JPH0559099A - Rat protease inhibitor - Google Patents

Abstract

PURPOSE:To provide the title inhibitor having a specific amino acid sequence, having lipoprotein-binding protease-inhibitory activity, thus useful for screening human lipoprotein-binding protease-inhibitors for myocardial infarction therapies, etc. CONSTITUTION:The whole RNA is first extracted from rat liver by a conventional means, and using this as template, cDNA is synthesized using a reverse transcriptase and amplified by PCR technique using as PCR primer part of a gene coding lipoprotein--binding protease inhibitor (LACI). Second, the resulting DNA is treated with a restriction enzyme and incorporated into a vector to prepare a probe. Third, using this probe, the rat cDNA library is screened by plaque hybridization to select positive clone, from which a DNA is then extracted, put to restriction enzyme treatment, bound to a vector, and introduced into e.g. Escherichia coli to make a transformation. Thence the resulting transformant is cultured, thus giving the objective rat protease inhibitor having an amino acid sequence of the formula.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rat proteainase inhibitor, more specifically to a rat lipoprotein-binding protease inhibitor (LACI), and a gene (cDNA) encoding the same.

[0002]

2. Description of the Related Art It is said that the coagulation cascade in mammals has two pathways, that is, intrinsic (extrinsic) and extrinsic (extrinsic). Antithrombin III, which inhibits the activity of factor Xa and thrombin in the presence of hepari, is well known as an inhibitor of the exogenous coagulation reaction.
The specific inhibitor for factor a has been unknown for a long time. Factor VIIa activates Factor IX or Factor X by binding to cofactor Tissue Factor (TF), but it is usually extrinsic that TF leaks from tissues into the bloodstream when tissue damage occurs. It is a trigger for sexual coagulation.

However, it has long been known that there is an inhibitor of the activation reaction by tissue factor in plasma,
Rapaport et al., Extrinsic pathway inhibitor (EPI)
(Rao, LVM and Rapaport, SI, Blood, 69, 645 (1988))
, Broze, Jr. et al., Lipoprotein-associated coagul
ation inhibitor (LACI) (Broze, GJJr., et al., Blood
d, 71, 335 (1988)), and the research was advanced. In the present invention, this is called a lipoprotein-binding protease inhibitor or simply LACI.

The group of Broze, Jr. et al. Screened a human liver and placenta cDNA library in 1988 and isolated the LACI gene (Wun, TC. Et al.
J. Biol. Chem., 263, 6001 (1988)). According to the amino acid sequence deduced from the gene, this polypeptide has three Kuni
a tz-type protease inhibitor domain, and N
It has been revealed that it has a unique structure in which an extremely large amount of acidic amino acids and basic amino acids are contained near the terminal and near the C terminal, respectively. In addition, it was confirmed that the expression of this gene in animal cells (C127) inhibits the factor VIIa / TF complex and the factor Xa, and that the first and second Kunitz-type protease inhibitor domains have different functions. We obtained various findings. (Girard, TJ et al., Nature, 338, 518 (198
9)). Further, this recombinant LACI was shown to suppress transient pulmonary thrombus formation and fibrinogen reduction dependent on TF in a model experiment using rabbits, and its potential as a drug is discussed. It has become to
(Day, KC et al., Blood, 76, 1538 (1990)).

On the other hand, the physiological action of LACI is often unknown, and its relation to disease has not been clarified. It is said that a hypercoagulable state exists before the occurrence of clinically important thrombosis-related diseases such as myocardial infarction, pulmonary infarction, and cerebral infarction, and the importance of measuring coagulation / fibrinolytic markers is important. It is increasing. LACI is an inhibitor of the specific VIIa factor-TF complex that was discovered for the first time, and future research progress is expected.

[0006]

The LACI that has been studied so far is only derived from human plasma or human cultured cells, and is a rodent useful as a bovine or experimental animal suitable as a research material for coagulation factors. There are few reports on LACI. We have LA of rat plasma
CI-like activity was measured and the VLDL, LDL, and H
We have analyzed the quantitative fluctuation of each DL fraction (Thrombos
is and Haemostasis 65, 950 (1991)).

It is estimated that TF has a large species difference among coagulation-related proteins, and that LACI binding to TF also has a species difference. In fact, in experiments using recombinant human LACI, prolongation of prothrombin time in various animals was observed. Significant differences have been shown (Day.KC et al., Supra). This means that in experiments using rodents, especially rats, rat LAC
It means that it is necessary to use I.

[0008]

In order to use LACI of rat, which is an experimental animal whose coagulation kinetics can be easily observed, as an experimental material, the present inventors isolated a gene encoding it and analyzed its structure. The present invention was completed by conducting an intensive research after analyzing that it was necessary to produce a recombinant protein based on the gene sequence. That is, the present invention is rat LACI consisting of the following amino acid sequence (hereinafter, simply referred to as sequence 1). (N-terminal) Met Thr Asn Lys Leu Lys Lys Asp His Ala Phe Trp Ala Ala Val Cys Leu Leu Leu Ser Ile Val Pro Glu Leu Leu Asn Ala Leu Pro Glu Glu Asp Asp Asp Thr Ile Asn Thr Asp Ser Glu Leu Arg Pro Met Lys Pro Leu His Thr Phe Cys Ala Met Lys Ala Glu Asp Gly Pro Cys Lys Ala Met Ile Arg Ser Tyr Tyr Phe Asn Met Asn Ser His Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn Arg Phe Asp Thr Leu Glu Glu Cys Arg Lys Thr Cys Ile Pro Gly Tyr Lys Lys Thr Thr Ile Lys Thr Thr Ser Gly Ala Glu Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Phe Met Thr Arg Tyr Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Gln Phe Lys Tyr Gly Gly Cys Leu Gly Asn Ser Asn Asn Phe Glu Thr Leu Glu Glu Cys Arg Asn Thr Cys Glu Asp Pro Val Asn Glu Val Gln Lys Gly Asp Tyr Val Thr Asn Gln Ile Thr Val Thr Asp Arg Thr Thr Val Asn Asn Val Val Ile Pro Gln Ala Thr Lys Ala Pro Ser Gln Trp Asp Tyr Asp Gly Pro Ser Trp Cys Leu Glu Pro Ala Asp Ser Gly Leu Cys Lys Ala Ser Glu Lys Arg Phe Tyr Tyr Asn Pro Ala Ile Gly Lys Cys Arg Gln Phe Asn Tyr Thr Gly Cys Gly Gly Asn Asn Asn Asn Phe Thr Thr Lys Gln Asp Cys Asn Arg Ala Cys Lys Lys Asp Ser Ser Lys Lys Ser Ser Lys Arg Ala Lys Thr Gln Arg Arg Arg Lys Ser Phe Val Lys Val Met Tyr Glu Asn Ile His (C-terminal) The present invention is also a gene sequence encoding the rat LACI of Sequence 1, particularly a rat LACI gene consisting of the following nucleotide sequence (hereinafter, simply referred to as Sequence 2).

(5 ′ side) ATG ACT AAC AAA CTG AAG AAA GAC CAC GCC TTC TGG GCG GCT GTG TGT CTG CTG CTT AGC ATT GTT CCT GAG CTT CTT AAT GCT CTG CCC GAG GAA GAC GAT GAT ACA ATA AAC ACA GAT TCC GAG CTG CGG CCA ATG AAA CCG TTG CAT ACA TTC TGT GCA ATG AAG GCG GAA GAC GGG CCC TGC AAA GCA ATG ATA CGG AGT TAT TAT TTC AAT ATG AAC AGT CAC CAG TGT GAA GAA TTT ATA TAC GGG GGA TGC AGA GGA AAC AAG CAC TTT GAT ACT CTG GAA GAG TGT AGG AAA ACA TGC ATA CCA GGT TAT AAA AAG ACA ACT ATA AAG ACA ACA TCT GGA GCA GAA AAG CCA GAT TTC TGC TTC TTG GAA GAG GAT CCT GGA ATC TGC CGA GGT TTC ATG ACC AGG TAT TTT TTT TTT AAC AAC CAG TCA AAG CAG TGT GAA CAA TTC AAG TAC GGC GGT TGC CTG GGC AAC AGC AAC AAC TTT GAG ACC TTG GAG GAG TGC AGG AAC ACC TGT GAG GAT CCA GTG AAT GAG GTA CAG AAG GGT GAC TAC GTA ACT AAT CAAATT GTA ACT GAT CGC ACT ACT GTA AAT AAC GTG GTG ATT CCT CAG GCT ACC AAA GCG CCC AGC CAA TGG GAC TAT GAT GGC CCT TCA TGG TGT CTT GAA CCA GCA GAC AGT GGA TTA TGT AAA GCC AGT GAG AAA AGA TTC TAC TAC AAC CC A GCC ATT GGG AAA TGC CGT CAA TTT AAC TAC ACT GGA TGT GGG GGA AAT AAT AAT AAT TTT ACT ACC AAA CAA GAT TGT AAT AGA GCA TGT AAA AAA GAT TCC TCC AAA AAA AGC TCA AAA AGA GCA AAG ACC CAA AGA AGA AGG AAG TCT TTC GTG AAA GTC ATG TAC GAA AAT ATT CAT (3 ′ side) Further, the present invention is a gene sequence such as a plasmid containing the rat LACI gene.

Furthermore, the present invention comprises an operation of transforming a host cell with a gene sequence containing the rat LACI gene,
Rat LACI including the operation of culturing the host cell
Is a manufacturing method. The present invention will be described in detail below.

Rat LA newly provided by the present invention
CI is a protein containing 302 amino acids and has a methionine (Me) group at its N-terminus, as shown in Sequence 1.
t). Rat LA provided by the present invention
The CI includes not only a protein having the same primary structure as Sequence 1 but also a rat LACI-like protein to which other amino acids or peptides at the N-terminal side or the C-terminal side, and further, a protein is added.

In addition, one or more amino acids in the 302 amino acid sequence are deleted or replaced with other amino acids within the range in which the rat LACI activity is not lost, or the original 302 amino acids in the range in which the activity is not lost. One or more amino acids may be inserted into the amino acid sequence of.

The rat LACI of the present invention can be produced by the genetic engineering method described later.

The gene encoding rat LACI has the gene described above for rat LAC when its codon is translated.
All genes that express I are included. An example of such a gene is the rat LACI gene consisting of the nucleotide sequence shown in Sequence 2. By the way, the rat LACI gene consisting of the base sequence shown in Sequence 2 has the same base sequence as the LACI gene existing in the rat genome, and a part thereof is used as a probe to prepare a rat gene library. -It is particularly preferable when the (full-length) rat LACI gene is prepared from-etc.

The rat LACI gene provided by the present invention can be prepared from the rat gene library as described above, but may be prepared by a chemical synthesis method. Further, the rat LACI gene may be composed of deoxyribonucleic acid or ribonucleic acid, but is excellent in stability when genetically used to produce rat LACI. The rat LACI gene consisting of deoxyribonucleic acid is preferred.

The gene sequence containing the rat LACI gene is, for example, a gene sequence containing the nucleotide sequences of the 5'and / or 3'untranslated regions as shown in the Examples below, or a promoter sequence, etc. And a vector for cloning / expression of rat LACI gene, etc., which results in expression of rat LACI gene and is capable of producing rat LACI. The shape of these vectors may be in the form of a fuzz or a plasmid, but a plasmid is preferable in view of long-term stability and the like. For example, these vectors
From the viewpoint of self-replication and the like, a plasmid-like one is usually used.

The gene sequence containing the rat LACI gene is, for example, rat L if it is the expression vector described above.
In addition to the ACI gene, a promoter for controlling expression, a gene for replicating the gene sequence itself, and a terminator.
It preferably contains a region, a stop codon, a translation initiation sequence and the like. Here, the promoter is appropriately determined in relation to the host used to express rat LACI, and for example, if animal cells are used as host cells, SV40
Examples thereof include a promoter.

The method of preparing a gene sequence containing the rat LACI gene described above will be briefly described in the case of preparing a vector for expressing rat LACI. The promoter, rat LACI gene, stop codon, and terminator The ratter LAC is continuously connected to the miner area from the 5'side.
It can be exemplified that the I gene is ligated so as to express rat LACI, and further the gene for self-replication is ligated to form a ring. The bond between the bases is T ₄ DN
A ligase or the like can be used.

Rat LACI can be produced by transforming an appropriate host cell with the gene sequence containing the rat LACI gene described above and culturing it. The host cell may be selected from those capable of functioning as a promoter that controls the expression of the rat LACI gene contained in the gene sequence containing the rat LACI gene.
In other words, the promoter may be selected according to the host cell used.

Natural LACI produced in rat is expected to be a glycoprotein to which a sugar chain is added. Therefore, when producing a rat LACI to which a sugar chain is added, it is preferable to use a CHO cell or a COS cell or the like to which a sugar chain can be added. When such an animal cell is used as a host cell, rat LACI produced in the cell is released into the culture medium, which is preferable in the subsequent extraction and purification operations. Here, the SV40 promoter described above can be exemplified as a promoter capable of functioning in these cells.

The transformation of the host cell and the culture operation of the transformed host cell may be carried out according to a usual method. When the promoter used has a property of functioning by receiving an external stimulus, the promoter used during the culture operation causes the promoter to function from beginning to end. If it is of a nature, rat LACI can be produced by simply continuing the culture operation.

[0022]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of probe Total RNA was extracted from rat liver according to a standard method, and total RNA was extracted.
CDNA was synthesized using NA as a template. Specifically, RN
A (1 μg / μl) 8 μl to oligo dT (0.3 μg / μl) 10 μl
And 50 μl of distilled sterilized water were added and treated at 70 ° C for 10 minutes. Placenta-derived RNase inhibitor (114 U / μl)
4 μl, 5x concentrated buffer mixture (0.25M Tris-HCl (pH 8.
3), 0.73M KCl, 15mM MgCl ₂ , 5mM DTT) 20μl, 1
5 μl of 0 mM dNTP and 4 μl of reverse transcriptase (34.8 U / μl) were added and incubated at 37 ° C. for 30 minutes.
DNA was synthesized.

A 10-fold concentrated buffer mixture (0.1 M Tris-HCl (pH 8.3), 0.5 M KCl, 15 mM MgCl was added to 5.3 μl of the above reaction solution.
_2. Add 5 μl of 0.1% gelatin (w / v) and sterile distilled water,
The total volume was 50 μl. Two types of PCR primers
(40 pmole / μl) 1.25 μl each and Taq DNA polymerase
(5 U / μl) 0.25 μl was added and PCR was performed. As a PCR primer, Wun, TC. Et al. (J. Biol. Chem., 263,
6001 (1988)) of the four oligonucleotides corresponding to 431 position from 413 th, ^{5 'GAATTCTTGAAAGTCTGGAAGAGT}
G ^{3 '} , ^5' GAATTCTTGAAAGTCTGGAAGAATG ^{3 '} , ^5' GAATTCTTGA
AAGTCTGGAGGAGTG ^{3 '} , ^5' GAATTCTTGAAAGTCTGGAGGAATG ^{3 '}
When four types of oligonucleotides corresponding to 637 position from 619 ^{th, 5 'GAATCCCCAGTGTCTCAAAATTGTT 3', 5} 'GAAT
CCCCAGTGTCTCAAAATTATT ^{3 ', 5'} GAATCCCCAGTGTCTCAAAGTT
GTT ^{3 ′} and ^{5 ′} GAATCCCCAGTGTCTCAAAGTTATT ^{3 ′} were chemically synthesized and used. The reaction cycle is 94 ° C (1 minute), 25 ° C (1 minute), 72 ° C (1 minute), 10 cycles, and 95 ° C (1 minute), 45 ° C (1 minute), 72 ° C (3 minutes). The above cycle was repeated 20 times.

After the reaction was completed, the amplified DNA was converted into Eco RI-B.
After digestion with am HI, it was incorporated into the pUC13 vector to complete the preparation of the probe.

Example 2 Rat cDNA Library
The rat cDNA library (manufactured by Clontech) incorporated into the λgt10 vector was purchased and screened by plaque hybridization using the above probe. Specifically, from pUC13 vector to Eco RI
The probe sequence was excised with -Bam HI, and this was labeled with radioactivity using a multipriming kit (manufactured by Amersham) and α- ³² PdCTP according to a standard method. Screening 3 × 10 ⁶ clones from the cDNA library (hybridization conditions 7% PEG, 10% SDS, 60%
℃, filter cleaning conditions 0.1 x SSC, 0.1% SDS, 60
As a result, three positive clones were obtained. These were considered to be the same clones because they had the same restriction enzyme cleavage mode. CDN for one of these
The entire sequence of the A insert was analyzed by the dideoxy method (Sanger, F. et al., Pr.
oc. Natl. Acad. Sci. USA 74, 5463 (1977)). As a result, a rat LACI gene (cDNA) containing a 5'-untranslated region lacking the 3'-end was prepared. This fragment corresponds to the nucleotide sequence from 1 to 836 of the full-length rat LACI gene shown in FIGS.

Example 3 Cloning of the portion containing the 3'end of mRNA In order to clone the portion containing the 3'end of rat LACI mRNA, Frohman, FA and Martin, GR (Techniqu) were used.
The principle of the Nested PCR method developed by e 1, 165 (1989)) was applied. First, rat liver mRN was prepared by the method described in Example 1.
CDNA was synthesized from A. However, in this example, Xho I-Sal I was added to the 5'side of oligo dT instead of oligo dT.
Oligonucleotides (adapter-d
T), using a 5 ^{'GACTCGAGTCGACATCGTTTTTTTTTTTTTTTTT 3'.}

Nested PC using the obtained cDNA as a template
R was performed. Oligonucleotides first round of PCR is equivalent to 639 th of the base sequence from 616 th rat LACI gene, the ^{5 'TCCAGTGAATGAGGTACAGAAGGG 3',} Xho
I-Sal I adapter sequences are linked in -, ^{5 'GACTCGAGTCGA}
Between CATCG ^{3 ',} the second round of PCR rat LACI
Oligonucleotides corresponding to 748 position from 748 th gene, the ^{5 'AAGCTTGGATCCATGGTGTCTTGAACCAGCAGAC 3',}
Performed with the above adapter.

The composition of the reaction solution for the first PCR was 1 μl of the above template cDNA, 5 μl of a 10-fold concentrated buffer solution, and 10 mM dN.
TP 1 μl, 2 kinds of PCR primers (40 pmole / μ
l) 1.25 μl each, Taq DNA polymerase (5 U / μl) 0.25 μl
The temperature condition was set so that the cycle of 94 ° C (1 minute), 45 ° C (2 minutes), 72 ° C (3 minutes) was repeated 40 times. The second PCR was carried out using 1 μl of the above reaction product as a template instead of cDNA under the same reaction solution composition and temperature conditions as in the first PCR.

[0030] The second round PCR products agarose - Sugeru electrophoresed, ^{5 'GAGAAAAGATTCTACTACAACCCAG 3'} consists of 791 th oligonucleotide (rat LACI 815
It was confirmed by hybridization that the target DNA was amplified.

The PCR product was cleaved with Sal I and Bam HI and Blu
Cloning was completed by incorporating it into the escript II SK + vector. The nucleotide sequences of 7 independent clones prepared as described above and having a fragment overlapping with the 3'side of the rat LACI gene fragment obtained in Example 2 were determined, and the consensus sequence was determined to be the final nucleotide. It was an array.

Clarified by Example 2 and this example,
The nucleotide sequence of rat LACI gene is shown in FIGS. 1 and 2.

Example 4 Preparation of full-length rat LACI gene From the recombinant phage DNA obtained in Example 2, Eco
The cDNA insert was prepared by RI digestion and used as pUC119.
The vector was inserted into the Eco RI site.

On the other hand, a Bam HI-Sal I fragment was prepared from the recombinant plasmid DNA obtained in Example 3, and this was prepared.
It was inserted into the BamHI-SalI site of pUC119 vector.

Next, a Pst I restriction site was introduced into the overlapping region of each cDNA insert by the following method. First, from the 752nd base (T) in FIG.
Of 774 th (G) corresponding to a sequence, 763-th base substitution oligonucleotides (A) is T, ^{5 'TGTCTTGA}
Chemically synthesized ACCTGCAGACAGTGG ^{3 ',} this template primer - as each of pUC119 black - introducing a mutation into training rats LACI gene (fragment), the same oligonucleotide pro - used as a blanking hybridization -It was confirmed that the mutation was introduced by performing a cation test and a Pst I digestion experiment.

Subsequently, the above-mentioned mutation-introduced plasmid DNA containing the 5'side fragment was digested with Eco RI-Pst I to obtain about 770 bp.
Was prepared. The mutation-introduced plasmid containing the 3'side fragment was similarly digested with Eco RI-Pst I, and the above-mentioned cDNA of about 770 bp was inserted.

[0037] In order to remove the introduced mutation, oligonucleotides corresponding to 774 position from 752 th base in FIG. 2 (T) (G), 5 'TGTCTTGAACCAGCAGACAGTGG 3'
Was chemically synthesized, and using this as a template primer, the mutation was introduced again to prepare a plasmid (pUC119) containing the full-length rat LACI gene (translation region).

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, rat LACI, a rat LACI gene encoding the same, a gene sequence containing the rat LACI gene, an operation of transforming with a gene sequence containing the rat LACI gene, and a transformed host cell are cultured. It is a method for producing rat LACI including the operation of

The present invention is, for the first time, to clarify the primary structure of rat LACI and the nucleotide sequence of the gene encoding it, whereby rat LCI can be genetically engineered.
It becomes possible to manufacture ACI.

According to the rat LACI of the present invention, it is possible to know the original physiological activity of LACI using a rat useful as an experimental animal system. Further, in rat LACI produced by genetic engineering, it becomes possible to freely produce rat LACI with or without addition of a sugar chain. Therefore, it is also possible to investigate the role of sugar chain in LACI. Useful. In addition, if a factor that inhibits the activity of rat LACI can be screened in an experimental system using rats, it becomes an important finding for the screening of human LACI inhibitor.

The rat LACI gene is the rat LACI
Has an especially important meaning in the production of For example, if rat LACI is to be obtained from a rat, a large amount of rat is required and a complicated purification operation must be performed. Moreover, when obtained in this way, it is expected that operational loss will occur, and it will be difficult to prepare rat LACI in large amounts, and contamination with other contaminating proteins and the like is unavoidable. .. On the other hand, if the rat LACI gene of the present invention is used for genetic engineering production, a large amount of rat LACI can be produced without contamination with other rat proteins.

The rat LACI gene provided by the present invention guarantees the production of rat LACI in heterologous cells such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, insect cells and mammalian cells. It is possible to freely produce rat LACI with or without added sugar chains. Furthermore, it can be used as a material for preparing a rat LACI derivative or as an antisense regulator.

[Brief description of drawings]

1 shows rat L prepared in Example 4. FIG.
It shows a part of the base sequence of the ACI gene.
The first base (G) to the 810th base (A) are shown. In the figure, the upper symbol in the second row is D
It shows the base of NA, and the symbol at the bottom shows the amino acid encoded by the codon located at the top (the symbol has the same meaning as that used in normal biochemistry). .. The numbers in the figure indicate the number of the base when the base at the 3'end of the obtained gene is counted as 1. The symbol * in the figure indicates that it is continuous with * in FIG. In FIG. 1, the 1st base (G) to the 88th base (A) are the 5'-side untranslated region, and the bases after the 89th base (A) are the translated region. The initiation codon ATG is determined in consideration of homology with human LACI and the like.

FIG. 2 is a rat L prepared in Example 4.
It shows a part of the base sequence of the ACI gene.
The 811th base (C) to the 1228th base (A) are shown. In the figure, the meanings of the symbols and the like written in the two columns are the same as the meanings of the symbols in FIG.
It is shown to be continuous with * inside. In FIG. 2, 9
The base before the 94th base (T) is a translation region and is 995
The bases after the th base (T) are the 3'untranslated region.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/02 C 8214-4B // A61K 37/64 ACB 8314-4C C12N 5/10 9/99

Claims (6)

[Claims] 1. A rat protease inhibitor having the following amino acid sequence. (N-terminal) Met Thr Asn Lys Leu Lys Lys Asp His Ala Phe Trp Ala Ala Val Cys Leu Leu Leu Ser Ile Val Pro Glu Leu Leu Asn Ala Leu Pro Glu Glu Asp Asp Asp Thr Ile Asn Thr Asp Ser Glu Leu Arg Pro Met Lys Pro Leu His Thr Phe Cys Ala Met Lys Ala Glu Asp Gly Pro Cys Lys Ala Met Ile Arg Ser Tyr Tyr Phe Asn Met Asn Ser His Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn Arg Phe Asp Thr Leu Glu Glu Cys Arg Lys Thr Cys Ile Pro Gly Tyr Lys Lys Thr Thr Ile Lys Thr Thr Ser Gly Ala Glu Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Phe Met Thr Arg Tyr Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Gln Phe Lys Tyr Gly Gly Cys Leu Gly Asn Ser Asn Asn Phe Glu Thr Leu Glu Glu Cys Arg Asn Thr Cys Glu Asp Pro Val Asn Glu Val Gln Lys Gly Asp Tyr Val Thr Asn Gln Ile Thr Val Thr Asp Arg Thr Thr Val Asn Asn Val Val Ile Pro Gln Ala Thr Lys Ala Pro Ser Gln Trp Asp Tyr Asp Gly Pro Ser Trp Cys Leu Glu Pro Ala Asp Ser Gly Leu Cys Lys Ala Ser Glu Lys Arg Phe Tyr Tyr Asn Pro Ala Ile Gly Lys Cys Arg Gln Phe Asn Tyr Thr Gly Cys Gly Gly Asn Asn Asn Asn Phe Thr Thr Lys Gln Asp Cys Asn Arg Ala Cys Lys Lys Asp Ser Ser Lys Lys Ser Ser Lys Arg Ala Lys Thr Gln Arg Arg Arg Lys Ser Phe Val Lys Val Met Tyr Glu Asn Ile His (C-terminal) 2. A rat proteainase gene encoding the amino acid sequence of claim 1. 3. The rat protease inhibitor gene according to claim 2, comprising the following nucleotide sequence. (5 'side) ATG ACT AAC AAA CTG AAG AAA GAC CAC GCC TTC TGG GCG GCT GTG TGT CTG CTG CTT AGC ATT GTT CCT GAG CTT CTT AAT GCT CTG CCC GAG GAA GAC GAT GAT ACA ATA AAC ACA GAT TCC GAG CTG CGG CCA ATG AAA CCG TTG CAT ACA TTC TGT GCA ATG AAG GCG GAA GAC GGG CCC TGC AAA GCA ATG ATA CGG AGT TAT TAT TTC AAT ATG AAC AGT CAC CAG TGT GAA GAA TTT ATA TAC GGG GGA TGC AGA GGA AAC AAG AAC CGA TTT GAT CTG GAA GAG TGT AGG AAA ACA TGC ATA CCA GGT TAT AAA AAG ACA ACT ATA AAG ACA ACA TCT GGA GCA GAA AAG CCA GAT TTC TGC TTC TTG GAA GAG GAT CCT GGA ATC TGC CGA GGT TTC ATG ACC AGG TAT TTT TAT AAC AAC CAAC TCA AAG CAG TGT GAA CAA TTC AAG TAC GGC GGT TGC CTG GGC AAC AGC AAC AAC TTT GAG ACC TTG GAG GAG TGC AGG AAC ACC TGT GAG GAT CCA GTG AAT GAG GTA CAG AAG GGT GAC TAC GTA ACT AAT CAA ATT GAT GTA ACT CGC ACT ACT GTA AAT AAC GTG GTG ATT CCT CAG GCT ACC AAA GCG CCC AGC CAA TGG GAC TAT GAT GGC CCT TCA TGG TGT CTT GAA CCA GCA GAC AGT GGA TTA TGT AAA GCC AGT GAG AAA AGA TTC TAC TAC AAC CCA GCC ATT GGG AAA T GC CGT CAA TTT AAC TAC ACT GGA TGT GGG GGA AAT AAT AAT AAT TTT ACT ACC AAA CAA GAT TGT AAT AGA GCA TGT AAA AAA GAT TCC TCC AAA AAA AGC TCA AAA AGA GCA AAG ACC CAA AGA AGA AGG AAG TCT TTC GTG AAA GTC ATG TAC GAA AAT ATT CAT (3 'side) 4. A gene sequence containing the rat proteaase inhibitor gene according to claim 2. 5. The gene sequence according to claim 4, which is a vector for cloning or expression of the rat proteaase inhibitor gene. 6. A method for producing a rat protease inhibitor, which comprises an operation of transforming a host cell using the gene sequence according to claim 4 and an operation of culturing the host cell.