produced. As a specific method, it is sufficient to apply an ordinary method such as a gene experiment protocol described in “Guide to Neoplastic Chemistry Experiment” (Chemical Doujinsha).

Examples of the DNA base sequence include, for example, Methanococcus thermolystroficus (Methanan).
ococcus thermolithotrophi
cus) -derived PPIase is described in JP-A-10-91.
Japanese Patent Application No. Hei 9-93, published in Japanese Patent Application Publication No.
No. 350623. Methanococcus thermolysotrophycus (Methanococcu)
s thermomotropicus) -derived chaperonin, as SEQ ID NO: 1 in the sequence listing,
The amino acid sequence is shown as SEQ ID NO: 2 (Furutani, M. et al., J. B.
iol. Chem. , 1998, 273, 2839.
9). In addition, the DNA sequence of PPIase derived from Methanococcus thermolystrophicus was replaced by SEQ ID NO: 3 in the sequence listing.
And its amino acid sequence is shown in SEQ ID NO: 4.

Specifically, as the archaea, Methanococcus thermolystroficus (Methanococ) is used.
cus thermolitrophicus)
The following method can be adopted as a method for obtaining this chaperonin using That is, the plasmid pETTSI deposited with the National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-16438 is
A plasmid containing a gene encoding a thermolystrophycus chaperonin, which is used as a template and amplified by a PCR method using a primer containing an appropriate restriction enzyme site according to a conventional method. A gene fragment obtained from the PCR product is inserted into a plasmid containing a resistance marker, and Escherichia coli and yeast are transformed with the recombinant plasmid. After culturing the transformant and disrupting the cultured cells, chaperonin of Methanococcus thermolystrophycus can be obtained by a conventionally known purification method such as ion exchange chromatography and hydrophobic chromatography.

As a method for confirming the molecular chaperone,
Since molecular chaperones have ATPase activity, La
nzetta P .; A, Anal. Biochem,
No. 100, pp. 95-97, by using the ATPase activity measurement method.

The molecular chaperone is composed of subunit 14
~ 16 units react as oligomers forming a donut-shaped two-stage structure. That is, the denatured protein binds to the oligomer, and as a result of the folding reaction, the protein returns to a normal structure, and then is released from the oligomer molecule chaperone.

In order to form and maintain the oligomer structure of the above molecular chaperone and to exhibit the activity, usually, nucleotides such as magnesium chloride (Mg ²⁺ ), ammonium sulfate, and ATP (adenosine triphosphate) are used in a solution.
In addition, potassium chloride is required. In particular, ATP
It is an essential factor when dissociating molecular chaperones and proteins. In addition, the above ammonium sulfate is a factor necessary for oligomer formation and maintenance, and potassium chloride is required for expression of the activity. The concentration of magnesium chloride, ammonium sulfate, ATP, and potassium chloride used in the reaction solution is not particularly limited. For example, magnesium chloride (Mg ²⁺ ) is 1 to 200 mM, ammonium sulfate and potassium chloride are 100 to 500 mM, and ATP is a molecule. The concentration is preferably equal to or higher than the concentration of the chaperone, for example, in the range of 0.5 to 10 mM.

The above-mentioned PPIase activity can be confirmed by a coupling assay with an α-chymotrypsin reaction. For example, in a HEPES buffer (pH 7.8), N-su in which the N-terminal is modified with an N-succinyl group and the C-terminal is modified with p-nitroanilide, respectively.
c-Ala-Ala-Ala-Phe-pNA or N-
Using a peptide of suc-Ala-Ala-Leu-Phe-pNA (final concentration: 17 μM) as a substrate, a sample is injected into a cell, incubated at 25 ° C., and the reaction is started by adding a chymotrypsin solution. The enzyme activity can be measured by monitoring the increase in absorption at 390 nm due to the release of pNA under stirring throughout. As the peptide serving as the substrate, trade name “Ns
uc-Ala-Ala-Pro-Phe-p-nitr
Oanilide "(manufactured by Sigma) can also be used.

The protein folding factor is enclosed in a blood test container together with a blood coagulation promoter. The blood coagulation promoter is a peptide and / or a peptide-like agent, and is preferably a blood coagulation factor and a bond between Arg and any amino acid residue of a peptide chain and / or Lys and any amino acid residue. A hydrolase that hydrolyzes a bond with a group. In the present specification, the term “peptide and / or peptide-like blood coagulation promoter” means a peptide or protein blood coagulation promoter, and includes other components such as sugar chains, lipids, and metal ions. Include those exhibiting a blood coagulation-promoting action by binding or forming a complex or the like with these other components.

Examples of the blood coagulation factor include blood coagulation factor X and blood coagulation factor Xa.
Examples of the hydrolase include serine proteases such as trypsin, thrombin, and snake venom thrombin-like enzymes; thiol proteases such as cathepsin B and ficin; metal proteases such as kininase I;
In particular, serine protease is preferably used.

When the amount of the peptide and / or the peptide-like blood coagulation promoter is small, blood coagulation time may be prolonged or blood coagulation may be incomplete, and when the amount is large, the test value may be adversely affected. So 1 mL of blood
0.1 to 100 units (IU) per unit are preferred,
0.5-50 units (IU) are more preferred.

The blood test container used in the present invention comprises a bottomed tube whose one end is open and the other end is closed, and a plug that seals the opening of the bottomed tube. Examples of the bottomed tube include those made of plastic and glass,
Examples of the material include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; thermosetting resins such as unsaturated polyester resins, epoxy resins, and epoxy-acrylate resins; cellulose acetate, propionic acid Modified natural resins such as cellulose, ethyl cellulose and ethyl chitin; silicate glass such as soda lime glass, phosphosilicate glass, borosilicate glass and the like; glass such as quartz glass; and any of those containing these as main components are used.

The bottomed tube may be partially colored on its outer surface for identification, and may be provided with a scale for measuring the amount of blood collected. It is preferable that the bottomed tube is coated with a silicon coating on the inner surface of the bottomed tube before enclosing the blood coagulation promoter and the protein folding factor.

The plug is not particularly limited as long as it seals the opening of the bottomed tube, and includes, for example, a butyl rubber plug having high gas barrier properties.

The method for producing the blood test container of the present invention can be prepared, for example, as follows.
That is, the blood coagulation promoter is diluted with a suitable buffer, and this is used as a blood coagulation promoter. As the buffer, an optimal buffer for the blood coagulation promoter is appropriately selected. For example, a buffer generally used such as a phosphate buffer, a Tris buffer, a Good buffer, and a glycine buffer is used. A liquid is preferably used. The three-dimensional structure of the blood coagulation promoter in the solution is normalized by adding the protein folding factor to the blood coagulation promoter and incubating for a predetermined time.

The molar concentration of the protein folding factor in the above solution is 0.05% of the molar concentration of the blood coagulation promoter.
It is preferably up to 500 times. If it is less than 0.05 times, the expected effect cannot be obtained, and if it exceeds 500 times, a specific reaction at the time of examination may be prevented. More preferably,
It is 0.2 to 100 times the molar concentration of the blood coagulation promoter.

The blood coagulation promoting solution containing the protein folding factor is sprayed on the inner surface of the bottomed tube by a spray or the like,
The blood test of the present invention can be performed by drying by freeze-drying and pressureless drying, or by injecting a blood coagulation promoting solution containing a protein folding factor into a bottomed tube, and then sealing the bottomed tube opening. Container. It is preferable to fill the blood test container with a serum separating agent since the workability during the test can be improved.

The blood test container of the present invention utilizes the function of the protein folding factor to maintain and repair the three-dimensional structure of the protein, and the three-dimensional structure of the blood coagulation promoter which is easily inactivated by the influence of heat or the like. And maintain the function of the blood coagulation promoter. as a result,
A blood test container that can coagulate blood in a short time and that has stable quality for a long period of time can be produced.

[0045]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Reference Example 1 describes an example of a method for preparing a thermophilic archaebacteria-derived molecular chaperone (chaperonin), but is not limited thereto. Other folding factors can be prepared by known methods.

Reference Example 1Molecular chapero from thermophilic archaea
Preparation of (chaperonin)  (1)Construction of expression plasmid and transfer to E. coli
Formation  Methanococcus thermolystroficus (Metha
nococcus thermolithotroph
icus) fragment of the gene encoding the chaperonin
FERM P to the Institute of Biotechnology and Industrial Technology, AIST
Plasmid pETT deposited as -16438
Using SI as a template, the forward
Immer and reverse primer of SEQ ID NO: 6 in the sequence listing
It was amplified by the PCR method used.

The fragment amplified and recovered by the PCR method using the above primers is digested with BamHI and NcoI, and then treated with the same restriction enzyme-treated expression vector pET.
Ligation to -11d (Stratigen). Thereafter, this was transformed into BL21 (DE3) strain (manufactured by Stratigen), and an LB medium plate containing 100 μg / mL ampicillin (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl / 1 L)
After culturing at (pH 7.0), colonies were picked up and used as glycerol stock.

(2)Chaperonin from recombinant Escherichia coli
Preparation  LB medium containing 100 μg / mL ampicillin (tripe
Ton 10g, yeast extract 5g, NaCl 10g / 1L
(PH 7.0)) 200 mL of the above expression plasmid
Retain BL21 (DE3) strain (recombinant eubacterium)
Ears were inoculated and precultured at 37 ° C. for 16 hours. This culture
The solution was inoculated into 6 liters of the above LB medium,
The main culture was carried out using a thermometer. Three hours after the start of the main culture, I
PTG (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1 mM
Was added and the culture was continued for 4 hours. After culture is complete,
The bacteria were collected by heart separation and stored frozen at -30 ° C.

Next, the recovered recombinant eubacterium was placed in a 50 m
MTris-HCl buffer (pH 7.5 / 5 mM Mg
(Containing Cl ₂ ) and disrupted by ultrasonication. The supernatant was collected by centrifugation and subjected to heat treatment in a water bath at 65 ° C. for 90 minutes to denature and aggregate the contaminating proteins, and removed by centrifugation. Ammonium sulfate was added to the obtained supernatant to a concentration of 1.3 M, and after performing ammonium sulfate salting-out, the supernatant was washed with TSKgel Ether-5PW.
The product was purified by hydrophobic chromatography using (manufactured by Tosoh Corporation). A fraction containing a protein having a size corresponding to chaperonin was collected, and 25 mM HEPES was collected.
(PH 6.8) -KOH, dialyzed against 5% glycerol buffer. The dialysis solution is TSKgel SuperQ-5P
The product was purified by anion exchange chromatography using a W column (manufactured by Tosoh Corporation). After collecting the fraction containing the protein of the size corresponding to the chaperonin,
This is concentrated by ultraconcentration, and the concentrated solution is TSKgel
Gel filtration using a G3000SWXL column (manufactured by Tosoh Corporation) (flow rate: 0.5 ml / min, developing solution: 25 mM)
HEPES-KOH (pH 6.8), 25 mM MgC
Purification by l _2, 5% Glycerol), to obtain a fraction containing chaperonin derived from Methanococcus thermophila resources Toro ficus.

The molecular weight of chaperonin was determined by SDS-
It was about 60 KDa in PAGE, and was confirmed by tracing a fraction having this molecular weight. Also, since chaperonin has ATPase activity, Lil
l, R. Dowhan, W .; & Wickner, W.C.
The activity was evaluated by using the ATPase activity measurement method described in Cell, No. 60, pages 271-280.

This fraction contains magnesium chloride, AT
P and ammonium sulfate were added at 50 mM and 0.25 respectively.
mM and 300 mM.
Incubated for hours. This solution was subjected to gel filtration again to obtain a 16-mer fraction of chaperonin. This fraction was used in the following Examples and Comparative Examples.

Example 1 200 mg of the chaperonin hexamer prepared in Reference Example 1
And thrombin (Mochida Pharmaceutical Co., Ltd.) 15 mg (1600 U)
Of 50 mM HEPES-KOH buffer / (50 mM
PH 7.4 containing MgCl ₂ and 0.25 mM ATP)
To a total amount of 10 g. After spraying 10 mg of this aqueous solution into a tubular container whose inner surface is coated with silicon,
It was air-dried to prepare a blood test container.

Immediately after the preparation, the blood coagulation time was measured according to the following method for the blood test containers after storage at 25 ° C. for 7 days, 14 days, and 30 days. In addition, as a temperature load test, the prepared blood test containers were placed at 25 ° C. for 10 minutes and at 40 ° C. for 10 minutes, respectively.
For 30 minutes at 60 ° C. and 30 minutes at 60 ° C., and the blood coagulation time was measured in the same manner. Table 1 shows the results.

[0054]Blood clotting time measurement method  8 mL of blood of a healthy person is collected in the blood test container, and collected.
Measure the time required for blood to clot from the end of the collection
Was. Blood coagulation can be determined by tilting the blood test container
Does not move and blood does not flow even if it is kept upside down
It was time. Then, the coagulated blood was heated at 25 ° C. and 1300
G (2500 rpm) for 5 minutes to separate
The presence or absence of fibrin in the serum was visually observed.

Example 2 Methanococcus thermolysomicus used in Example 1
methanococcus voltae (Methanococcus voltae)
The investigation was carried out according to Example 1 except that chaperonin derived from C. cubae (ccus voltae) was used. The results are shown in Table 1.

Example 3 Methanococcus thermolysophilus used in Example 1
5 mg of PPIase derived from the same fungus obtained according to the preparation method described in Japanese Patent Application No. 9-235999, instead of chaperonin derived from T. tropics.
50 mM MgCl in M HEPES-KOH buffer
_The study was carried out in the same manner as in Example 1 except that ₂ and 0.25 mM ATP were not contained. The results are shown in Table 1.

Example 4 In place of thrombin used in Example 1, 1 mg (80%) of human-derived blood coagulation factor Xa (manufactured by SERBIO) was used.
0U) Except that it was used, the study was conducted in the same manner as in Example 1. The results are shown in Table 1.

Comparative Example 1 15 mg of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to 50 mM HE.
It was dissolved in a PES-KOH buffer to make a total amount of 10 g. 10 mg of this aqueous solution was sprayed on a tubular container whose inner surface was coated with silicon, and then air-dried to prepare a blood test container. The examination was carried out in the same manner as in Example 1. The results are shown in Table 1.

Comparative Example 2 In place of thrombin used in Comparative Example 1, 1 mg (80%) of human-derived blood coagulation factor Xa (manufactured by SERBIO) was used.
0U) The study was performed in the same manner as in Comparative Example 1 except that it was used. The results are shown in Table 1.

[0060]

[Table 1]

[0061]

As described above, the present invention can coagulate blood in a short time by using a peptide and / or a peptide-like blood coagulation promoter and a protein-folding factor in combination, and The present invention can provide a blood test container in which a blood coagulation accelerator having a stable quality over a long period of time is enclosed.

[0062]

[Sequence list] <110> SEKISUI CHEMICAL CO., LTD. Marine Biotechnology Institute Co., Ltd. <120> Blood test container <130> 99P01525 <150> JP P1998 -144507 <151> 1998-05-26 <160> 6 <210> 1 <211> 1680 <212> DNA <213> Methanococcus thermolithotrophicus DSM 2095 strain <220> <221> CDS <222> 32..1663 <400 > 1 tctaatacac ttataaaaaa ggggtgaaat c atg gca gct aac cag cca gta 52 Met Ala Ala Asn Gln Pro Val 1 5 gta gta tta cct gaa aac gta aaa aga ttt atg gga aga gat gct caa 100 Val Val Leu Pro Glu Asn Val Lys Arg Phe Met Gly Arg Asp Ala Gln 10 15 20 aga atg aac atc tta gca gga aga att atc ggt gag aca gta aga tcc 148 Arg Met Asn Ile Leu Ala Gly Arg Ile Ile Gly Glu Thr Val Arg Ser 25 30 35 aca tta ggt cca aag gga atg gac aaa atg tta gta gat gat tta ggt 196 Thr Leu Gly Pro Lys Gly Met Asp Lys Met Leu Val Asp Asp Leu Gly 40 45 50 55 gac att gtt gtt aca aat gac ggt gtt aca atc tta aaa gaa atg agt 244 Asp Ile Val Val T hr Asn Asp Gly Val Thr Ile Leu Lys Glu Met Ser 60 65 70 gta gaa cac cca gca gct aaa atg tta att gaa gtt gca aaa aca caa 292 Val Glu His Pro Ala Ala Lys Met Leu Ile Glu Val Ala Lys Thr Gln 75 80 85 gaa aaa gaa gtt gga gac ggt aca aca aca gct gtt gtt atc gct ggt 340 Glu Lys Glu Val Gly Asp Gly Thr Thr Thr Ala Val Val Ile Ala Gly 90 95 100 gaa tta tta aga aaa gct gag gaa ttg tta gac caa aac gta cac cca 388 Glu Leu Leu Arg Lys Ala Glu Glu Leu Leu Asp Gln Lys Val His Pro 105 110 115 aca att gtt att aaa ggt tac cag tta gca gtt caa aaa gca caa gaa 436 Thr Ile Val Ile Lys Gly Thr Gln Leu Ala Val Gln Lys Ala Gln Glu 120 125 130 135 gta tta aaa gaa ata gca atg gat gta aaa gca gac gat aaa gaa ata 484 Val Leu Lys Glu Ile Ala Met Asp Val Lys Ala Asp Asp Lys Glu Ile 140 145 150 tta cac aaa ata gca atg acc tca att act gga aaa ggt gca gaa aaa 532 Leu His Lys Ile Ala Met Thr Ser Ile Thr Gly Lys Gly Ala Glu Lys 155 160 165 gct aaa gaa aaa ttg gga gaa atg atc gtt gaa gct gtt act gca gtt 580 Ala Lys Glu Lys Leu Gly Glu Met Ile Val Glu Ala Val Thr Ala Val 170 175 180 gta gac gaa agt gga aaa gtt gac aaa gat tta ata aaa atc gag aag 628 Val Asp Glu Ser Gly Lys Val Asp Lys Asp Leu Ile Lys Ile Glu Lys 185 190 195 aaa gaa gga gct tca gtt gac gaa acc gaa tta atc aat ggt gta tta 676 Lys Glu Gly Ala Ser Val Asp Glu Thr Glu Leu Ile Asn Gly Val Leu 200 205 210 215 ata gac aaa gaa aga gta agc cca caa atg cct aag aag ata gaa aac 724 Ile Asp Lys Glu Arg Val Ser Pro Gln Met Pro Lys Lys Ile Glu Asn 220 225 230 gca aag atc gca tta tttg aac tgc cca atc gaa gtt aaa gaa aca gaa 772 Ala Lys Ile Ala Leu Leu Asn Pro Ile Glu Val Lys Glu tha Glu 235 240 245 aca gat gca gaa att aga atc aca gat cct aca aaa tta atg gaa ttc 820 Thr Asp Ala Glu Ile Arg Ile Thr Asp Pro Thr Lys Leu Met Glu Phe 250 255 260 att gaa caa gaa gaa aaa atg tta aaa gat atg gta gat acc ata aaa 868 Ile Glu Gln Glu Glu Lys Met Leu Lys Asp Met Val Asp Thr Ile Lys 265 270 275 gct tca gga gca aac gtt tta ttc tgc caa aaa ggt atc gat gacAla Gln His Tyr Asn Val Leu Phe Cys Gln Lys Gly Ile Asp Asp Leu 280 285 290 295 gca caa cac tac tta gca aaa gaa gga atc ctt gca gta aga aga gtc 964 Ala Gln His Tyr Leu Ala Lys Glu Glu Ile Leu Ala Val Arg Arg Val 300 305 310 aaa aaa tca gac atg gaa aaa tta tca aaa gct act gga gct aac gta 1012 Lys Lys Ser Asp Met Glu Lys Leu Ser Lys Ala Thr Gly Ala Asn Val 315 320 325 gta aca aac atc aaa gac tta aaa gct gaa gat tta ggg gaa gca gga 1060 Val Thr Asn Ile Lys Asp Leu Lys Ala Glu Asp Leu Gly Glu Ala Gly 330 335 340 att gta gaa gaa aga aaa att gct gga gac gca atg atc ttc gtt gaa 1108 Ile Val Glu Lys Ile Ala Gly Asp Ala Met Ile Phe Val Glu 345 350 355 gaa tgc aaa cat cca aaa gca gta aca atg tta ata aga ggt aca aca 1156 Glu Cys Lys His Pro Lys Ala Val Thr Met Leu Ile Arg Gly Thr Thr 360 365 370 370 375 gaa cac gta att gaa gaa gta gca aga gct gta gat gat gct att gga 1204 Glu His Val Ile Glu Glu Val Ala Arg Ala Val Asp Asp Ala Ile Gly 380 385 390 gtt gta gca tgt acc atc gaa gac ggt aag atc gtt gca ggt ggt gga 1252 Val Val Ala Cys Thr Ile Glu Asp Gly Lys Ile Val Ala Gly Gly Gly 395 400 405 gct gca gag ata gaa tta gca atg aaa tta aga gac tac gct gaa gga 1300 Ala Ala Glu Ile Glu Leu Ala Met Lys Leu Arg Asp Tyr Ala Glu Gly 410 415 420 gta agt gga aga gaa caa ttg gca gtt aga gca ttt gca gat gct tta 1348 Val Ser Gly Arg Glu Gln Leu Ala Val Arg Ala Phe Ala Asp Ala Leu 425 430 430 435 gaa gta gca aga aca tta gca gaa aac gca ggt tta gat gca att 1396 Glu Val Val Pro Arg Thr Leu Ala Glu Asn Ala Gly Leu Asp Ala Ile 440 445 450 455 gaa atg ctc gtt aaa tta aga gct aaa cat gca gaa gga 1 at aac gca Glu Met Leu Val Lys Leu Arg Ala Lys His Ala Glu Gly Asn Asn Ala 460 465 470 tac tat ggg tta aac gtc ttc aca ggc gat gtt gaa aac atg act gaa 1492 Tyr Tyr Gly Leu Asn Val Phe Thr Gly Asp Val Glu Asn Thr Glu 475 480 485 aac gga gta gtt gaa cca tta aga gtt aaa act caa gct ata caa tca 1540 Asn Gly Val Val Glu Pro Leu Arg Val Lys Thr Gln Ala Ile Gln Ser 490 495 500 gct aca gaa gct aca gaa atg tta tta aga ata gac gat gta atc gct 1588 Ala Thr Glu Ala Thr Glu Met Leu Leu Arg Ile Asp Asp Val Ile Ala 505 510 515 gca gaa aaa tta agc ggc gga tct ggc gga gac atg gga gac atg ggc 1636 Ala Glu Ser Gly Gly Ser Gly Gly Asp Met Gly Asp Met Gly 520 525 530 535 gga atg gga ggt atg ggc gga atg atg taattccccc tacgtcc 1680 Gly Met Gly Gly Met Gly Gly Met Met 540 <210> 2 <211> 544 <212> PRT <213> Methanococcus thermolithotrophicus DSM 2095 strain <400> 2 Met Ala Ala Asn Gln Pro Val Val Val Leu Pro Glu Asn Val Lys Arg 1 5 10 15 Phe Met Gly Arg Asp Ala Gln Arg Met Asn Ile Leu Ala Gly Arg Ile 20 25 30 Ile Gly Glu Thr Val Arg Ser Thr Leu Gly Pro Lys Gly Met Asp Lys 35 40 45 Met Leu Val Asp Asp Leu Gly Asp Ile Val Val Thr Asn Asp Gly Val 50 55 60 Thr Ile Leu Lys Glu Met Ser Val Glu His Pro Ala Ala Lys Met Leu 65 70 75 80 Ile Glu Val Ala Lys Thr Gln Glu Lys Glu Val Gly Asp Gly Thr Thr 85 90 95 Thr Ala Val Val Ile Ala Gly Glu Leu Leu Arg Lys Ala Glu Glu Leu 100 105 110 Leu Asp Gln Lys Val Hi s Pro Thr Ile Val Ile Lys Gly Thr Gln Leu 115 120 125 Ala Val Gln Lys Ala Gln Glu Val Leu Lys Glu Ile Ala Met Asp Val 130 135 140 Lys Ala Asp Asp Lys Glu Ile Leu His Lys Ile Ala Met Thr Ser Ile 145 150 155 160 Thr Gly Lys Gly Ala Glu Lys Ala Lys Glu Lys Leu Gly Glu Met Ile 165 170 175 Val Glu Ala Val Thr Ala Val Val Asp Glu Ser Gly Lys Val Asp Lys 180 185 190 Asp Leu Ile Lys Ile Glu Lys Lys Glu Gly Ala Ser Val Asp Glu Thr 195 200 205 Glu Leu Ile Asn Gly Val Leu Ile Asp Lys Glu Arg Val Ser Pro Gln 210 215 220 Met Pro Lys Lys Ile Glu Asn Ala Lys Ile Ala Leu Leu Asn Cys Pro 225 230 235 240 Ile Glu Val Lys Glu tha Glu Thr Asp Ala Glu Ile Arg Ile Thr Asp 245 250 255 Pro Thr Lys Leu Met Glu Phe Ile Glu Gln Glu Glu Lys Met Leu Lys 260 265 270 Asp Met Val Asp Thr Ile Lys Ala Gln His Tyr Asn Val Leu Phe Cys 275 280 285 Gln Lys Gly Ile Asp Asp Leu Ala Gln His Tyr Leu Ala Lys Glu Glu Glu 290 295 300 300 Ile Leu Ala Val Arg Arg Val Lys Lys Ser Asp Met Glu Lys Leu Ser 305 310 315 320 Lys Ala Thr Gly Ala A sn Val Val Thr Asn Ile Lys Asp Leu Lys Ala 325 330 335 Glu Asp Leu Gly Glu Ala Gly Ile Val Glu Glu Arg Lys Ile Ala Gly 340 345 350 Asp Ala Met Ile Phe Val Glu Glu Cys Lys His Pro Lys Ala Val Thr 355 360 365 Met Leu Ile Arg Gly Thr Thr Glu His Val Ile Glu Glu Val Ala Arg 370 375 380 Ala Val Asp Asp Ala Ile Gly Val Val Ala Cys Thr Ile Glu Asp Gly 385 390 395 400 Lys Ile Val Ala Gly Gly Gly Ala Ala Glu Ile Glu Leu Ala Met Lys 405 410 415 Leu Arg Asp Tyr Ala Glu Gly Val Ser Gly Arg Glu Gln Leu Ala Val 420 425 430 Arg Ala Phe Ala Asp Ala Leu Glu Val Val Pro Arg Thr Leu Ala Glu 435 440 445 445 Asn Ala Gly Leu Asp Ala Ile Glu Met Leu Val Lys Leu Arg Ala Lys 450 455 460 His Ala Glu Gly Asn Asn Ala Tyr Tyr Gly Leu Asn Val Phe Thr Gly 465 470 475 475 480 Asp Val Glu Asn Met Thr Glu Asn Gly Val Val Glu Pro Leu Arg Val 485 490 495 Lys Thr Gln Ala Ile Gln Ser Ala Thr Glu Ala Thr Glu Met Leu Leu 500 505 510 Arg Ile Asp Asp Val Ile Ala Ala Glu Lys Leu Ser Gly Gly Ser Gly 515 520 525 Gly Asp Met Gly Asp Met G ly Gly Met Gly Gly Met Gly Gly Met Met 530 535 540 <210> 3 <211> 608 <212> DNA <213> Methanococcus thermolithotrophicus <220> <221> CDS <222> 82..543 <400> 3 tgtaataaat ccattaatcg tggtagcaat aataaaatat atataccaat acagtttgag 60 ttatgatatc gacataagca t gtg att ttt ttg gta gat aaa gga gtt aaa 111 Val Ile Phe Leu Val Asp Lys Gly Val Lys 1 5 10 ata aaa gta gac tac ata ggt aaa ctt gat gt gat gt gat gt gat gt gat gt ag Lys Val Asp Tyr Ile Gly Lys Leu Glu Ser Gly Asp Val Phe Asp 15 20 25 act tcc ata gaa gag gta gca aaa gaa gct gga ata tac gcg cct gat 207 Thr Ser Ile Glu Glu Val Ala Lys Glu Ala Gly Ile Tyr Ala Pro Asp 30 35 40 aga gaa tat gaa cct cta gag ttc gtt gta gga gaa ggt cag ttg att 255 Arg Glu Tyr Glu Pro Leu Glu Phe Val Val Gly Glu Gly Gln Leu Ile 45 50 55 caa ggt ttt gaa gaa gct gtt tta gacg gaa gtc ggg gac gaa aaa 303 Gln Gly Phe Glu Glu Ala Val Leu Asp Met Glu Val Gly Asp Glu Lys 60 65 70 acc gta aaa atc cct gca gag aaa gca tac ggt aac aga aat gaa atg 351 Thr Val Lys Ile Pro Ala G lu Lys Ala Tyr Gly Asn Arg Asn Glu Met 75 80 85 90 tta ata cag aaa ata cca aga gat gct ttt aaa gaa gct gat ttt gaa 399 Leu Ile Gln Lys Ile Pro Arg Asp Ala Phe Lys Glu Ala Asp Phe Glu 95 100 105 cct gaa gaa gga atg gta atc ctt gca gaa gga att cct gct aca ata 447 Pro Glu Glu GIy Met Val Ile Leu Ala Glu Gry Ile Pro Ala Thr Ile 110 115 120 acc gaa gtt aca gat aat gag gta act tta gac ttt aac cat gaa ctt 495 Thr Glu Val Thr Asp Asn Glu Val Thr Leu Asp Phe Asn His Glu Leu 125 130 135 gca gga aaa gat tta gta ttt aca att aaa att att gaa gtt gtc gaa 543 Ala Gly Lys Asp Leu Val Phe Thr Ile Lys Ile Ile Glu Val Val Glu 140 145 150 taattatcat tataacattc cgccgagatt aaataaaatc acttgtgatt ttatatcttt 603 acccg 608 <210> 4 <211> 154 <212> PRT <213> Methanococcus thermolithotrophicus <400> 4 Val Ile Phe Leu Val Asp Lys Gly Val Asp Tyr Ile 1 5 10 15 Gly Lys Leu Glu Ser Gly Asp Val Phe Asp Thr Ser Ile Glu Glu Val 20 25 30 Ala Lys Glu Ala Gly Ile Tyr Ala Pro Asp Arg Glu Tyr Glu Pro Leu 35 40 45 Glu Phe Val Val Gly Glu Gly Gln Leu Ile Gln Gly Phe Glu Glu Ala 50 55 60 Val Leu Asp Met Glu Val Gly Asp Glu Lys Thr Val Lys Ile Pro Ala 65 70 75 80 Glu Lys Ala Tyr Gly Asn Arg Asn Glu Met Leu Ile Gln Lys Ile Pro 85 90 95 Arg Asp Ala Phe Lys Glu Ala Asp Phe Glu Pro Glu Glu GIy Met Val 100 105 110 Ile Leu Ala Glu Gry Ile Pro Ala Thr Ile Thr Glu Val Thr Asp Asn 115 120 125 Glu Val Thr Leu Asp Phe Asn His Glu Leu Ala Gly Lys Asp Leu Val 130 135 140 Phe Thr Ile Lys Ile Ile Glu Val Val Glu 145 150 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223 > Sequence Listing Free Text Forward Primer <400> 5 ggccatggca gctaaccagc ca 22 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Sequence Listing Free Text Reverse Primer <400> 6 ccggatcctt acatcattcc gcccatac 28

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Jun Doi 2-1 Momoyama, Shimamotocho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd. (72) Masahiro Furuya 2-1 Hyakuyama, Shimamotocho, Mishima-gun, Osaka Sekisui Inside Chemical Industry Co., Ltd. (72) Inventor Toshiki Kawabe 2-1 Shimomotocho, Mishima-gun, Osaka Prefecture Sekisui Chemical Industry Co., Ltd. (72) Inventor Tatsuo Yamamoto 2-1 Shimomotocho, Moyama, Mishima-gun, Osaka Sekisui Chemical (72) Inventor Hironobu Isagawa 2-1 Momoyama, Shimamoto-cho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd.

Claims (4)

[Claims] 1. A blood test container comprising a bottomed tube whose one end is open and the other end is closed, and a stopper that seals the opening of the bottomed tube, comprising a protein folding factor, a peptide and / or a peptide. A blood test container containing a peptide-like blood coagulation promoter. 2. The blood test container according to claim 1, wherein the peptide and / or the peptide-like blood coagulation promoter is blood coagulation factor X or blood coagulation factor Xa. 3. The peptide and / or peptide-like blood coagulation promoter is a hydrolase that hydrolyzes a bond between Arg and any amino acid residue of a peptide chain and / or a bond between Lys and any amino acid residue. The blood test container according to claim 1, wherein 4. The protein folding factor is a molecular chaperone and / or peptidylprolyl-cis-trans-.
4. The blood test container according to claim 1, which is isomerase.