KR970002916B1 - Novel dna, recombinant plasmid, and transformed cell - Google Patents

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Description

New DNA Encoding Polypeptides, Recombinant Plasmids Containing DNA and Transgenic Cells Containing Recombinant Plasmids

1 is a schematic showing the restriction endonuclease diagram of the recombinant plasmid pMKT7 of the present invention.

2 is an explanatory diagram showing a PCI cDNA nucleotide sequence according to the present invention, wherein the nucleotides are numbered starting with No. 1 in the translation start codon ATG.

3 is a sketch showing the restriction enzyme diagram of the plasmid pMKTX1 for polypeptide expression of the present invention.

4 shows the results of electrophoresis and western blotting of SDS-polyacrylamide gel (10% gel) of the polypeptide of the present invention, and lane 1 of E. coli JM05 / pMKTXl cultured with IPTG was added. Cell extracts were obtained by electrophoresis, lane 2 was recorded by electrophoresis of cell extracts of E. coli JMl05 / pMKTX1 cultured without addition of IPTG, and lane 3 was recorded by E. coli JMl05 / pKK233- cultured with addition of IPTG. It was produced by electrophoresis of the cell extract of cell 2, lane 4 was obtained by electrophoresis of the cell extract of E. coli JMl05 / pKK233-2 incubated without adding IPTG, and lane 5 was formed by electrophoresis of PCI. .

FIG. 5 is a graph showing that E. coli extracted from the DEAE-Toyo Pearl column in Reference Example 2 was eluted with a straight NaC1 gradient method to collect the shade fractions.

Figure 6 is a graph showing that the shade fraction is collected by eluting the peptide of the present invention from the antibody column in Reference Example 2.

The present invention provides a novel DNA encoding a polypeptide having anticoagulant activity, such as placental coagulation inhibitor (hereinafter referred to as `` PCI '') obtained from human placental tissue, recombinant plasmid containing DNA, recombinant plasmid It relates to a transformed cell containing a divided application of 1988 patent application No. 1424.

Heparin, heparin cofactor-II, antithrombin-III, α ₂ -macroglobulin, α ₁ -trypsin inhibitor, Ci-esterase inhibitor, protein C and the like are known as anticoagulants. However, although only heparin is practically useful, heparin has side effects that cause bleeding, and therefore, strict restrictions on administration and dosing methods. Therefore, heparin was not satisfactory as an anticoagulant in stable cells.

Under the circumstances described above, the present applicant has already succeeded in the separation and purification of PCI from the human placenta and applied for it (Japanese Patent Application No. 217512/1985).

PCI is a substance having the following properties and useful as a medicine.

(1) Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state):

34,000 ± 2,000

(2) Isoelectric point (isoelectric column electrophoresis using ampolite):

4.7 ± 0.1

(3) Stability:

(a) Deactivation in heat treatment at 50 ° C. for 30 minutes

(b) stable at pH 4-10

(c) stable for 30 min at 37 ° C. in plasma

(4) Action:

(a) prolongation of calcium home coagulation

(b) prolong prothrombin time

(c) prolong the activated partial tromboplastin time

(5) Analysis of amino acids:

Assay of amino acids in the presence of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1/2 cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine.

Applicant has prepared a monoclonal antibody specific for PCI and has already applied for it (Japanese Patent Application No. 269588/1986). By using this monoclonal antibody, it became possible to perform high sensitivity analysis, purification, etc. of PCI.

However, some problems have arisen because the human tissues limited to human placenta are indispensable as raw materials for obtaining PCI at present. For example, since the amount of PCI obtained in human tissues is limited, human tissues have difficulty in stable supply of raw materials when collected and used as raw materials. In addition, the potential risk of pathogenic viruses in human tissues cannot be ignored.

Therefore, it has been desired to develop a cheaper price, a larger quantity, a more stable and safe method of supplying PCI, or a material having a PCI-like action.

The present inventors have extensively researched to solve the above problems, and as a result, PCI peptide-encoded DNA fragments can be obtained from human placental cDNA libraries using PCI-specific antibodies as probes, and DNA fragments can be recombined. One plasmid was used to transform microbial cells and to express PCI genes, thus discovering that a PCI-like polypeptide could be prepared, and the present invention was completed.

The invention amino acid sequence

To provide a novel DNA encoding a polypeptide having a recombinant DNA, a recombinant plasmid containing a DNA, a transformed cell containing the recombinant plasmid.

Since the polypeptide according to the present invention exhibits strong anticoagulant activity, the anticoagulant containing it as an active ingredient can be used for various diseases caused by deterioration of blood coagulation activity, for example, brain, heart and peripheral, such as thromboses, cerebral infarction and myocardial infarction. It is useful for the prevention and treatment of DIC (distributed intravascular coagulation) in blood vessels.

On the other hand, the polypeptide according to the present invention is a safe substance that does not have antigenicity against human because it has similar properties to the polypeptide of human placental placental inhibitor (PCl). Although PCI is useful as an anticoagulant, there is a drawback in that it cannot produce large amounts due to the difficulty in using the human placenta. In contrast, the polypeptides according to the invention can be produced in large quantities at low cost.

These and other objects, aspects, and advantages of the present invention will become more apparent with reference to the following description and appended claims, the accompanying drawings.

1 is a schematic showing the restriction endonuclease diagram of the recombinant plasmid pMKT7 of the present invention.

2 is an explanatory diagram showing a PCI cDNA nucleotide sequence according to the present invention, wherein the nucleotides are numbered starting with A in the translation initiation codon ATG.

3 is a sketch showing the restriction enzyme diagram of the plasmid pMKTX1 for polypeptide expression according to the present invention.

4 shows the results of electrophoresis and western blotting of SDS-polyacrylamide gel (10% gel) of the polypeptide according to the present invention. Lane 1 was cultured by adding IPTG to E. coli (E. coli) was obtained by electrophoresis of cell extract of JMl05 / pMKTX1, lane 2 was recorded by electrophoresis of cell extract of E. coli JMl05 / pMKTX1 cultured without addition of IPTG, and lane 3 was cultured by addition of IPTG. coli JMl05 / pKK233-2 was produced by electrophoresis of cell extracts, lane 4 was obtained by electrophoresis of cell extracts of E. coli JMl05 / pKK233-2 cultured without addition of IPTG, and lane 5 was PCI. It is formed by electrophoresis.

FIG. 5 is a graph showing that E. coli extracted from the DEAE-Toyo Pearl column in Reference Example 2 was eluted with a straight NaC1 gradient method to collect the shade fractions.

Figure 6 is a graph showing that the shade fractions are collected by eluting the peptide of the present invention from the antibody column in Reference Example 2.

DNA, recombinant plasmids and transformed cells encoding polypeptides according to the invention are prepared, for example, by the following method.

That is, (1) antibody positive clones are screened from human placental cDNA libraries using PCl-specific antibodies.

(2) Recombinant DNA is prepared from antibody positive clones and is isolated, and the recombinant phage DNA is treated with restriction endonucleases to attach cDNA fragments from the recombinant phage DNA and bind to plasmid vectors. (3) The host cell is transformed with the resulting cDNA recombinant plasmid to obtain the transformed cell of the present invention. The transformed cells of the present invention thus obtained are cultured to obtain a recombinant plasmid of the present invention containing the DNA fragment of the present invention from the cells and cultured. DNA fragments according to the invention can be obtained by cleaving a recombinant plasmid with appropriate restriction endonucleases.

Each said process is demonstrated below, respectively.

(1) Screening of antibody positive clones from human placental cDNA library:

cDNA library produces mRNA in human placenta and reverse transcribase

ptase) and appropriate vector DNA. Commercially available cDNA libraries can optionally be used, for example, human placental cDNA libraries (λgtll) (manufactured by Clontech Laboratories, Inc.).

The cDNA library, in which λgtll was created as a vector, is described in Huynh, T.V., Young, R. A. and Davis, R.W. (1985) In: ####: A Practical Approach, vo1. 1, (D. M. Glover, ed), pp 49-78, IRL Press, Oxford, can be used to screen specific antibodies as probes to isolate clones to specific antibodies.

Primary antibodies used as probes include PCI-specific antibodies, namely anti-PCI rabbit polyclonal antibodies, anti-PCI mouse polyclonal antibodies and anti-PCI monoclonal antibodies. Of these, anti-PCI monoclonal antibodies, in particular anti-PCI mouse monoclonal antibodies, are preferred. The antibody may be any one of serum, ascites, hybridoma culture, and purified immunoglobulin forms.

The detection of the primary antibody binding to the antigen, wherein the labeled with radioactive iodine ⁽¹²⁵ I) of protein A or radioactive iodine ⁽¹²⁵ I) Auto the radio and using an anti-immunoglobulin antibody labeled with our method or a peroxidase-labeled immune It can be carried out by enzymatic antibody method using a globulin antibody or an anti-immunoglobulin antibody labeled with alkaline phosphatase.

If desired, anti-PCI monoclonal antibodies can be prepared, for example, by the method in Japanese Patent Application No. 269588/1986 described above. That is, mice are immunized with purified PCI and extracted from human placenta. Splenocytes are collected from mice and then fused to mouse myeloma cells. The cell-fused cells are cultured using a HAT selective medium to grow only hybridomas. The hybridoma culture thus grown is screened by enzyme immunoassay using PCI as an antigen to obtain a hybridoma capable of producing monoclonal antibodies specific for PCI. Monoclonal antibodies are obtained from cultures in which the resulting hybridomas are cultured or ascites fluid from mice inoculated with hybridomas.

(2) Preparation of PCI cDNA Recombinant Plasmids:

Recombinant λgtll phage DNA was antibody-positive isolated according to a method proposed by Bernard Perba1, PREPARATION OF λ PHARGE DNA in A PRACTICAL GUIDE TO MOLECULAR CLONING, pp 175-184, Awilley-Interscience Publication (1984), NewYork, USA. Extracted from clones into purified form. cDNA can be isolated from vector DNA in which purified recombinant λgtll phage DNA was digested with restriction endonuclease ECoRI. The resulting cDNA is recombined with various cloning plasmid vectors obtained by digestion with ECoRI to obtain a recombinant plasmid. Examples of plasmid vectors that can be used include pBR322, pBR325, pUC18, pUC118, pTZ18R and the like.

(3) Transformation of host cells by PCI cDNA plasmid and production of recombinant plasmid of the present invention and DNA of the present invention:

The resulting PCI cDNA recombinant plasmid is transformed into a host cell by introducing a genetic label with the recombinant plasmid into the maximum available variety of host cells. E. coli is preferred as the host cell. As various strains of the E. coli K12 strain, for example, HB101, C600K, JM01, JMl05, X1776, MV1304 and the like are used. Competent cell methods by calcium treatment may be used for the introduction of recombinant plasmids.

The transformed cells are cultured in selection medium suitable for the vector plasmid gene label and the recombinant plasmid of the present invention is collected from the cells.

When pUC118 or pTZ18R is used as a vector, single-stranded DNA can be prepared by infecting the helper phage Ml3K07 with transformant E. coli containing the recombinant vector. The resulting single-stranded DNA was found in dideoxynucleotide chain termination methods [Sanger, F., Nicklen, S. and Coulson, A.R. : DNA Sequencing with Chain Terminating Inhibitors, Proc, Natl. Aca. Sci. USA ##, 5463-5467 (1977)] may determine the DNA base sequence.

The nucleotide sequence, nucleotides of the portion that can be coated with a polypeptide is, for example, as follows.

The DNA fragment of the present invention is not limited to the nucleotide sequence as long as it can encode the above-described amino acid sequence. If the recombinant plasmid of the present invention has a nucleotide sequence capable of encoding the above-described amino acid sequence and is replicable, the recombinant plasmid of the present invention is E. coli, B. a. It can be prepared by combining with vector DNA derived from B. subtilis, yeast and the like.

Polypeptides according to the invention are prepared by culturing transformant cells containing the recombinant plasmid of the invention and collecting the polypeptide from the resulting culture. However, in order to efficiently produce the polypeptide of the present invention, following (1)-(6),

(l) promoter

(2) ribosomal binding sites

(3) initiation codon

(4) DNA having an amino acid sequence codeable nucleotide sequence of a polypeptide of the present invention

(5) termination codons and

(6) transcription terminator

It is necessary to construct an expression recombinant plasmid containing plasmid and transform the host cell using this plasmid cloning vector.

Vector hosts for obtaining expression plasmids of PCI cDNA include single cell microorganisms such as bacteria, in particular E. coli, B. a. Serotolis or streptomyces are preferred. When E. coli is selected as the host, various strains of the E. coli K12 strain, such as HB01, C600K, JM1

01, JM105, JM109, X1776, MV1304 and the like are used.

As a DNA used as a vector, a plasmid is preferable. For example, when using E. coli as a host, the plasmid DNA has the sequence of DNA necessary for the propagation of the plasmid in E. coli cells, such as the DNA sequence of the starting region of the ColEl plasmid replication, and will also act as a promoter and transcription terminator. Have another DNA sequence that can More preferably, it contains a gene which becomes the selectable label of the transformed E. coli. For example, promoters, such as λPL, lac, trp, tac, trc, and lpp, and transcription terminators include rrnB ribosomal RNA, and the selected gene markers are ampicillin-resistant gene, kanamycin-resistant gene, tetracycline-resistant gene, Chloramphenicol-resistant genes; and the like. These genes can be used alone or in combination.

DNA conjugates having a nucleotide sequence capable of encoding the amino acid sequence of the polypeptide of the present invention, ie, the DNA fragment of the present invention, may cleave the above-described vector DNA with an appropriate restriction endonuclease and add a suitable linker as necessary. The resulting DNA fragments can then be performed by binding to the vector DNA digested with appropriate restriction endonucleases. For example, in the case of DNA having the nucleotide sequence shown in FIG. 2, Nco I and Sac I, Nco I and Sph I, Nco I and Hind III, and the like can be cited as restriction endonucleases.

Introduction of the resulting plasmid for expressing the PCI cDNA into the host cell by methods such as the competent cell method, the protoplasts method, the calcium coprecipitation method, the electropulse method, and the like can be obtained by the transformed cells capable of effectively producing the polypeptide of the present invention. Lose.

Polypeptides of the invention can be prepared by culturing the resultant transformed cells and extracting and separating the polypeptides from the cultured cells and / or production cultures.

The culture of the transformed cells is used in various natural and synthetic media. The medium preferably contains a simple element such as sucrose, an alcohol or an organic acid, a protein mixture, a nitrogen source such as an amino acid or an ammonium salt and an inorganic salt. It is also desirable to add vitamins and antibiotics corresponding to the genetic labels. If the plasmid is allowed to modulate control of expression, it is necessary to make manipulations to induce gene expression during incubation. After incubation, the resulting culture broth is separated into a culture medium and cultured cells by centrifugation. When the polypeptide according to the present invention accumulates in cultured cells, the cells are lyzed or crushed by freeze thawing, sonication, French press, enzyme treatment, homogenization, and the like, and the polypeptide according to the present invention is, for example, EDTA, It is necessary to dissolve with surfactant, urea, guanidine hydrochloride and the like.

The resulting culture or cultured cell extract containing the polypeptide according to the invention is chromatographed on one of the various columns to obtain the polypeptide in purified form. As column chromatography, ion exchange chromatography and column chromatography can be used alone or in combination.

The polypeptide according to the present invention thus obtained has the following properties.

(1) amino acid sequence

Using the "JEOL JAS-570K Sequence Analyzer" ("JEOL JAS-57OK sequence analyzer" manufactured by JEOL Ltd.), the N-terminal amino acid sequence was analyzed by converting the N-terminal region into PTH amino acid. The amino acid sequence of 10 from the polypeptide N-terminus of was determined as follows.

As a result, the polypeptide amino acid sequence according to the present invention is estimated according to the DNA fragment nucleotide sequence of the present invention.

(2) molecular weight:

35,805 (calculated from amino acid sequence)

34,000 ± 1,000 (SDS-polyacrylamide gel electrophoresis, reduced state)

(3) isoelectric point (isoelectric point electrophoresis using ampolite)

5.0 ± 0.1

(4) Stability:

Stable for 30 minutes at 37 ° C in plasma

(5) Action:

(a) prolonged calcium resolidification time

(b) prolong the protrumbin time;

(c) prolong the active partial tromboplastin time

The polypeptide according to the present invention described above is useful as an anticoagulant because it has excellent anticoagulant activity. As an active ingredient, the dosage form which uses the polypeptide which concerns on this invention is an injection solution. Injectables are preferably administered in the form of lyophilized powder, dissolved in distilled water for injection, physiological saline or the like whenever necessary. Suitable route of administration is intravenous administration.

The administration of the polypeptide may vary depending on the symptoms of the disease, the weight of each patient and the like, but is generally in the range of 10 μg to 10 mg / Kg days. The polypeptide of the present invention did not show any abnormality when used in the above administration range and is safe.

Since the present invention has been described generally, the present invention may be specifically understood with reference to the following specific examples, but the present invention is not limited to the following examples, which are merely illustrative purposes.

(Example :)

The invention is illustrated by the following reference examples and examples.

Reference 1: Anti-PCl Monoclonal Antibody Preparation

(1) Purification of antigen (PCl):

(a) Finely cut the five human placenta (about 2,500g), remove the membrane, etc., and wash it thoroughly with saline solution. The separated placenta is crushed with a Waring blender and then added to 500 mM Tris-HCl buffer (pH 7.4) 21 and regrind with `` polytron ''. The resultant homogenate was centrifuged at 7,000 r.p.m for 15 minutes to allow for immersion. The mixture obtained by adding back 50 mM Tris-HCl buffer (pH 7.4) 21 to the collected acupuncture is homogenized with "polytron" and then centrifuged at 7,000 r.p.m for 15 minutes to obtain washing acupuncture.

The process is repeated several times until blood components are removed to finally give about 930 g of rinsing sap.

(b) 50 mM containing 50 mM EDTA is added to about 900 g of tris-hydrochloric acid buffer (pH 7.4) to the immersion obtained in step (a) and homogenized with Waring blender. The obtained hodogenate was shaken overnight at 4 ° C, and then centrifuged at 7,000 r.p.m for 15 minutes to obtain 2 L of extract.

(c) Solid ammonium sulfate is added to the extract obtained in the step (b) to bring its concentration to 35% saturation. The resulting mixture was allowed to stand at 4 ° C. for 30 minutes to several tens of hours, followed by centrifugation at 7,000 r.p.m for 15 minutes to separate the supernatant. Ammonium sulfate was added to this supernatant to bring it to 85% saturation concentration. The resulting mixture was left at 4 ° C. for 2 hours and then centrifuged at 7,000 r · p. The resultant solution was dissolved in a small amount of 20 mM Tris-hydrochloric acid buffer and sufficiently dialyzed overnight at 4 ° C for the same buffer. The precipitate formed during the dialysis was removed by centrifugation at 7,000 r.p.m for 15 minutes to give 390 ml of dialysate.

(d) The dialysate thus obtained was adsorbed onto DEAE-Toyo Pearl (Ø5.5x19 cm) equilibrated with 20 mM Tris-HCl buffer (pH 74) and washed well with the same buffer. Each of 41 identical buffers containing 0 to 0.3 M sodium chloride was eluted at a rate of 20 ml per minute according to the linear concentration gradient method. The active fractions were eluted at a concentration of about 0.15 M sodium chloride, resulting in 380 ml of water-soluble active fractions.

(e) The resulting active fractions were dialyzed overnight at 4 ° C. in 0.1 M phosphate buffer (pH 7.0) and a `` Blue Sepharose '' column (ø2.5 cm x 12 cm) pre-equilibrated with the same buffer. Passed. Pass fractions (480 ml) showing absorption at A <sub>280</sub> were collected and concentrated using `` Diaflow Membrane Filter YM-10 ''("DIAFLOW Membrane Filter YM-10", trade name).

(f) The concentrate obtained in step (e) was subjected to gel filtration using Sephadex G-100 (`` Sephadex G-100 '', trade name ø4.5 x 75 cm), and 8 mL per fraction with saline. The active fractions 88-104 were collected and concentrated by ultrafiltration to give 14.5 ml of PCI (protein weight: 136.1 mg, Lowry method).

In addition, the amount of protein obtained in each purification step is as follows.

(2) Preparation of immune spleen cells:

The purified PCI (100 μg) was suspended in Freund's complete supplement and administered intraperitoneally to BALB / C mice.

Two doses of PCI (50 μg / administration) and coemulsifier are administered twice, followed by a final immunization with only 50 μg of PCI.

After 3 days, the mice were killed, spleens were taken out, cut to constant length, and filtered through a 100-mesh nylon net to obtain isolated splenocytes.

(3) hybridoma preparation:

Red blood cells were hemolysed by adding a stock solution (155 mM ammonium chloride) to the immunized splenocytes thus obtained, and then washed three times with Iscoves denatured double course medium (IMDM). Wash three times with IMDM. The number of each cell was measured. The splenocytes and PAI cells were centrifuged at 5: 1. 45% obtained by distilling the supernatant, separating the resulting sap, and then diluting polyethylene glycol (PEG) 4,000 with culture medium

0.5 ml of solution is added dropwise to dissolve. The resulting mixture was allowed to stand at 37 ° C for 30 seconds, and then 1 ml of IMDM was added dropwise for 1 minute or longer, and then 10 ml of IMDM was added dropwise for 5 minutes or longer so that the final volume of the centrifuge tube was 40 ml. The resulting mixture was centrifuged at l, 000r.p.m for 8 minutes.

Acupuncture was suspended with IMDM added with 10% calf serum (FCS). The suspension was centrifuged again and the supernatant of the product was discarded.

Thus obtained acupuncture was prepared by adding 10 ⁻⁴ M hypoxantine, 4 × 10 ⁻⁷ M aminopterin, and 1.6 × 10 ⁻⁵ M thyrindine (HAT-). Resuspend in IMDM with% FCS and place 100 μl into each well of a 96-well microtiter plate. 50 μl of medium was added to each well every 3 to 4 days to observe cell proliferation.

It was confirmed that only hybridomas proliferated by HAT selective action.

(4) Antibody Production Hybridoma Screening:

Cultures in wells in which hybridomas were grown were collected and examined for the presence of antibody-producing hybridomas by enzyme immunoassay. First, PCI was added to each well of a 96-well microtiter plate ("Immunoplate I", manufactured by NUNC Company) at a rate of 0.1 μg / 100 μl / well and allowed to stand for 18 hours at 25 ° C., followed by 100 μl of the sample culture. It is injected at a rate of / well and reacted at 25 ° C. for 2 hours. After three washes of the culture with phosphate buffered saline containing 0.05% "Tween 20" [PBS-Tween], horse radish peroxidase conjugated goat anti-mouse IgG, KPL Laboratories , Inc.) is added at a rate of 100 μl / well, and after 2 hours the cultures are washed three times with PBS-twin. To each well was added 0.1 M citric acid-sodium hydroxide buffer (pH 5.0) [Sigma Chemical Company] containing 0.001% H ₂ O ₂ solution and 0.4 mg / ml orthophenylenedia cotton, and the absorbance of the culture in each well was 492 nm. Measured at the wavelength.

Coloring was observed only in wells in which antibodies were present against PCI, so cells were collected from the wells.

(5) Cloning of hybridomas to produce monoclonal antibodies specific for PCI:

Peritoneal cells collected by injecting IMDM into the mouse abdominal cavity are used as feeder cells.

Peritoneal cells suspended at 1 × 10 ⁵ cells per ml in 10% FCS-added IMDM are poured into each well of a 96-well microtiter plate. The next day, 5 antibody-producing hybridomas are prepared per ml and 100 μl are poured into each well. Every three days, the culture medium was replaced with fresh, identical culture medium, and culture supernatants were continuously collected from cells in which hybridomas were grown to appropriate volumes. Antibody production confirmation is performed in the same manner as described above. Positive well cultures were cloned again to obtain hybridomas that produce anti-PCI monoclonal antibodies. Six types of hibari choma were obtained, and PCI-H 39 (Japanese non-performance FERM BP-1701), PCI-H 46 (FERM BP-1702), and PCI, respectively, depending on the type of anti-PCI monocloyl antibody produced. -H 167 (FERM BP-1703), PCl-H 169 (FERM BP-1704), PCI-H 176 (FERM BP-1705) and PCI-H 180 (FERM BP-1706).

(6) Anti-PCl Monocloyl Antibody Preparation:

Pristane (Aldrich Chemical Co. Inc.) was injected into at least 7 week old mice BALB / C. After about 1 week, the hybridomas obtained above were intraperitoneally administered at a rate of 1 × 10 ⁶ per mouse. After about 10 days, the ascites were collected from the abdominal cavity of the mouse, and the supernatant was collected by centrifugation at 3,000 rpm for 10 minutes, and 5 ml of ammonium sulfate was added until it became 50% saturated concentration, and then allowed to stand overnight at 4 ° C. Centrifuge at 3,000 rpm for 15 minutes, and dissolve the saline in 0.1 mM Tris-HCl buffer (pH 8) and dialyzate with the same buffer. The resulting dialysate is subjected to column chromatography "Protein A Sepharose CL-4B" (produced by Phannacia AB) equilibrated with the same buffer.

Anti-PCI monoclonal antibodies are obtained by subjecting the monocloyl antibody to elution with 0.1 M glycan-0.15 M sodium chloride buffer (pH 2.7). When using PCI-H39, 14.2 mg of PCI-A39 is obtained. 20.2 mg of PCI-A46 for PCl-H46, 22.9 mg of PCI-A 167 for PCI-H167, 25.0 mg of PCI-A169 for PCI-H169 and 8.6 mg of PCI-A180 for PCI-H180 are obtained.

Example 1 PCI cDNA Cloning

(1) Screening of Human Placental cDNA Libraries:

(a) cDNA library:

The human placental cDNA library (cDNA from Clontech Laboratories, Inc.) was prepared from an average of 1.8 kb of placental placenta mRNA using reverse transcriptase and bound to the λgtll pie EcoRI site via an EcoRI linker. The library was a recombinant λgtll phage consisting of 1.0 × 10 ⁶ independent clones.

(b) Host E. coli cell Y1090 (ATCC 37197) was treated with LB agar plates containing ampicillin (100 μg / ml) and maltose (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of sodium chloride, 2 g of maltose, Stir in agar 15g, distilled water 11: pH 7.5) and incubate overnight at 37 ° C. The single colony thus obtained was transplanted into LB medium containing ampicillin (100 µg / ml) and maltose (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of sodium chloride, 5 g of maltose, 11: pH 7.5), Shake and incubate overnight at 37 ° C.

(c) Infection of phage library

0.1 ml of the host E. coli Y1090 cells (0.2 ml) incubated overnight with λ diluent (10 mM Tris-hydrochloric acid buffer, 10 mM magnesium chloride, pH 7.5) 3.7-5.5 × 10 ⁵ pfu / ml The mixture was allowed to stand at room temperature for 20 minutes to adsorb phage to host cells. 2.5 ml of LB top layer agar medium containing maltose and maintained at 45 ° C. (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of sodium chloride, 2 g of maltose, 7.2 g of distilled water, pH 7.5) was added and mixed The resulting mixture is placed on an LB agar plate containing 9 cm diameter maltose and incubated at 42 ° C. for 3 hours 30 minutes.

(d) Transfer to nitro cellulose filter

Sterilized Nitro Cellulose Filters with 10 mM Isopropyl-β-D

Saturated with thiogalactopyranoside (IPTG) and then dried. The filter is dried and incubated at 42 ° C. for 3 hours 30 minutes and placed on LB agar plates containing fresh λgtll phage plaques. Incubate for 3 hours 30 minutes at 37 ° C and then remove the filter. Plates are stored at 41 ° C. with the resulting phage plaques. The filter is washed with TBST (10 mM Tris-HCl buffer, 150 mM sodium chloride, 0.05% "Tween 20": pH 8.0) and then blocked with 1% procin serum albumin / TBST for 30 minutes at room temperature.

(e) primary antibody binding

The filter is placed in the primary antibody solution and allowed to react by shaking for 30 minutes at room temperature. As a primary antibody, the anti-PCI mouse monoclonal antibody (obtained in the reference example) dissolved in TBST is allowed to stand at room temperature for 30 minutes, and a mixed solution in which other antibodies are absorbed is used. As anti-PCl mouse monoclonal antibody mixture, PCI-A39 + PCI-A46 + PCI-A180 (1: 1: 0.5), bovine serum albumin 1mg / ml and E. coli extract (Promega Corporation) 0.25 µg / ml to 0.04 μg / ml containing material were used.

After the primary antibody reaction, the filters are washed three times for 10 minutes each with TBST.

(f) secondary antibody binding

The filter is transferred to a secondary antibody solution and reacted under stirring at room temperature for 30 minutes. As a secondary antibody, the combined mouse murine lgG (H + L) [Promega Corporation product] bound with alkaline phosphatase was used diluted to 1 / 7,500 of its original concentration in TBST.

The filter is washed three times with TBST for 10 minutes each and once with AP buffer (100 mM Tris-HCl buffer, 100 mM sodium chloride, 5 mM magnesium chloride: pH 9.5).

(g) color development

The filter is immersed in a chromogenic substrate solution obtained by mixing 33 μl of nitroblue tetrazolium solution (50 mg / ml) with 66 μl of 5-bromo-4-chloro-3-indolyl phosphate (50 mg / ml).

After 1 hour of color development at room temperature, the filter is transferred to reaction complete solution (20 mM Tris-HCl buffer, 5 mM sodium ethylenediamine tetraacetate: pH 8.0) to complete color development.

(h) Positive Plaque Preparation and Purification

Plaques were collected by agar medium for the positive spots showing color development, and transferred to 0.1 ml of TMG buffer (10 mM Tris-HCl buffer, 10 mM magnesium chloride, gelatin 100 µg / ml pH: 7.4). 2 drops of chloroform are added dropwise and centrifuged for 5 min at 4,000 r.p.m at 4 ° C. One chloroform is added to the resulting supernatant, and the prepared mixture is stored at 4 ° C.

The screening described above was carried out with about 3 × 10 ⁶ phage plaques, resulting in 26 positive plaques.

Dilute the phage solution to an appropriate amount in each of the 5 week plaques showing a deep color change and then screen the purified phage twice.

(2) Preparation of Recombinant Phage DNA

Host E. coli cell Y1088 (ATCC 337195) is infected with the resulting 5 weeks of recombinant phage and phage is induced at 42 ° C. λ phage preparation [Bernard Perbal, PREPARATION OF λ PHAGE DNA in A PRACTICAL GUIDE TO MOLECULAR CLONLNG, PP 175-184, A Wilcy-Interscience Publication (1984), New York, USA], by small volume preparation, bulk preparation, liquid culture Recombinant phage of 10 ⁹ pfu / ml was obtained via bulk preparation.

10 ¹¹ pfu / ml by polyethylene glycol precipitation according to λ DNA preparation [Bernard Perbal, PURIFICATION OF λ DNA NA A PRACTICAL GUIDE TO MOLECULAR CLONING, pp 184-187, A Wiley-Interscience Publication (1984), New York, USA]. The concentrated phage solution was subjected to glycerol stepwise centrifugation [Bernard Perbal, Shigeyasu Kobayashi Translation; Purctical Handbook of Gene Manipulation Experiments, pp 175 (1985), The Jatec Publishing Co.

Recombinant phage DNA was prepared in the same manner as in the preparation of λDNA using purified recombinant phage, and about 30-160 µg of DNA was obtained from 300 ml of culture.

(3) subcloning of cDNA

pUC118 (manufactured by Takara Shuzo Co., Ltd) is used as the vector. pUC118 is cleaved with EcoRI. Five weeks of recombinant λgtll phage DNA and DNA ligation kit (Takara Shuzo Co., Ltd.) were used to connect to cleaved pUC118, respectively.

When the recombinant vectors thus obtained were introduced into the host E. coli cell MV1304 (manufactured by Takara Shuzo Co., Ltd.), five recombinant vectors pMKTl, pMKT3, pMKT5, pMKT7, pMKT9 were obtained.

Restriction endonucleases or various restriction endonucleases are used and prepared. All recombinant vectors have a cleavage site of Pst I, Sac I, Sph I and Hind III in common. The inserted fragment is about 15 kb in length. Restriction endonucleases of pMKT7 with the longest fragment inserted are also shown in FIG. The quality of pMKT7 is about 4.8 kb.

(4) Identification of PCI cDNA Nucleotide Sequences

The resulting PCI cDNA recombinant vector was treated with various endonucleases and exonuclease III-mungbeannucleases, respectively, to shorten the strand of cDNA. Short strand plasmid was reconstructed as pUC118 as a vector. The resulting short stranded plasmid was used to transform host E. coli cell MV1304. Single-stranded DNA was prepared from phage particles grown by infecting helper phage M13K08 (manufactured by Takara Shuzo Co, Ltd.) in the obtained transformed cell culture medium, and the nucleotide sequence was determined by the deoxy nucleotide method.

Nucleotide sequence is shown in FIG. It was possible to obtain cDNA with a nearly full length of 1567 (Figure 2).

The 319 amino acid sequence of the polypeptide according to the present invention was found to have a 957 bp sequence starting with the fourth base G and ending with the 960th base C code.

E. coli MV1304 / pMKT7, which contains a plasmid coding full open reading frame of the polypeptide of the present invention, has been deposited as FERM BP-1262 at the Institute of Microbiological Technology at the Japan Institute of Commerce and Industry, and also as KFCC-10631 at the Korean spawn association. Deposited.

(Example 2)

Construction of plasmid for expressing PCI cDNA and transformation of host cell by plasmid

(1) Selection of Expression Vectors

As shown in Figure 2, the PCI cDNA translation initiation site ATG is located within the recognition site of restriction endonuclease Nco I. Peptide coding sites of the present invention are readily expressed by linking DNA fragments with an expression vector having an Nco I site downstream of a strong promoter.

Therefore, the expression vector pKK233-2 (Amann, E. and Brosius, J. T.), which has the trc promoter, the strong promoter of E. coli, and the ribosome binding site of LacZ and the translation initiation site ATG downstream of the 8 base of the ribosome binding site. , Gene, ##, 183-190, 1985). The translation initiation site ATG of pKK233-2 is present in the restriction endonuclease NcoI recognition site.

(2) Construction of PCI cDNA Expression Plasmid

Plasmid pMKT7 containing the PCI cDNA prepared in Example 1 was digested with restriction endonucleases Nco I and Hind III, and low melting point agarose gel electrophoresis [Bernard Perbal, Shigeyasu Kobayashi translation, Practical Handbook of Gene Manipulation Experiments , pp 211-222 (1985), isolated a 1308 (second) bp DNA fragment containing the region encoding the polypeptide of the invention. Pre-limiting endonucleases

The vector pKK233-2 cut with Nco I and Hind III and this DNA fragment were linked using a DNA ligase kit (manufactured by Takara Shuzo Co., Ltd.) to construct a recombinant vector pMKTX1 for expression (FIG. 3).

3) The method proposed by Vistas and Shamanis for the plasmid pMKTX1 constructed in step (2) [Hanahan, D .: DNA Cloning: A Practical Approach, Vol. 1, (D.M. Glover, ed.), Pp 121, (1985) IRL Press, Oxford]. Host E. coli JM105 [C. Yanisch-Perron, J. Vieira and J. Messing: Improved M13 phage C1oning Vectors and Host Strains-Nucleotide Sequences of the M13mp18 and pUC19 Vectors, Gene, 33, 103-119 (1985)]. Transformed cells E. coli JMl05 / pMKTX1 is selected on LB agar plates (1% bacto-trypton, 0.5% bacto-east extract, 1% sodium chloride, 1.5% agar) containing 100 μg / ml of ampicillin.

The transformed cells E. coli JM105 / pMKTX1 were deposited as FERM BP-1407 at the Japan Institute of Commerce, Industry and Technology, Microbial Technology Research Institute, and re-deposited as KFCC-10630 at the Korean spawn association.

(Reference Example 2)

Confirmation of Polypeptide Expression of the Present Invention by Western Blotting Method

E. coli JMl05 / pMKTX1 prepared in Example 2 was incubated overnight at 37 ° C. on an LB agar plate containing 100 μg / ml ampicillin. 50 ml of SOC medium (20 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 mM sodium chloride, 2.5 mM calcium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM glucose, 50 µg / ml ampicillin), which were divided into 500 ml Erlenmeyer flasks. Distilled water 11: pH 7.0). Transformed cells were incubated at 37 ° C. for 2 hours with shaking. Isopropyl-β-D-thiogalactopyranoside is added to a final concentration of 1 mM and incubated for another 2 hours with shaking. The cells are collected and suspended in 0.2 ml of TBS (10 mM Tris-HCl buffer, 150 mM sodium chloride pH 8.0). Lisozyme, ribonuclease and deoxyribonuclease are added at each 100 μg / ml rate. The resulting suspension is frozen with dry ice / ethanol and then thawed. The freeze and thaw process is repeated and the suspension is left for 15 minutes at room temperature. EDTA is added to a final concentration of 20 mM to obtain a cell lysate. Centrifuge the cell lysate through a "Millipore" filter to obtain an E. coli JM05 / pMKTX1 extract.

15 μl of the extract thus obtained is reduced to β-mercaptoethanol and then subjected to SDS-polyacrylamide electrophoresis (10% gel). Western blotting is performed using anti-human placenta derived PCI monoclonal antibody PCI-A46 and a protoblot immunoassay system (promega Biotec Corp.). As a result, the expression of the polypeptide of the present invention was confirmed to have the same molecular weight as that of human placenta derived PCI (FIG. 4).

(Reference Example 3)

Polypeptide Production of the Invention

1) Culture

Transformed cells E. coli JMl05 / pMKTX1 is incubated overnight at 37 ° C. on an LB agar plate containing 100 μg / ml of ampicillin. The resulting colonies were inoculated into LB medium (proposed agar from LB agar) containing 50 µg / ml of 5 ml of ampicillin, each of which was divided into four L-tubes. 50 ml of MMCA medium (10.5 g of potassium diphosphate, 4.5 g of potassium phosphate, 1.0 g of ammonium sulfate, 0.5 g of sodium citrate), injected into a 500 ml Erlenmeyer flask (40), 0.5 ml of each culture incubated overnight at 37 ° C. under shaking. 0.2 g of magnesium sulfate, 2.0 g of glucose, 5.0 μl of hydrogen thiamine chloride, 5.0 g of casamic acid, 50 μg / ml ampicillin, distilled water 11: pH 7.0). The transformed cells were incubated at 37 ° C. under agitation for 4 hours, and then cultured under shaking for 2 hours and 30 minutes by adding isopropyl-β-D thiogalactopyranoside to a final concentration of 1 mM.

(2) extraction and purification

(a) Cells are collected and suspended in 50 ml of 25 mM Tris-HCl buffer (pH 7.4) containing 25 mM EDTA. The suspension is centrifuged to obtain washed cells. Washed cells are suspended in 75 ml of the same buffer, lysed, and centrifuged to obtain supernatant. The residue is resuspended in 75 ml of the same buffer, lyophilized and centrifuged to give the added supernatant. Combine the two supernatants and remove impurities with 30% saturated ammonium sulfate. Creating solution

Dialysis was performed on 25 mM Tris-HCl buffer (pH 7.4) to give 244 ml of E. coli extract.

(b) The E. coli extract obtained in step (a) is adsorbed onto a DEAE-Toyopearl column (ø1.6 × 10 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.4). The column is washed thoroughly with the same buffer. Elution at a rate of 3 ml per fraction was carried out using 240 ml of the same buffer in each portion containing 0 to 0.3 M sodium chloride, and a fraction containing the polypeptide according to the invention was obtained in a total volume of 39 ml. . In some cases, the amount of each fraction is measured by the Enzyme-Linked Immunosorbent Assay (ELISA) method.

(c) The fraction containing the polypeptide of the present invention obtained in the above step (b) was charged to a monoclonal antibody PCI-A46 antibody column (ø3 × 4 cm), and to the borosilicate activated cepharose 4B. Combine. The column was thoroughly washed with 0.1 M Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, followed by elution at 2 ml per fraction with 50 ml of 0.1 M sodium acetate buffer (pH 50) containing 0.5 M sodium chloride. In total, 20 ml of a fraction containing the polypeptide according to the present invention was obtained. This fraction was concentrated by "Immersable CX-10" (Millipore) and purified by dialysis with 25 mM Tris-HCl buffer (pH 7.4) containing 0.15 M sodium chloride (protein 7.3 ml) (protein Weight 4.7 mg, determined by BIO-RAD PROTEIN ASSAY). The obtained polypeptide according to the present invention is considered to be a single component containing no impurities from showing a single band in SDS-polyacrylamide gel electrophoresis. In addition, the quantity of the protein obtained by each purification process is as follows.

(Reference Example 2)

Properties of Polypeptides According to the Invention

(1) N-terminal amino acid sequence determination

2 mg of the polypeptide according to the invention was dialyzed against distilled water, lyophilized, and then converted to PTH amino acid using JEOL JAS-570K sequence analyzer and analyzed. The analysis results are shown in the table below.

* nd. : Not detected

From the above results, the polypeptide N-terminal amino acid sequence according to the present invention was determined as follows.

Ala -Gln-Va1- Leu -Arg -Gly-Thr -Va1-Thr -Asp-

(2) molecular weight measurement

After reduction of the polypeptide of the present invention to 2-mercaptoethanol, the molecular weight was determined by the Lambri method [Lammli UK: Nature, , 680-685 (1970)] was measured by 12.5% SDS- polyacrylamide gel electrophoresis. Used as molecular weight markers: phosphorylase b (mw 94,000), bovine serum albumin (mw 67,000), egg white albumin (mw 43,000), carbonic anhydrase (mw 30,000), soybean trypsin inhibitor (mw 20,100) and α-lactoalbumin (mw 14,400).

The polypeptide molecular weight of the present invention is calculated to be 34,000 ± 1,000.

(3) isoelectric point measurement

As for isoelectric point, "Amphorine Pagplate"("AMPHOLINEPAGPLATE", a brand name, pH 3.5-9.5, the product of LKB) is used, and the Rigley method (wrigley, CW: Methods Enzymol., , 559-564 (1971)], such as polyacrylamide gel was measured by all the focusability.

The polypeptide isoelectric point according to the invention was calculated to be 5.0 ± 0.1.

(4) stability determination

The stability of the polypeptides according to the invention is determined in plasma.

5 μl of the polypeptide (60 μg / ml) according to the invention is mixed with 100 μl of the human site. The resulting mixed solution is left to stand at 37 ° C. for 0 to 30 minutes in each set time. 200 μl of 0.0125M calcium chloride is added to each mixture and the extended solidification time is measured according to the calcium resolidification method. The measurement results are shown in the following table, and the extent of each extension represents 100% of the coagulation time when the coagulation time when the polypeptide of the present invention is not added.

The polypeptide according to the present invention remains stable for 30 minutes at 37 ° C. in a gallbladder.

(5) Determination of anticoagulant activity

(Iii) Calcium re-solidification time measurement

In a small glass test tube treated with silicone, 100 μl of the solution containing the polypeptide of the present invention and 100 μl of standard plasma are mixed. The mixture is allowed to stand at 37 ° C. for 3 minutes, and then 100 µl of 0.025 M aqueous calcium chloride solution is added and mixed well, and the time until the blood coagulates is measured.

In the table below, the measurement results set the coagulation time when no polypeptide according to the present invention was added to 100%, and show the percent extension of the coagulation time.

(Ii) protrumbin time measurement

50 μl of the solution containing the polypeptide according to the invention, 20 μm tris-hydrochloric acid complete solution containing 0.15 M sodium chloride (pH 7.4) and thromboplastin (“Lyoplastin”, diluted to a concentration of 0.1 mg / ml with 20 mM calcium chloride aqueous solution). Trademark: Mochida Phannaceutical Co., Ltd.) 100 μl are mixed in a glass cuvette. The resulting mixture is allowed to stand at 37 ° C. for 3 minutes and then 200 ml of standard plasma diluted twice with 0.15 M saline is added. The solidification time is measured using "COAGTECH TE-600" (trade name: Erma Inc., Tokyo).

The results measured in the table below show the percent solidification time extension as 100% of the solidification time when the polypeptide according to the present invention was not added.

(Iii) measurement of active partial tromboplastin time

50 μl of a solution containing the polypeptide according to the invention and “activated thrombofax” (“ACTIVATED THROMBOFAX”, trade name Ortho Pharmaceutical Corp.) are mixed in glass cuvette. The resulting mixture is allowed to stand at 37 ° C. for 2 minutes and then 100 μl of plasma is added. The mixture thus obtained is allowed to stand for at least 4 minutes, and 200 µl of 0.0125 M calcium chloride solution is added, and the solidification time is measured using "COAGTECH TE-600" (trade name Erma Inc., Tokyo).

In the table below, the measurement product shows the percent solidification time extension as 100% of the solidification time when the polypeptide according to the present invention was not added.

(Reference Example 4)

Comparison of anticoagulant activity between polypeptide and PCI according to the present invention

The anticoagulant activity of the polypeptide according to the present invention and the anticoagulant activity of human placenta derived PCI were compared. One hundred microliters of each sample of the different concentrations described above are mixed into 100 microliters of 0.5 mg / ml PT reagent (`` Lyoplastin '' (trade name: manufactured by Mochida Pharmaceuticals)). After 3 minutes, 200 μl of standard site diluted twice with physiological saline is added and the coagulation time is measured.

As shown in Table 1 below, the polypeptide according to the present invention and the human placenta derived PCI showed the same coagulation time (PT) extension effect.

TABLE 1

Anticoagulant Activity of Polypeptides and Human Placenta Derived PCI According to the Present Invention

Reference Example 5

Inoculate and dissolve 2 ml of distilled water. The solution thus obtained is filled into sterile glass bottles and pre-frozen at -30 ° C to -40 ° C for 2 hours. It is then produced by primary drying for 35 hours at −30 ° C. to ± 20 ° C. and 0.05 to 0.1 Torr, secondary drying for 5 hours at 30 ° C. and 0.01 to 0.05 Torr, and injecting into a glass bottle.

Claims (6)

DNA fragment having a nucleotide sequence capable of encoding the following amino acid sequence: Or a DNA fragment having a nucleotide sequence complementary to the nucleotide sequence. The DNA fragment of claim 1, wherein the nucleotide sequence has the following sequence; A DNA fragment having a nucleotide sequence complementary to said nucleotide sequence. A DNA fragment having a nucleotide sequence capable of encoding the following amino acid sequence, Or a DNA fragment having a nucleotide sequence complementary to the nucleotide sequence; And a recombinant plasmid consisting of a replicable vector. The recombinant plasmid of claim 3, wherein the recombinant plasmid is sequentially added in the downstream direction of transcription to the following (1)-(6): (1) Promoter (2) Ribosome binding site: (3) initiation codon: (4) DNA having a nucleotide sequence capable of encoding the following amino acid sequence: (5) end codon; And (6) Recombinant plasmid consisting of a transcription terminator. A DNA fragment having a nucleotide sequence capable of coordinating the following amino acid sequence, Or a DNA fragment having a nucleotide sequence complementary to the nucleotide sequence; And a recombinant plasmid consisting of replicable vector DNA, wherein the host tax table is E. coli MV1304 / pMKT7, E. coli JM105 / pMKTX1, hybridoma PCI-H39, hybridoma PCI-167, hybridoma PCI-H169 Or transformed cells that are hybridoma PCI-H180. The transformed cell of claim 5, wherein the following (1)-(6) sequentially in the transcription downstream direction; (1) promoter: (2) Ribosome binding site: (3) initiation codon: (4) DNA having a nucleotide sequence capable of encoding the following amino acid sequence (5) termination codons; And (6) The host cell containing a recombinant vector consisting of a transcription terminator is E. coli MV1304 / pMKT7, E. coli JM105 / pMKTX1, hybridoma PCI-H39, hybridoma PCI-H39, hybridoma PCI-H46 Transformed cells, hybridoma PCI-H167, hybridoma PCI-H169 or hybridoma PCI-H180.