TW555764B - Human bikunin - Google Patents

Abstract

The instant invention provides for proteins, polypeptides, nucleic acid sequences, constructs, expression vectors, host cells, pharmaceutical compositions of, and methods for using human placental bikunin, serine protease inhibitor domains, and fragments thereof.

Description

555764 A7 B7 V. Description of the invention (1) Field of the invention The composition of the present invention relates to a protein domain which can inhibit serine protease activity. The present invention also relates to a nucleic acid construct, a vector, and a host cell for producing a serine protease inhibitory protein, a pharmaceutical composition containing the protein, and uses thereof. BACKGROUND OF THE INVENTION The problem raised Blood loss is a serious complication of major surgery, such as open heart surgery and other complicated steps. Cardiac surgery patients are the bulk of blood loss. Blood transfusions are at risk for disease infection and adverse reactions. In addition, donated blood is expensive and demand often exceeds supply. Pharmacological methods to reduce blood loss and the need for transfusion results have been described (Overview by Scott et al., Ann Thorac. Surg. 50 · 843-851, 1 " 0). Protein Serine Protease Inhibitor Aprotinin, a Kunitz family bovine serine protease inhibitor, is the active ingredient of the drug Trasylol®. Trasylol® has been reported to be effective in reducing blood loss during surgery (Royston et al., Lancet ii: 1289-1291, 1987; Dietrich et al., Thorac. Cardiovasc. Surg. 37: 92-98, 1989; Fraedrich et al., Thorac. Cardiovasc. Surg. 37: 89-91, 1989); W. van Oeveren et al. (1987), Ann Thorac. Surg. 44, pp 640-645; Bistrup et al., (1988) Lancet I, 366-367) but has also reported adverse effects, including hypotension and flushing (Bohrer et al., Anesthesia 45: 853-854, 1990) and allergic reactions (Dietrich et al., Supra ). However, for patients who have been previously exposed to aprotinin, it is not recommended to use -4. This paper size is applicable to Chinese national standard ^ (CNs) A4 (210/297 mm 1 (Please read the precautions on the back first) —Installation — write this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (2) Aprotinin (Dietrich et al Above). Trasylol® has been used to treat hyperfibrinolytic bleeding and traumatic hemorrhagic shock. Aprotinin is known to inhibit many serine proteases, including trypsin, chymotrypsin, plasmin, and Kallikrein and its therapeutic application in the treatment of acute pancreatitis, various symptoms of shock, fibrinolytic hemorrhage and myocardial infarction (Trapnell et al., (1974) Brit J_ Surg. 61 ·· 177 J. McMichan et al., (1982) Circulatory Shock 9: 107; Auer et al., (1979) Acta Neurochir. 49: 207; Sher (1977) Am J. Obstet. Gynecol. 129: 164; Schneider (1976) ), Artzneim. -Firsch 26: 1606). It is generally known that Trasylol® can reduce blood loss in vivo through the inhibition of kallikrein and plasmin. It has been found that aprotinin (3_58, Argl5, Alal7, Ser42) and natural aprotinin are more effective in comparison. In order to improve the inhibitory efficacy of plasma kallikrein (WO 89/10374). Problems related to aprotinin Since aprotinin is derived from bovine, once a human patient is again exposed to the drug, there is a limited risk of developing an allergic reaction Therefore, human functional equivalents equivalent to aprotinin will be most useful and desirable because of the low risk of allergies. Inhibitory enzymes should be administered repeatedly at high doses, and also in rats and dogs. Has renal toxicity (Bayer, Trasylol®, Inhibitor of proteinase; Glasser et al., In "Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Kongress", Bergmann, Munchen, 1972 pp 1612-1614). There is a hypothesis Attribute this effect to the accumulation of aprotinin in the kidneys. -5- (Please read the precautions on the back first-loading-I write this page)

、 1T-line · This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 × 297 mm) 555764 A7 B7___ printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (3) Near the small tube filled with ground, this It is due to its high net positive charge (W0 93/14120). Therefore, an object of the present invention is to identify human proteins with functional activity similar to aprotinin. An object of the present invention is also related to the following human proteins, which are less charged, but exhibit the same, highly similar or improved protease specificity as aprotinin, especially in the inhibition of plasmin and kallikrein In terms of effectiveness. Such inhibitors can then be used repeatedly as a drug in human patients with a lower risk of adverse immune response and reduced renal toxicity. SUMMARY OF THE INVENTION The present invention proposes a purified human serine protease inhibitor that specifically inhibits kallikrein, and is isolated from human placental tissue via affinity chromatography. The present invention proposes a newly identified human protein, referred to herein as human placenta dicominin, which contains two seritinase inhibitor parts of the Kunitz family. In a specific embodiment, the present invention includes proteins having the following amino acid sequence: ADRERSIMDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNM 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFP RWYFDVERQ CNMLN CNNFIFI In a preferred specific example, the present invention proposes a natural human placenta diconine with the following amino acid sequence: -6-(Please read the note on the back first to write this page) -Packing-,?! Country; quasi (CNS) A4 specification (210X297 mm Ding " 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (4) ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD DSVPSSED RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK 17 0 (SEQ ID NO: 52) In one aspect, the biological activity of the protein of the invention is that it can bind and substantially inhibit trypsin, human plasma and tissue Biological activity of peptide-releasing enzyme, human plasmin and factor Xlla. In a preferred embodiment, the present invention proposes a natural human placenta diconine protein in a glycosylated form. In a further specific example, The present invention includes a natural human dicominin protein that has been shaped so that it contains at least one cysteine-cysteamine • acid disulfide bond. In a preferred embodiment, the protein contains at least one intra-chain cysteine- Cysteine disulfide bond formed between cysteine pairs selected from: CYS11-CYS61, CYS20-CYS44, CYS36-CYS57, CYS106-CYS156, CYS115-CYS139, and CYS131-CYS152, of which cysteine Amino acids are numbered according to the amino acid sequence of natural human placenta diconicin. General artisans can confirm that the protein of the present invention can be folded into an appropriate three-dimensional configuration, so that the biological activity of natural human diconicin can be maintained. None of the cysteine-cysteine disulfide bonds in the natural bond, more than one or all of them exist. In yet another preferred embodiment, the protein of the present invention is suitably folded and formed with all appropriate natural cysteine-cysteine disulfide bonds. The active protein of the present invention can be obtained from the purification of human tissue, such as placenta, or via synthetic protein chemistry techniques, as described in the following examples. It is also understood that the protein of the present invention can be obtained by molecular biology techniques, in which the paper size of the self-replication is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the note on the back first ^^ write this page) -Line 555764 A7 B7 V. Description of the invention (5) The carrier produced by (5) can express the protein of the present invention from the transformed cells. This protein can be made non-secreted or secreted from the transformed cells. In order to help the secretion of the self-transformed cells, enhance the functional stability of the translated protein, or help the folding of the dicominin protein, certain signal peptide sequences can be added to the NH2-terminal site of the natural human dicominin protein. In a specific example, the present invention therefore proposes a natural human dicominin protein having at least a portion of a complete natural signal peptide sequence. Thus a specific example of the present invention is to propose a native human Corning double prime which has at least part of the signal peptide, having the following amino acid sequence: AGSFLAWLGSLLLSGVLA _1 ADRERSIHDFCLVSKWGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100 NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 15 0 ACMLRCFRQQENPPLPLGSKVWLAGAVS 179 (SEQIDNO: 2) in a In a preferred specific example, the present invention proposes a natural human placenta diconicin protein, which has the amino acid sequence of SEQ ID NO: 52, and the complete leader fragment has the following amino acid sequence. MAQLCGL RRSRAFLALL GSLLLSGVLA- 1 (SEQ ID NO: 53) In another specific example, the present invention proposes a biconictin protein having a part of the complete leader sequence, which has the amino acid sequence of SEQ ID NO: 52, and the complete leader The fragment has the following amino acid sequence: MLR AEADGVSRLL GSLLLSGVLA -1 (SEQ ID NO: 54) -8-This paper size applies to China National Standard (CNS) A4 (210X 297 mm) (Please read the notes on the back first Copybook ) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 555764 A7 B7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (6) In the preferred naming system used here, the amino acid name is +1 Yes It is located at the NH2-terminus of the natural human placenta diconicin amino acid sequence. We can easily understand that functional protein fragments can be derived from natural human placenta dikonin, which can maintain at least part of the biological activity of natural placenta dikonin, and can act as serine protease inhibitors. . In a specific example, the protein of the present invention contains a fragment of natural human placenta diconicin, which contains at least one functional class-Kunitz moiety, and has an amino acid of natural human placental diconicin amino acid 7-159. The sequence is hereinafter referred to as "diconine (7-159)". Accordingly the present invention comprises a protein having the following amino acid sequences: IHDFCLVSKWGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100 NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQ 159 (SEQIDNO: 3) wherein amino acids corresponding to the amino acid sequence named placental natural pigment of bis Corning. Another functional variation of this specific example may be a fragment of natural human placenta diconicin, which contains at least one functional class-Kunitz-like moiety, with natural human placental diconicin amine 11-156 amine Base acid sequence, Shuang Kang Ningsu (11-156) CLVSKWGRCRASMPRWWYNYNDGDGQQVVYGGCDGNSNN 50 YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100 NYEEYCTANAVTGPCRASFPRWYFDVERNSCNN part of the plate can also be confirmed by the natural placenta, etc. (Conventional bi-single plate can be confirmed by the two-class compound): ACMLNO 156. China National Standard (CNS) Α4 specification (210X 297 mm) (Please read the note on the back π ^ write this page)-Binding · Threading · 555764 A7 _B7__ V. Fragment of Invention Description (7) > In particular, the present invention proposes a protein having a first type of -Kunitz partial amino acid sequence, which is composed of the amino acid sequence of natural human placenta bisconine amino acid 7-64, hereinafter referred to as " bi Corning (7-64) ". Therefore, a specific example of the present invention includes a protein containing at least one -Kunitz-like moiety, which has an amino acid sequence: IHDFCLVSKWGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKCATV 64 (SEQ ID NO: 4) wherein the amino acid number is equivalent to the natural human placenta double corning Amino acid sequence. Another type of protein of the present invention may be the first type-Kunitz part, which is composed of the amino acid sequence of natural human placenta diconine amino acid 11-61, and "diconine (11-61)" has the following Amino acid sequence: CLVSKWGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKC 61 (SEQ ID NO: 5) The present invention also proposes a protein with a -Kunitz-like partial amino acid sequence, which is composed of the natural human placental diconine amino acid 102-159 amino acid sequence , Hereinafter referred to as " Shuang-Cornin (102-159). Therefore, a specific example of the present invention includes a protein containing at least one Kunitz-like moiety, which has the following amino acid sequence: YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQ 159 (SEQ ID NO: 6) The amino acid number is equivalent to the amino acid sequence of natural human placenta diconicin-10- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back ^ | ^ Write this page)…, printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs __Lk 555764 A7 B7 Member of the Central Standards Bureau of the Ministry of Economic Affairs The Industrial and Consumer Cooperatives printed the fifth and eighth column of the invention description. Another type of this part can be a kind-Kunitz part, which consists of the amino acid sequence of natural human placenta bis-conine amino acids 106-156. Corningin (106.156) π has the following amino acid sequence: CTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRC 156 (SEQ ID NO: 7) Therefore, ordinary skilled artisans can understand that fragments of natural human dicominin protein can be made, which can be made It retains at least some of the biological activity of natural proteins. This kind of tablet can also be combined in different directions or multiple combinations to generate different proteins. It retains some of the natural human dikonin protein, which is the same or more biologically active. We should be able to confirm The biologically active protein of the present invention may contain more than one Kunitz part of this class, and combined with additional class-Kimitz parts from other sources. The biologically active protein of the present invention may contain more than one Kunitz class Fractions, combined with additional protein fractions from other sources, with various biological activities. The biological activity of the protein of the invention, It can be combined with other known proteins to provide a multifunctional fusion protein with predictable biological activity. Therefore, in a specific example, the present invention includes an amino acid sequence containing at least one amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7 Identical or functionally equivalent proteins. An open reading architecture that terminates early stop codons can still guide the synthesis of a functional protein. The present invention includes this different termination, and in a specific example, a protein with the following amino acid sequence can be provided: 11 _ This paper size applies to China National Standard (CNS) A4 specifications (210'〆297 mm) (please first Read the note on the back ¥ to write this page)-Packing · ， 11 lines _ 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (9) ADRERSIHDFCLVSKW (^ C ^ ASMPRWWYN \ nTCSCQIJ ^ GGCIX3SrS) S ^ 50 YLTKEECIJCKCAIVIENATQMATSRNAADSSVPSAPRRQDS 92 (SEQ ID NO: 8) In a specific example, the present invention proposes a natural human dicominin protein that is substantially purified or produced by a recombinant, which has a complete leader sequence fragment, and at least some A complete natural transmembrane region. Therefore, a specific example of the present invention is to propose a natural human dicominin with a complete leader sequence and at least a part of the transmembrane region (underlined), which is Amino acid sequence: DEST 'MLR AEADGVSRLL GSLLLSGVLA -1 2) PCR MAQLCGL RRSRAFLALL GSLLLSGVLA -1 3) XcDNA MAQLCGL RRSRAFLALL GSLLLSGVLA- 1 DADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 2) ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 3) ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 1) YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 2) YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 3) YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 DNYEEYCTANA VTGPCRASFP RWYFDVERNS CMNFIYGGCR GNKNSYRSEE 150 2) NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 3) NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 DACMLRCFRQQ ENPPLPLGSK VWLAGLFVM VLILFLGASM VYLIRVARRN 200 2) ACMLRCFRQQ ENPPLPLGSK VWLAGLFVH VLILFLGASM VYLIRVARRN 200 3) ACMLRCFRQQ ENPPLPLGSK WVLAGLFVM VLILFLGASM VYLIRVARRN 200

DQERALRTVWS SGDDKEQLVK NTYVL 2) QERALRTVWS FGD 3) QERALRTVWS SGDDKEQLVK NTYVL 5 3 5 2 12 2 2 2 where sequence 1) is EST derived from consistent SEQ ID NO: 45, 2) is a PCR pure line SEQ ID NO · 47, and 3 ) Yes; l cDNA is pure SEQ ID NO: 49. In a preferred embodiment, the protein of the present invention contains one of the amino acids of SEQ ID NO: 45, 47, or 49, wherein the protein is at the end of the last Kunitz part -12- Chinese paper standard (CNS) A4 Specifications (210X297mm) (Please read the note on the back ΐ ¾ 舄 this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 555764 A7 B7 V. The description of the invention (1〇) Dissociation. The invention also includes proteins in which the signal peptide has been deleted. Therefore, the present invention proposes a protein having the amino acid sequence of SEQ ID NO: 52, and is followed by a transmembrane amino acid sequence: EST VWLAGLFVM VLILFLGASM VYLIRVARRN 200 EST QERALRTVWS SGDDKEQLVK NTYVL 225 (SEQ ID NO ·· 69) transmembrane amine Base sequence: PCR VWLAGLFVM VLILFLGASM VYLIRVARRN 200 PCR QERALRTVWS FGD 213 (SEQ ID NO: 68) or transmembrane amino acid sequence: λ cDNA VWLAGLFVM VLILFLGASM VYLIRVARRN 200 λ cDNA QERALRTVWS SGDDKEQLVK NTYVL 225 (SEQ ID NO: 67) The protein amino acid sequence clearly shows the skilled person that a suitable nucleic acid sequence can be applied in molecular biotechnology to produce the protein of the invention. Therefore, a specific example of the present invention proposes a nucleic acid sequence that can guide the synthesis of human dicominin (SEQ ID NO: 9) with the DNA sequence of FIG. 3, which can be translated into the natural human placenta dicominin of FIG. 3. Amino acid sequence (SEQ ID NO. · 10). In another specific example, the present invention proposes a consensus nucleic acid sequence (SEQ ID NO: 51) of FIG. 4C, which guides the synthesis of the amino acid sequence (SEQ ID NO: 45) of FIG. 4D.

In a preferred specific example, the present invention proposes a guide for synthesizing tools. Figure 4F -13- This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) (please read the note on the back to write this page) * · AFin ϋϋ lh9-loaded ·

1T line 555764 A7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs ---__ B7 V. Description of the invention (11) DNA sequence of natural human placenta diconicin nucleic acid sequence (SEQ ID NO: 48), which guides the synthesis of SEq ID no: 49 protein sequence. In another specific example, the present invention proposes the nucleic acid sequence (SEQ ID NO: 46) of FIG. 4E, which guides the synthesis of the protein sequence of SEQ ID NO: 47. We can easily confirm that certain dual gene mutations and inherent substitutions in nucleic acid sequences can be made, which can still generate the protein amino acid sequences covered by the present invention. The skilled artisan can confirm that certain natural dual gene mutations of the protein of the present invention and the amino acid substitution effect inherent in the protein of the present invention do not significantly change the biological activity of the protein, and are also encompassed by the present invention. The present invention also proposes a pharmaceutical composition ' containing human placenta diconine and fragments thereof, which can be used to reduce blood loss in patients undergoing surgery during the week of surgery. The present invention also proposes a method for reducing blood loss of a patient during the operation period during the operation. This method is to administer the effective serine protease inhibitor of the present invention to the patient in a biocompatible solvent. The present invention also proposes a variant of placenta diconicin, and the above-mentioned specific Kunitz formula, which contains an amino acid substitution that can change the specificity of a protease. The preferred positions of the substitutions are shown below, such as positions Xaa1 to Xaa32 in the natural placenta diconine amino acid sequence. The substitutions from Xaa1 to Xaa16 are also better for the diconicin (7-64) variant, and the substitutions from Xaa17 to Xaa32 are better for the diconicin (102-159) variant. Therefore, the present invention includes specific proteins with the following amino acid sequences: (Please read the notes on the back to write this page first) < fi ϋ Lr. Binding · 14- Specifications (210X297 mm) 555764A7B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (12)

Ala Asp Arg Glu Arg Ser lie Xaa ^ · Asp Phe l〇Cys Leu Val Ser Lys Val Xaa ^ Gly Xaa ^ Cys 20 Xaa4 Xaa ^ Xaa6 Xaa7 Xaa8 Xaa9 Trp Trp Tyr Asn 3〇Val Thr Asp Gly Ser Cys Gin Leu Phe Xaa ^ 40 Tyr Xaa 11 Gly Cys Xaa xaa 13 14 ser Asn Asn 50 Tyr XaaThr Lys Glu Glu CyGlu Leu Lys Lys 60 Cys Ala Thr Xaa16 Thr Glu Asn Ala Thr Gly 70 Asp Leu Ser Thr Ser Arg Asn Ala Ala Asp 80 Ser Ser Val Pro Ser Ala Pro Arg Arg Gin 90 Asp Ser Glu His Asp Ser Ser Asp Met Phe lOO〇Asn Tyr Xaa17 Glu Tyr Cys Thr Ala Asn Ala ll〇Val Xaa18 Gly Xaa19 Cys Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 120 XaaTrp Tyr Phe Asp Val Glu Arg Asn Ser 130 Cys Asn Asn Phe Xaa2 ^ Tyr Xaa27 Gly Cys Xaa28 140 Xaa29 Xaa30 Lys Asn Ser Tyr Xaa31 Ser Glu Glu 150 Ala Cys Met Leu Arg Cys Phe Arg Xaa〇ln 160 Glu Asn Pro Pro Leu Pro Leu Gly Ser Lys 170 Val Val Val Leu Ala Gly Ala Val Ser 179 (SEQ ID NO: 11) · Where Xaa1-Xaa32 each independently represents a naturally occurring amino acid residue, with the exception of Cys, with the limitation that at least one of the amino residues Xaa ^ Xaa32 One different from natural Sequence of equivalent amino acid residues. In this specification, the so-called " naturally occurring amino acid residue " is used to indicate any of the 20 generally generated amino acids, namely Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Try and Val 0 Substituting more than one amino acid at more than one position shown above may change the specificity of the natural placenta diconine inhibitor Sex outline, or individual class-Kunitz part, Shuang Corning Su (7-64) or Shuang Corning Su (102-159), so its -15- (please read the note on the back II to fill-pack- : Write this page) Order-line paper size applies to Chinese National Standard (CNS) A4 (210X297mm) --- 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (13) Different Inhibit other serine proteases, such as enzymes in the complement cascade, TF / F Vila, FXa, thrombin, neutrophil elastase, cathepsin G or protease-3, but it is not limited to this. Examples of preferred variants of Corningin include wherein Xaa1 is an amino acid residue selected from the group consisting of His , Glu, Pro, Ala, Val or Lys, especially where Xaa1 is His or Pro; or where Xaa2 is an amino acid residue selected from the group consisting of Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala, Lys, especially J is where Xaa2 is Val or Thr; or where Xaa3 is an amino acid residue selected from the group consisting of: Arg, Pro, lie, Leu, Thr, especially where Xaa3 is Arg or Pro; or where Xaa4 Is an amino acid residue selected from the group consisting of: Arg, Lys and Ser, Gin, particularly where Xaa4 is Arg or Lys; or wherein Xaa5 is an amino acid residue selected from: Ala, Gly, Asp, Thr, Especially where Xaa5 is Ala; or where Xaa6 is an amino acid residue selected from the group consisting of Ser, lie, Tyr, Asn, Leu, Val, Arg, Phe, especially where Xaa6 is Ser or Arg; or where Xaa7 is An amino acid residue selected from the group consisting of: Met, Phe, lie, Glu, Leu, Thr and Val, in particular where Xaa7 is Met or lie; or where Xaa8 is an amino acid residue selected from the following: Pro, Lys, Thr, Gin, Asn, Leu, Ser or lie, especially where Xaa8 is Pn > or lie; or where Xaa9 is an amino acid residue selected from the group consisting of Arg, Lys or Leu, In particular, where Xaa9 is Arg; or where Xaa1G is an amino acid residue selected from Val, He, Lys, Ala, Pro, Phe, Trp, Gin, Leu, and Thr, especially where Xaa10 is Val; or where Xaa11 is an amino acid residue selected from the group consisting of: Gly, Ser, and Thr, particularly where Xaa11 is Gly; or wherein Xaa12 is an amino acid residue selected from: Asp 'Arg, Glu, Leu, Cln, Gly , Especially where Xaa12 is -16- (please read the note on the back first Ϊ --- write this page), 11-line-this paper size applies the Chinese National Standard (CNS) Α4 specification (210 × 297 mm) 555764 A7 B7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (14) Asp or Arg; or Xaa13 is an amino acid residue selected from the following: Gly and Ala; or where Xaa14 is an amino group selected from the following: Acid residues: Asn or Lys; or wherein Xaa15 is an amino acid residue selected from the group consisting of: Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val, and Lys, especially where Xaa15 is Leu or Lys; or Where Xaa16 is an amino acid residue selected from the group consisting of Val, Gin, Asp, Gly, lie, Ala, Met and Val, In particular, where Xaa16 is Val or Ala; or where Xaa17 is an amino acid residue selected from the group consisting of His, Glu, Pro, Ala, Lys, and Val, especially where Xaa17 is Glu or Pro; or where Xaa18 is selected from The following amino acid residues: Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala or Lys, in particular where Xaa18 is Thr; or where Xaa19 is an amino acid residue selected from the group: Arg, Pro , Lie, Leu or Thr, especially where Xaa19 is Pro; or where Xaa20 is an amino acid residue selected from the group: Arg, Lys, Gin, and Ser, especially where Xaa2G is Arg or Lys; or where Xaa21 is selected From the following amino acid residues: Ala, Asp, Thr or Gly, especially where Xaa21 is Ala; or where Xaa22 is an amino acid residue selected from the group consisting of Ser, lie, Tyr, Asn, Leu, Val, Arg or Phe, especially where Xaa22 is Ser or Arg; or where Xaa23 is an amino acid residue selected from the group consisting of: Met, Phe, lie, Glu, Leu, Thr or Val, especially where Xaa23 is Phe or lie; Or where Xaa24 is an amino acid residue selected from the group consisting of Pro, Lys, Thr, Asn, Leu, Gin, Ser, or lie, especially Xaa24 is Pro or lie; or where Xaa25 is an amino acid residue selected from the group: Arg, Lys or Leu, particularly where xaa25 is Arg; or where Xaa20 is an amino acid residue selected from the group of Val, lie, Lys, Leu, Ala, Pro, Phe, Gin, Trp, and Thr, especially where Xaa26 is Val or lie; or where Xaa27 is selected from the following -17- (Please read the note on the back first—: copy Page), 1T line-Ξ- y I ϋ-ρ · This paper size applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 555764 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 15) the amino acid residues listed: Gly, Ser and Thr, especially where Xaa27 is Gly; or where Xaa28 is an amino acid residue selected from the group consisting of Asp, Arg, Glu, Leu, Gly or Gin, particularly Where Xaa28 is Arg; or where Xaa29 is an amino acid residue selected from the group consisting of: Gly and Ala; or where Xaa3G is an amino acid residue selected from the group consisting of: Asn or Lys; or wherein Xaa31 is selected from the group consisting of Amino acid residues: Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val and Lys, especially where Xaa31 is Arg or Lys; Wherein Xaa32 is selected from the group consisting of an amine acid residues: Val, Gin, Asp, Gly, lie, Ala, Met, and Thr, in particular wherein Xaa32 is Gin or Ala. BRIEF DESCRIPTION OF THE DRAWINGS The present invention takes into account the following detailed description and patent application park, and can be understood more in conjunction with the accompanying drawings, in which: Figure 1 shows the nucleotide sequence (SEQ ID NO ·· 12) of EST R3 5464 and this DNA sequence Translation (SEQ ID NO: 13), in which an open reading architecture is generated, and some sequences are similar to aprotinin. The translation product contains 5 of the 6 cysteine acids in the correct spatial position, which is characteristic of the inhibitor region of the class-Kunitz (indicated in bold). The place normally occupied by the remaining cysteine (codon 38) is replaced by amphetamine (shown by the symbol). Figure 2 shows the nucleotide sequence of EST R74593 (SEQ ID NO: 14) and a translation of this sequence (SEQ ID NO: 15), in which an open reading framework is generated and interacts with the Kunitz class of the serine protease inhibitory portion With homogeneity. The translation product contains 6 cysteine acids in the correct spatial position, which is characteristic of the class of Kunitz-like inhibitors (shown in bold). However, this translation framework sequence includes stop codons on codons 3 and 23. -18- This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the notes on the back-Pack-: Write this page)-Line-1 555764 Α7 Β7 Central Bureau of Standards, Ministry of Economic Affairs Printed by Employee Consumer Cooperatives 5. Description of the invention (16) Figure 3 shows the deduced nucleic acid sequence (SEQ ID NO: 9) of human placenta dikonin, marked with "consistent", and translated with 丨 丨 translation 丨丨 The amino acid sequence of the translated protein matches (SEQ ID NO: 10). The ESTs H94519 (SEQ ID NO: 16), N39798 (SEQ ID NO: 17), R74593 (SEQ ID NO: 14), and R35464 are also compared together. (SEQ ID NO: 12). The nucleotides underlined in the consensus sequence correspond to the positions of the PCR primers described in the examples. In the translated consensus sequence, the amino acids underlined are homogeneous residues and have been identified by The purified natural human placenta diconicin was confirmed by amino acid sequence. Nuclear: y: acid and amino acid code as a standard one-letter code, "N " indicates an unspecified nucleic acid in the nucleic acid code, and" " Indicates a stop codon in an amino acid sequence. Figure 4A shows the original covering of a series of ESTs, with certain nucleic acid sequences homologous to ESTs encoding human placenta diconine or a portion thereof. Shown for reference are the relative positions of succinicin (7-64) and succinicin (102-159), labeled KID1 and KID2, respectively. Figure 4B shows a wider range of ESt coverage with additional ESTs. The number on the upper X-axis refers to the length of the test base pair, starting from the first test base of the most 5, est sequence. The length of each horizontal line is proportional to the length of individual ESTs base pair length (including gaps). The EST registration number is shown to the right of its individual EST line. Figure 4C shows the equivalent arrangement of the oligonucleotide sequences of the overlapping ESTs outlined in Figure 4B. The upper sequence (SEQ ID NO: 51) marked with diconicin represents a consistent oligonucleotide sequence derived from overlapping nucleotides at various positions. Numbers refer to base pair positions within the EST map. In the EST R74593, the oligos in bold and underlined: y: acid (at positions 994 and 1005) is in R74593 -19- This paper size applies Chinese National Standard (CNS) M specification (21〇 × 297 public envy ) (Please read the notes on the back to fill out this page) -5-t »

T Liang 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. The base embeddings observed in the description of invention (17) are consistently lacking in other repeated ESTs. Fig. 4D shows the amino acid translation (SEQ ID NO: 45) of the diconiconin consensus oligonucleotide sequence shown in Fig. 4C. FIG. 4E shows the nucleotide sequence (SEQ ID NO: 46) and equivalent amino acid translation (SEQ ID NO: 47) of the placenta diconine coding sequence, which are derived from humans based on PCR-based amplification Placental cDNA library. FIG. 4F shows the pure human placenta diconiconin-encoding pure nucleotide sequence (SEQ ID NO: 48) and equivalent amino acid translation (SEQ ID NO: 49), which were isolated from human placenta by colony hybridization; I cDNA library. Figure 4G compares the oligonucleotide sequence translation of placenta diconine amino acid translation | J, repeated by EST (SEQ ID NO: 45), PCR-based colonization (SEQ ID NO: 47) And traditional; I colony hybridization (SEQ ID NO: 49). Figure 5 shows the purification of human placenta diconicin from placental tissue after filtration with Superdex 75 · gel. The plot is the overlay of a protein dissolution profile, detected by OD 280 nm (solid line), the activity of the dissociated protein in trypsin inhibition analysis (% inhibition, shown in circles), and kallikrein Inhibition activity of dissociated protein in analysis (% inhibition, expressed as a square). Figure 6 shows the purification of human placenta succintocin from placental tissue using C18 reverse phase chromatography. The plot is the overlay of a protein dissolution profile, measured at OD 215 nm (solid line), the activity of the dissociated protein in the trypsin inhibition assay (inhibition%, circled), and the kallikrein inhibition assay The activity of the dissociated protein in the method (% inhibition, shown as a square). -20- (Please read the Caution Pack on the back-write this page first) The size of the paper used in this edition applies to the Chinese National Standard (CNS) A4 (210X 297 mm) Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 555764 A7- ------ B7 ______ V. Description of the invention (18) Figure 7 shows a highly purified silver-stained sdS-PAGE gel (column 2) of placenta diconine, and it is indicated by the size of the ear canal. Molecular weight indicates the protein series (column 1). Move from top to bottom. Figure 8 shows the amount of trypsin inhibitory activity in the cell-free fermented gravy grown by yeast strain SC101 (Figure 8A) or WHL341 (Figure 8B), which has been stably transformed with a plastid (pS604). The performance of placenta diconine (102-159). Figure 9 shows the silver stained SDS-PAGE (left) of cell-free yeast gravy grown on yeast strain SC101 (recombinants 2.4 and 2.5) and western blot analysis (right) with pAb anti-placenta diconine. It stably transforms with plastids that can be commanded by bovine aprotin, or placenta diconicin (102-159). The movement is from top to bottom. Fig. 10 shows a silver-stained SDS-PAGE chart (column 2) of highly purified placenta diconine (1022-159), and a series of proteins (column 1) marked by molecular weights of the canal ear volume. Move from top to bottom. Figure 1 1 shows the northern blotting results of mRNAs of various human tissues. The tissues are coded by placenta digoxigenin (102-159) (Figure 11A) or placental digoxigenin (1-213) (Figure 11B). The labeled cDNA probe is hybridized. Move from top to bottom. The number to the right of each suction strip is the size of the adjacent RNA markers based on the test pair. The organs from which mRNA is derived are described below each aspiration column.

FIG. 12 shows an immunosucking map of placenta-derived diclosynin derived from placenta, in which rabbit antibodies generated using synthetic reduced placental dictocinin (7_64) (image A) or 102 2 159 (image B) were used. Serum. In each figure, the contents are: • Molecular weight markers (column 1); natural placenta dicominin isolated from human placenta (column 2); synthetic placenta dicominin (7-64) (column 3) and Synthetic Moon A -21-This paper size applies Chinese National Standard (CNS) A4 specification (21〇: 297 mm) ~~ ----- (Please read the notes on the back first and fill in this page) --- 555764 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 ___B7 V. Description of the invention (19) Panshuang Corningsu (102-159) (column 4). Suck on a Tricine 10-20% SDS-PAGE gel, and use protein A-purified primary polyclonal antibody (8 μg IgG in 20 ml 0.1% BSA / Tris-buffered saline (pH 7.5), followed by Goat anti-rabbit secondary antibody conjugated with alkaline phosphatase expands and moves from top to bottom. Figure 13 shows 3 micrograms of highly purified placenta diconine (1_170) derived from the baculovirus / Sf9 expression line, Coomassie blue-stained 10-20% Tricine SDS-PAGE gel image (column 2). The first column is the molecular weight marker. It moves from top to bottom. Figure 14 shows Sf9-derived human placenta double Corningin (1-17) (solid circle), synthetic placenta double-conine (102-159) (open circles), or aprotinin (open squares) at an increased concentration on human plasma activated partial prothrombin Comparison of the effects of kinase time. CaCl2 initiates coagulation. The protein concentration is plotted against the doubling of the coagulation time. The uncontrolled coagulation time is 308 seconds. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a newly identified human protein, which is referred to herein as Human Placenta Twin Corningin, which contains Kunitz Serine protease inhibitor portion. The present invention also includes a pharmaceutical composition containing placenta diconine and fragments thereof, which can reduce blood loss during surgery or in patients with major trauma. The invention also proposes reducing surgery Method for intermediate or severe blood loss during surgery in which a patient is administered an effective dose of a disclosed human serine protease inhibitor in a biocompatible vehicle. Placenta diconine, its isolated fraction The better application of morphing and other deformations is to reduce trauma or blood loss during surgery. These methods and -22- This paper standard applies Chinese National Standard (CNS) to specifications (2lGx297 public celebration) --- (Please First read the note on the back mi to fill in this page)-_ _ order line-1. 555764 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (2) The product can reduce or eliminate whole blood or blood donation or The demand for blood products can reduce the risk of infection and other adverse side effects and the cost of surgery. Because: Dagger: can be used to reduce blood loss in normal patients, that is, non-congenital or its Patients with a loss of coagulation factors before surgery. The decrease in blood loss can be seen in the reduction of blood loss during surgery, 'reduced fluid loss after surgery, or both. Better surgical applications include chest and abdominal surgery, and full or partial body replacement surgery And surgery for patients with ocular epithelial injury, but it is not limited to this. The preferred thoracic surgery procedures include aortic coronary circuit, resection of the heart and aortic aneurysm ', and esophageal varicose vein surgery, coronary circuit surgery, But it is not limited to this. Preferred abdominal surgery includes: liver transplantation, radical prostatectomy, colon diverticulitis surgery, large tumor resection, abdominal aortic surgery, and duodenal ulcer surgery, liver and spleen trauma The repair is not limited to this. Preferred uses of wound treatment include stabilizing the disaster site in patients with severe injuries such as amputations or major chest / abdominal wounds. In the application examples of reducing blood loss due to surgery, it is better to administer placenta succintin before and during surgery, the separated part, or other variants, and in the application examples of proper trauma placement, placenta Shuangkangsulin is deformed, and the separated part or other variants are administered as soon as possible after the injury, and should be contained in the emergency vehicle to be moved to the disaster site. Factor XII (also known as Hageman factor) is a serine protease that is found in the circulation in an enzyme prototype (80 kD) of approximately 29.40 μg / ml (see pixley, etal, (1993) Meth · in Enz ·, 222, 51-64), and are activated by tissue and plasma kallikrein. Once activated, it can participate in the intrinsic path of coagulation, which is activated when blood or plasma contacts "foreign" or anionic surfaces. First (please read the notes on the back to fill in, write this page) --Ί. -23 This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (21) Once activated, factor Xlla Many other plasma proteases can be re-dissociated and activated, including factor XI, prokinin and C1 of the complement system. Therefore factor XII is involved in causing a hypertensive response because activated kallikrein can dissociate kallikrein release Vasokinin (aColman, (1984) J. Clin. Invest., 73, 1249) 〇 Sepsis is a disease caused by bacterial infection caused by bacterial endotoxin or lipopoly St (LPS). Factor XII and LPS exposure can cause factor XII activation. Patients with sepsis often have symptoms of intravascular coagulation, which can also be caused by LPS activating factor XII. Septic shock is caused by bacterial infection, and Fever, low systemic vascular resistance, and low arterial blood pressure. This is a common cause of death in the intensive care unit in the United States, and 75% of patients who die from septic shock have persistent hypotension (see Parillo, et al · (I989) Ann Rev. Med., 40, 469-485) 〇 Adult respiratory distress syndrome is characterized by pulmonary edema, hypoxemia, and low lung compliance. The pathogenesis of the disease is currently unknown, however The proteolytic pathway of coagulation and fibrinolysis plays an important role (see Carvalho, et al. (1988) J. Lab Clin. Med., 112: 270-277). The protein of the invention is also a novel human Kunitz-type Peptide release enzyme inhibitor, activator of factor XII. Therefore, another object of the present invention is to propose preventive or therapeutic treatments for systemic inflammatory reactions, such as septic shock, adult respiratory distress syndrome (ARDS), ex Vigilance, multiple organ failure, and diffuse intravascular coagulation (DIC). Therapeutic or prophylactic administration of the peptides of the present invention can cause the regulation of these inflammatory conditions and benefit patients. Fibrinolytic enzymes are extracellular Degradation and matrix-metal protein-24- (Please read the notes on the back to fill in this page) Binding and binding The paper size is applicable to Chinese National Standard (CNS) Α4 size (210X 297 mm) 555764 Central Standard of the Ministry of Economic Affairs Printed by the Consumer Cooperatives of the Bureau A7 B7__ 5. Description of the invention (22) The activation of enzyme (MMP) cascade plays an important role. In summary, these proteases can mediate the movement of endothelial cells in angiogenesis / neoangiogenesis and Tissue invasion and cancer cells during metastasis. Neoangiogenesis can basically support tumor growth, and metastasis is a process that mediates tumor spread, and it is related to the patient's extremely poor prognosis. Many preclinical studies have suggested that Kunitz weak amino acid protease inhibitors with protease specificity similar to aprotinin can be used as cancer drugs. For example, aprotinin can reduce tumor growth and invasion, and increase tumor necrosis, when administered to hamsters with highly invasive fibrosarcoma, or to mice with similar malignant breast tumors. (Latner et al., (1974), Br. J. Cancer 30: 60-67; Latner and Turner, (1976), Br. J. Cancer 33: 535-538). In addition, 200,000 KIU aprotinin was administered from 1 to 14 days after inoculation with Lewis lung tumor cells; ip · to C57B1 / 6 Cr male mice could reduce lung metastasis by 50%, whereas for primary tumor masses No effect (Giraldi et al., (1977) Eur. J. Cancer, 13: 1321-1323). Similarly, after inoculation with Lewis tumor cells (13-16 days in 57BL / 6J mice, each administration of 10,000 KIU ip can inhibit lung metastasis by 90% without affecting primary tumor growth (Uetsuji et al., (1992) , Jpn. J. Surg. 22: 429-442). In this same study, administration of plasmin or kallikrein at the same dose schedule has been shown to increase the number of lung metastases. These results prompted the authors to suggest The administration of aprotinin to cancer patients during surgery can reduce the possibility of metastasis. Black and Steger (1976, Eur · J · Pharmacol., 38: 313-319) found that aprotinin can inhibit the transplantation in rats. The growth of murine Murphy-Strum lymphosarcoma, and the suggested effect involves the inhibition of the kallikrein-forming enzyme system. -25- This paper size applies Chinese National Standards (CNs) A4 (210X297 mm) (Please read first Note on the back fill in this page) • Binding line 555764 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of the invention (23) 10,000 KIU aprotinin was injected into the female ddY mouse twice a day for a total of 7 Week, rats were treated with 3-methylcholanthrene Causes a single homogenous squamous cell tumor, which can reduce the growth rate of primary tumors by as much as 90%. Tumor regression can be observed in some animals. Although many vehicle-treated animals die within 7 weeks. But All groups treated with aprotinin survived. Reduced tumor growth was associated with hyperkeratosis (Ohkoshi, Gann (1980), 71: 246-250). Clinically, the 26th place received aprotinin (iv ) In the surgical cure group, 70% of the patients in the surgical group survived for two years and the tumor no longer recurred, while 26 patients in the blank group showed only 38% survival at the same time, and a high proportion of tumors would relapse (Freeman et al. Br. Soc. Gastroenterol (1980) supplement A: 902). In a case study (Guthrie et al., Br. J. Clin. Pract (1981) 35: 330-332), bromocriptine and aprotinin were administered Remission in patients with advanced cervical cancer will cause remission. Aprotinin is administered in large doses of 500,000 KIU ip every 8 hours, plus aprotinin is administered in a continuous IV infusion at 200,000 KIU / 6 hours for 7 days, one month. Once. At the end of the 4th month because of the development of aprotinin Allergic reactions have therefore discontinued treatment. More recent evidence has further emphasized the role of plasmin as a target for these effects on metastasis. The mechanism of these effects is related to the fact that aprotinin blocks the possibility of invasion of cancer cell lines (Liu G., et al., Int J. Cancer (1995), 60: 501-506). Furthermore, since the protein of the present invention is also a strong inhibitor of plasmin and kallikrein, it can also be used as an anti-cancer agent. For example, its limitation of invasion via neoangiogenesis ' primary tumors can block the growth of primary tumors and block metastasis by the inhibition of tissue infiltration. The compound can be locally administered to tumor-26- This paper size is suitable for towels ^ " 5 ^^ 7 (CNS) 8-4 specifications (21GX 297 public attack) (Please read the notes on the back first and fill in this page) • Packing _, Order-line ----- H. 555764 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs. 5. Description of Invention (24) or royal medicine. In a better treatment model, the protein can be administered during surgery during mass tumor resection to reduce the risk of metastasis. During this treatment, the blood-control properties of the compound may have additional advantages in providing a clearer surgery and field. Another preferred mode of administration is combined therapy with MMP inhibitors or chemotherapy. Another additional and better drug administration model, 弋 可 疋 局 # for gene therapy, is designed to achieve placenta succintocin "selective expression" in tumor cells in the matrix and vascular bed connected to it. The preferred cancer type for the treatment target is solid tumors related to blood vessels, such as breast, colon, lung, prostate, and ovarian tumors, which exhibit high metastatic potential, and local delivery of high concentrations of protein is a feasible cancer, such as lung cancer. Lung Shao delivery, colon tumors are delivered via liver to liver metastases, or skin cancers such as head and neck Shao tumors or melanoma are delivered subcutaneously. Since the protein of the present invention is derived from the human body, it seems to be relatively unrelated to the type of allergic or allergic reactions observed, such as Guthrieetal, supra. In addition, the protein of the present invention is intended to reduce the use of thromboembolic complications associated with the activation of the intrinsic pathway of coagulation. These include avoiding pulmonary embolism in end-stage cancer patients, a common cause of death (Donati MB, (1994), Haemostasis 24: 128-131). Brain and spinal edema is due to traumatic brain or spinal plug damage, stroke Brain hemorrhage / bleeding of the brain and subarachnoid, surgical (including open heart surgery) infectious diseases such as encephalitis and meningitis' granulomatous diseases such as sarcoma and focal or diffuse tumors, and these are Causes of high illness rates and deaths after the incident. Vasodilatin is known experimentally to disintegrate blood brain barriers. 27- This paper size applies Chinese National Standard (CNS) A4 (21 × 297 mm) (please read the notes on the back first and fill in this page). · Thread 555764 A7 B7 V. Description of the invention (25) (Greenwoold J., (1991), Neuroradiology, 33: 95-100; Whittle et al., (1992), Acta Neurochir, 115, 53-59) And infusion of vasodilatin into the aorta can induce cerebral edema in autonomic hypertension rats (SHR) receiving general aortic obstruction (Kamiya, (1990), Nippon Ika Daigaku Zasshi. 57: 180- 191). High levels of vasodilatation kallikrein can be found in extracellular fluid after trauma in spinal models involving injured rats (Xu et al. (1991), J. Neurochem, 57: 975-980), and It is found in the plasma and tissues of rats with cerebral edema caused by cerebral hemorrhage. (Kamiya et al., (1993), Stroke, 24: 571-575). Vasokinin is released from serine proteases, including kallikrein, from high molecular weight kininogens (Coleman (I984) J. Clin Invest., 73: 1249) and serine protease inhibitor aprotinin was found SHR rats (Kamiya, (1 " 0), Nippon Ika Daigaku Zasshi 57: 180-191: Kamiya et al. (1993), Stroke, 24: 571-575) and rabbits ( Unterberg et al. (1986), J. Neurosurgery, 64: 269-276) block the magnitude of edema due to cerebral hemorrhage. These observations show that the cause of cerebral edema is the local proteolytic release of high-molecular-weight kininogen from kallikrein, such as vasodilatin, followed by an increase in blood-brain barrier permeability due to vasodilatin kinin. Therefore, placenta succintocin and its fragments can be used as drugs to prevent edema in patients at risk for this condition, especially patients with high mortality or risk of brain injury. This may include patients with head and spinal trauma, patients with multiple trauma, patients undergoing brain or spinal surgery, and related vascular or other general surgery, including open heart surgery, stroke, brain or subarachnoid hemorrhage, and brain Infectious diseases, brain granulomatous diseases, or -28- This paper size applies the Chinese National Standard (CNS) M specifications (210X 297 mm) (Please read the precautions on the reverse side-y write this page) Order economy Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Education 555764 A7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economics ______B7 V. Description of the Invention (26) Diffuse or focal tumors of the brain, or any condition such as multiple sclerosis (involving blood brain Patients suffering from disintegration of the barrier wall), or suffer from any other inflammation process of the brain or spine. Patients can receive placenta succintocin as an infusion or infusion, intravenously or intraperitoneally. Additional doses of placental bisconine can be administered intermittently over the next one to three weeks. Dose levels can be designed to achieve circulating concentrations higher than required to neutralize high plasma levels, or to form vasodilatin and other vasoactive peptides through the action of serine proteases, which are sufficient to reduce edema. Since the protein is derived from the human body, repeated administration during this treatment should not cause an immune response to the protein. Placenta diconine and its fragments should be available for monotherapy or prophylactic use, as well as for use with other drugs, such as neurotherapeutics and neuroprotective agents. Recent evidence shows that (Dela Cadena R. A. et al., (1995), FASEB J. 9: 446-452) contact activation pathways can contribute to arthritis and anemia and pathogenesis, and kallikrein inhibition Agents may have therapeutic benefits. Therefore, the protease inhibitor of the present invention can be used as a medicine for treating human arthritis and anemia according to its ability to inhibit human kallikrein. Treatment of male non-insulin type diabetes (NIDDm) patients with aprotinin can significantly improve overall glucose uptake and reduce insulin metabolic clearance (Laurenti et al., (1996), Diabetic Medicine 13: 642_645). Therefore, the human protein of the present invention can be used as a drug for treating NIDDM for long-term use. Treatment of patients at risk of preterm birth with urinary protease inhibitors for two weeks daily can significantly reduce recurrent uterine contractions (Kanayama et al., (1996), Eur J. Obstet. Gynecol. &Amp; Reprod. Biol. 67 ： 133- -29-This paper size is applicable to Chinese National Standard (CNS) M specification (21 × 297 mm) (Please read the notes on the back first and fill in this page) • Binding and Thread _ 555764 A7 B7 Central Standard of the Ministry of Economic Affairs Printed by the Bureau's Consumer Cooperatives V. Invention Description (27) 13 8). Therefore, the human protein of the present invention can be used to avoid premature birth. Aprotinin has been shown to stimulate the differentiation of mouse myoblasts in culture (Wells and Strickland, Development, (1994), 120: 3639-3647)), a process that can be inhibited by TGFb. TGF exists as an inactive polypeptide prototype, which is activated by restricted proteolysis. The mechanism of aprotinin action has been proposed to involve the inhibition of proteases, which are modified by TGFb pro-treatment into a mature activated form. TGFb has been shown to be positively regulated in various fibrous lesions and has long been regarded as a possible target in anti-fibrotic therapy. For example, in the rat model of pulmonary fibrosis, the concentration of TGF-b is comparable to the degree of inflammation of bleomycin-biosynthesis. In addition, the level of plasmin in pulmonary macrophage cells is consistent with the level of mature TGF-b, and the addition of plasmin inhibitor a-2 · anti-plasmin can eliminate macrophages. Post-translational activation of TGFb (Khel et al. (1996), Am. J. Respir. Cell Mol. Biol. 15-252-259). The data suggest that fibrinase can help lung macrophages to form active TGFb, and that this process plays a pathological role in the microinflammation of lung inflammation. Based on these observations, Shuangconin and its fragments can be used as therapeutic agents for various fibrotic disorders, including lung, liver, kidney, and skin (scleroderma) fibrosis. Silicated aprotinin has been shown to protect lethal doses of influenza virus or paracumulus virus-infected mice by more than 50% (Ovcharenko and Zhirnov, Antiviral Research, (1994), 23: 107-118). Treatment with silicified aprotinin may also note that the development of fatal hemorrhagic bronchopneumonia is suppressed and weight gain is normalized. Based on these observations, placenta succintocin and its fragments can be used as a therapeutic agent for various influenza-related influenza diseases. -30- (Please read the notes on the back first and fill in this page) Binding and Binding--1 — H _ This paper size is applicable to China National Standard (CNS) A4 (210X 297 mm) 555764 Zhongli Bureau of Standards, Ministry of Economic Affairs Printed by Employee Consumer Cooperatives V. Invention Description (28 The invention < Human placenta diconicin, isolated fractions and other variants hope to be used in medical / therapeutic applications, this is also the recommended application of natural skin suppressor f or its homologs with other inhibition profiles, especially for large Dosage users and ^. These can include diseases in which the use of human proteins can be demonstrated by their ability to inhibit human serine proteases, among which proteases such as proteases, plasmin, elastase, cathepsin 0, and protease-3 are included The diseases are as follows, but not limited to them: acute pancreatitis (pancreatic elastase and trypsin), inflammation, thrombocytopenia, preservation of platelet function, organ preservation, wound healing, various types of shock, including shock lungs' Endotoxin shock and postoperative complications; disorders of blood coagulation, such as excessive fibrinolytic bleeding; acute and chronic inflammatory reactions, especially the treatment and prevention of organic damage, such as pancreatitis and radiation treatment of enteritis Body-promoting inflammatory reactions such as immune vasculitis, glomerulonephritis and various types of arthritis, collagen disease, especially rheumatoid arthritis; related by metabolism < All types of arthritis (such as gout) from storage; degeneration of elastic components of connective tissue of organs, such as atherosclerosis (serum elastase) or emphysema (neutrophil elastase) ; Or respiratory pain syndrome, inflammatory bowel disease and psoriasis. The main accidental finding was that the synthetic peptides encoding dicominin (7_64) and dicominin (102_159) can correctly fold into the correct three-dimensional configuration of the biological activity of the active protease inhibitor (respectively Examples 1 and 2). Once folded, the reduction of each of the six cluster units of these dicominin fragments is consistent with the formation of three intra-chain disulfide bonds between the six cysteine residues of each fragment in each case. Another surprising discovery is the code Shuang Corning Su (7_64), Shuang Corning Su 31-This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the notes on the back to fill in this Page}-Binding-Thread 555764 A7 B7 V. Description of the Invention (29) — Synthetic peptide printed by (102-159) and Shuang Corningin (1_17〇) produced by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, for fibrin Lysin and kallikrein in tissues and plasma are highly inhibitory (examples 4, 3, and 10 respectively). Trasylol® inhibition of fibrin and kallikrein is considered to involve Trasylol® in the In the mechanism of reducing blood loss during surgery, our unexpected discovery of the specificity of the Kunitz part of the present invention makes it a suitable therapeutic agent for restricting bleeding during surgery or injury, or it can be applied to plasmin and / Or the inhibition of kallikrein is any other condition that is beneficial. Furthermore, we show in this disclosure (Example 10) placenta dikonin (1-17) is a potent inhibitor of factor XIa, and factor Xa Moderate inhibitor. Factor XIa The intrinsic pathway of blood coagulation plays a fundamental role in the interactive conversion of inactive factor IX into active factor IXa. Therefore, placenta dikonin can inhibit two critical enzymes in the intrinsic pathway, kallikrein And factor XIa. Consistent with these observations, we have also shown that placenta dikonin (1-170) is a potent inhibitor of activated partial thromboplastin time. This time is detected by the internal pathway Driven coagulation rate. On the other hand, we show that placenta diconine (1-17) is a very weak inhibitor of tissue factor VIIa complex, and it is suggested that it is not important in the regulation of external coagulation cascades. Based on these unexpected findings, placenta siroconine can be used as a drug in diseases in which activation of the intrinsic pathway of coagulation can significantly contribute to disease mechanisms. Examples of such diseases include posttraumatic shock and diffuse intravascular An important advantage of the Kunitz portion of the present invention is that it is a human protein and has a slightly less positive charge than Trasylol® (Example 1), which is due to the risk of kidney damage when administered in high doses of protein Reduced. Because it is derived from the human body, the protein of the present invention is cast-32. This paper scales Xianjia County (CNS) A4 size (210; ~ 297 mm) (Please read the notes on the back first and fill in this page)- Binding-Thread | 555764 Printed A7 ____B7 by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (30) The risk of non-desired immune response when administered to the human body will be significantly lower than when Trasylol® is administered at a similar dose In addition, it was found that in the test tube, dicominin (102-159), dicominin (7-64) and dicominin (1_170) are more potent plasma kallikrein inhibitors than Trasyl〇1®. . (Examples 3, 4 and 丨 0). Therefore, 'shuangkonin and its fragments are expected to be more effective in reducing patient blood loss (in vivo). Serine protease inhibitors should be administered in an amount sufficient to provide higher than normal plasma levels. To prevent the reduction of bleeding during and after coronary aortic circuit surgery (CABG), the protein of the present invention can be used in place of Trasylol®, taking into account differences in strength. The usage of Trasylol® is summarized in the Physicians Desk Reference, (1995), listed in Trasylol®, Supplement A. In short, for patients in the supine position, the placenta diconine is given slowly over a period of about 20 to 30 minutes after separation or other variants, this is after anaesthesia but before the sternum. Generally, a total dose of about 2 X 106 KIU (Kallikrein Inhibition Unit) and 8 X 106 KIU can be used, depending on factors such as patient weight and length of surgery. A preferred dose is one containing a total of 1 to 2 million kallikrein kallikrein inhibitor units (KIU). When the dose was complete, the group took a fixed infusion dose and continued this until the operation was complete and the patient left the operating room. Preferred fixed infusion doses are in the range of about 250,000 to 500,000 KIU per hour. The pump infusion dose is added to the infusion fluid in the cardiopulmonary return route. It replaces an infusion fluid before the cardiopulmonary circuit is set. The preferred pump irrigating dose is one in which it contains about 1 to 2 million KIU. The protein of the present invention can be used in pharmaceutical compositions which are blended by methods known in the art. This composition contains active ingredients, plus more than one pharmacologically. 33- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) '' —— ~-(Please read the notes on the back first and fill in (This page) Binding and binding --- 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (31) Can be used as a carrier, diluent, filler, cumulant and other excipients. Music mode and intended dosage form. Examples of therapeutically inert inorganic or organic carriers known to the artisan include: lactose, corn starch or derivatives thereof, talc = vegetable oil, waxy, lipid, polyhydric alcohols such as polyethylene glycol, sugars' alcohols, glycerol Etc., but not limited to this. Various preservatives, emulsifiers, dispersants, flavors, moisturizers, antioxidants, sweeteners, colorants, salts, buffers, etc. can also be added, as needed to help the% of the blender, or to Helps increase the bioavailability of the active ingredient, or produce a blend with acceptable flavor or odor in the oral dosage example. The inhibitor applied in the dagger can be in its own original compound form, or it can be pharmaceutically as required Acceptable salt form. The protein of the present invention can be administered alone, or in various combinations, and combined with other therapeutic components. The composition so blended can be administered in any suitable mode known to the artisan, and administered as an inhibitor Required choices.% Extracorporeal: Modes include intravenous (iv), subcutaneous (sc), intraperitoneal (ip), and intramuscular (im) pathways. Intravenous administration can be used to obtain the peak plasma concentration that the drug may require Acute control. In addition, the drug can be continuously administered at the desired rate through an intravenous catheter. Suitable solvents include sterile, pyrogen-free aqueous diluents, such as sterile water for injection, sterile Wash solution or sterile saline solution. The resulting composition is administered by intravenous injection or infusion before and / or during the operation. Intercepting the drug in the liposome will help improve the half-life and drug alignment. For inflamed phages, such as neutrophils and macrophages, it should also be possible to improve the selectivity of liposomes by targeting FLB. That is, the inclusion of ligands in lipids-34 This paper applies Chinese National Standard (CNS) A4 Specifications (210X 297mm) (Please read the notes on the back to fill in this page first) Binding · Binding----- 555764 A7 B7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (32) In vitro test The ligand is combined with target organs / tissues, such as GI tract and lung-specific macromolecules. In addition, im or sc storage injection can also be used, with or without drug encapsulation into degradable microspheres ( If it contains poly-DL-lactide-co-glycolide, or a protective blend containing collagen, to prolong the sustained drug release. To improve the suitability of the dosage form, it can also be stored by IP implantation Troughs and septa, such as percuseal System. The use of a syringe pen (such as Novo Pin or Q-pen) or a needleless injection syringe (such as from Bioject, Mediject or Becton Dickinson) can also achieve improvements in compliance and patient compliance. It can also achieve precisely controlled release, If an implantable pump is used, it is delivered to the desired location via a catheter. Examples include subcutaneously implanted osmotic pumps available from ALZA, such as ALZET osmotic f pumps. Nasal delivery can also be achieved by incorporating the drug into a bioadhesive Sex microparticle carrier < 200 mm), if it contains cellulose, polyacrylic acid vinegar or polycarbophil, and with appropriate absorption enhancer, such as phospholipid or alkenyl carnitine. Commercialized systems include a non-parenteral model of drug delivery to the circulation as described by Dan Biosys and Scions Nova. 0 Lung delivery represents drug administration to the circulation. The lower respiratory tract epithelium is highly permeable to a wide range of proteins with molecular weights up to about 20 kDa. Small-sized dry powder containing the drug in a suitable carrier such as mannitol, sucrose, or lactose, can also be delivered to remote lung surface with dry powder inhaler, such as Inhale ™, Dura ™, Fisons (SpinhalerTM) and Glaxo ( Rotahaler ™), or Astra (Turbohaler ™) propellant-based metered inhaler. Solution blends with or without liposomes can be delivered using an ultrasonic sprayer. Oral delivery can be achieved by incorporating the drug into lozenges, coated tablets, sugar-coated tablets, hard • 35- (Please read the notes on the back first and fill out this page) Packing ·, 11 -line —----- This paper size applies to China National Standard (CNS) A4 specification (210 × 297 mm) 555764 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (33) and soft gelatin capsules, solutions, emulsions, suspensions or casing capsules, Designed to release drugs in the colon with low digestive protein acid activity. The latter example includes ALZA's OROS-CT / Osmet ™ system and Scherer Drug Delivery Systems' PULSINCAP ™ system. Other systems use azo-crosslinked polymers that can be degraded by colon-specific bacterial azo reductases or pH-sensitive polyacrylate polymers that can be activated by an increase in pH in the colon . These systems can be used with a wide range of available absorption enhancers. Rectal delivery can be achieved by incorporating the drug into a suppository. For its better medical application, in order to reduce blood loss during the week of operation, the preferred mode of administration of the placenta shuangkonin variant is parenteral, preferably via the i.v. route of the central line. The dosage of the pharmaceutical composition to be applied depends on the recipient and the condition to be treated. The necessary amount can be determined without undue experimentation by methods known to the artisan. In addition, the necessary amount can be estimated based on the amount of target protease enzymes necessary to treat the condition, such as plasmin or kinin. To the extent that the active substances included in the present invention are indeed non-toxic, treatment preferably involves administering a dosage that is more than the optimally necessary active dosage. In addition, placenta succintin, which can be isolated from human substances through isolated parts or other variants, such as its associated proteases, can use affinity-based separation methods to defame antibodies that produce proteases, It can be further used to develop the tissue distribution and useful functions of placenta diconine. Seeking human sequence data According to the unique analysis of sequence entities, the NCBI (National Center for -36- this paper size is suitable for Ugj furniture standard (CNS) A4 specification (21 () > < 297 mm) (Please read the notes on the back to fill in this page) Binding and ordering 555764 Printed by A7 B7, printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Presentation sequence of Biological Information (Maryland) -tag database (hereinafter referred to as dbEST), which can be derived from the existence of human proteins that are functionally homogeneous with aprotinin. Using the TBlastN mutual division method (BLAST, or Basic Local Alignment Search Tool, using the method of August 1 (8: 111161 & 1., (1990)) · Mol Biol 215: 403_410, to find the questioned sequence and all sequences in the database, presented Similarity between any combination of proteins or nucleic acids), check the database for homologous nucleotide sequences with bovine pre-aprotinin, Trasylol® sequences. Many pure lines of this search can be selectively narrowed to two The special pure line may guide the synthesis of a deduced amino acid sequence, which is equivalent to a human protein functionally homologous to aprotinin. The selected nucleic acid sequences are R35464 (SEQ ID NO: 12) and R74593 ( SEQ ID NO: 14), which is generated from the human placental nucleic acid library. In R35464, the translated protein sequence (SEQ ID NO: 1 3) in the longest open reading framework is 6 in which 1 cysteine is missing, and Crucial for the formation of the Kunitz-part covalent structure, indicating that the nucleic acid sequence of R3 5464 does not generate a functional inhibitor. Similarly, the longest translation open reading from pure line R74593 (SEQ ID NO: 15) Translate It contains a stop codon at the 1 end of the region encoding a similar sequence of Kunitz, indicating that this sequence cannot be translated into a functional secreted Kunitz part. The importance of these sequences alone is unknown. It may represent a) the product of a pseudogene 'b) the region of the untranslated mRNA, or c) the product of the variable mRNA, which was incorrectly sequenced. Discovery of human bisinin In order to specifically isolate and determine the exact human sequence, cDNA primers are designed to be similar to the Kunitz proposed by the codes we proposed in R35464 and R74593 (please read the notes on the back first and fill in this page)

、 1T line-37- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) i Printed by 555764 A7 B7, Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. cDNA in column (35) of the invention description 5 'And 3. · sequence hybridization. The primers used to expand the fragment encoding similar sequences of R74593 Kunitz are CGAAGCTTCATCTCCGAAGCTCCAGACG (3 with Hindlll position, primer; SEQ ID NO: 3 3), and AGGATCTAGACAATAATTACCTGACCAAGGA (5 'primer with Xbal position, SEQ ID NO: 34). These primers can be used to amplify (30 cycles) a 500 bp product via PCR from Clontech's human placental cDNA library (MATCHMAKER, Cat # HL4003AB, Clontech Laboratories, Palo Alto, CA), which is subsequently cloned to Bluescript_SK + , And sequenced with Sequenase ™ Kit 2.0 T3 primers. Surprisingly, the sequence of the fragments obtained using our primers is different from the sequence listed in the pure line R74593 in the dDEST database. In particular, our new sequence contains an extra guanine nucleobase embedded in the putative stop codon 3, but at the 5 · end of the coding class 1 Kunitz sequence fragment (Figure 3). The extra g is embedded to shift the stop codon out of the class-Kunitz part of the translation architecture (G is at the 114th base pair of the correct sequence of R74594; Figure 3). Next, questioning dbEST for homogeneity with the Kunitz-like R74593 peptide sequence can generate H94519 (which is derived from the human retina library) and N39798. These sequences contain a Kuniz-like sequence which is almost identical to the Kuniz-like portion encoded in R35464, except that it contains all six characteristic cysteines. By comparing each nuclear acid sequence with R74593 (corrected for embedding G in b, p, 114) and R35464 outer cover, a consistent nucleotide sequence of human partial placental diconicin can be obtained (SEQ ID NO: 9; image 3 ). Translated Consistent Order-38-This paper size applies to Chinese National Standards (CNS> a4 size (210X 297mm) (Please read the notes on the back first and fill out this page) • Packing. Line 555764 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative printed A7 B7 V. Description of the invention (36) The column can generate an open reading structure extending from -18 residues to +179 (Figure 3; fully translated SEQ ID NO: 10), which is at amino acid residues 17_64 There are two kinds of Kunitz partial sequences in the region of 1012159. Questions about the R35464 sequence dbEST can make further efforts to obtain additional 5, sequences. Since the search for possible matches, it has additional 5, sequences, It is used to question dbEST again. In this iterative method, a series of overlapping 5 sequences can be identified, including pure lines H16866, T66058, R34808, R87894, N40851, and N39876 (Figure 4). Some of these sequences are arranged It can be suggested that the existence of '5' ATG can be used as the starting position of synthetically consistent translation protein sequences. From the selected data, it is currently possible to selectively screen and determine humans with homogeneous functions with aprotinin Nucleic acid and peptide sequences of proteins. A re-examination of dbEST shows that there are many new EST entities, shown in Figure 4B. Overlapping comparison of these additional ESTs allows us to construct a longer, consistent oligonucleotide Sequence (Figure 4C), which extends at the 5 and 3 ends beyond the original oligonucleotide sequence shown in Figure 3. In fact, the new sequence with a total length of ι · 6 仟 bases all the way to the 3 'poly-A tail Along the sequence, the increased number of overlapping ESTs at each base pair position can improve the level of confidence in certain regions, so that the sequence can overlap the 3 'end of EST R74593 (Figure 3). Many in this region Overlapping ESTs confirm the deletion of two key bases compared to R74593 (the positions are underlined in bold in Figure 4C, and the map positions are 994 and 1005). The new consensus in the Shuang Corning coding architecture The translation of the sequence (Figure 4D) can generate a placenta diconine (248 amino acid) pattern that is larger than the mature sequence (179 amino acids) encoded by the original consensus sequence (SEQ ID NO: 1),- 39- This paper size applies to China National Standard (CNS) A4 specification (2 丨 〇 > < 297 mm) " (Please read the notes on the back to fill out this page first) • Binding · Binding-Thread · 555764 A7 B7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (37) The stop codon in the internal structure of the oligonucleotide consensus sequence terminates. The increase in size is due to a frameshift in the 3 'coding region, which is generated by the removal of the two bases unique to EST R74593. The frameshift shifts the original consensus stop codon (Figure 3) out of the framework and reads it into a new framework that encodes an additional amino acid sequence. The new translation product (Figure 4D) is identical to the original protein consensus sequence (SEQ ID NO: 1) from +1 to +175 residues (encoding the Kunitz part), but contains a new C-terminal extension, presenting a The inferred length of the transmembrane portion of 24 residues (underlined in Figure 4D) is followed by a short cytoplasmic portion of 31 residues. The exact sequence around the starter methionine and signal peptide is somewhat hypothesized, due to the considerable heterogeneity among the ESTs that overlap in this region. Using Geneworks ™ to analyze protein sequences, the important aspartamine residues at residues 30 and 67 are consistent positions for the putative N-chain glycosylation. Asparagine 30 was not observed in the N-terminal sequencing of the full-length protein isolated from the human placenta, consistent with its glycosylation. Selection of human dicominin Corresponding to the human mRNA of the human dicominin ribonucleotide sequence inferred from the analysis indicated in Figure 3, its existence can be confirmed as follows. Nucleic acid primers that can be hybridized with Kunitz-coding cDNA sequence 5 of R35464 (Figure 3-bp 3-27 of the resulting nucleotide sequence): GGTCTAGAGGCCGGGGGGGTCTCTCCTCTGGCTGGGA (a 5 primer with Xbal position derived from R35464 sequence; SEQ ID NO: 3 5), and nucleic acid primers that can hybridize with R74593 Kunitz coding sequence (the consistent nucleotide sequence bp 680-700 in Figure 3) can be used for PCR amplification -40- (please read the notes on the back to fill in this Page) • Binding and Binding I " Γ. This paper size is applicable to China National Standard (CNS) A4 specification (210X297 issued:) 555764 Printed by A7 B7, Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. ) A fragment from the Clontech human placenta library. The size of this fragment (approximately 670 bp) can be expected to be generated from the CDNA-induced nucleotide sequence encoding the placenta diconine sequence (Figure 4A). Using the 5 'primer (Figure 4A, bp · 110) that can hybridize with the sequence of 5' 126 bp in the above-mentioned putative ATG start position in R87894, plus the 3 * primer as described above in R74593, it will be possible to clone from Clontech humans An enlarged fragment in the placenta library, which is a fragment of the expected size (about 872 bp) predicted by EST overlap comparison (shown in Figure 4). The sequenced 872 bp fragment showed a nucleotide fragment corresponding to EST R87894 bp 110 to 218 at its 5 'end, and a placental double at the 3 »end as indicated by the above-mentioned EST overlap comparison analysis (Figure 3). Consensus sequences for Corning bp 310 to 542. This 31-nucleotide sequence contains all the class-1 ^ 1111 seven parts (102-159) encoded by the placenta diconine. To obtain a cDNA encoding the entire extracellular region of the protein, use the following 5. PCR primers: CACCTGATCGCGAGACCCC (SEQ ID NO: 36) is designed to hybridize to the sequence in EST R34808 and uses the same EST 74593 3 'primer to expand (30 cycles) the 780 bp cDNA product in the human placental cDNA library. This product was purified by gel, cloned into TA vector (Invitrogen), and DNA sequencing by dideoxy method was performed with the following primers (Sanger F ·, et al ·, (1977) Proc. Natl · Acad. Sci ( USA), 74: 5463-5467): Carrier-specific: GATTTAGGTGACACTATAG (SP6) (SEQIDNO: 37) TAATACGACTCACTATAGGG (T7) (SEQIDNO: 38) -41-1 The paper size applies the Chinese national standard (CNS> A4 specification (210X 297) (Public Director) (Please read the notes on the back of IBP and fill out this page)

P • Assembling and Threading-Γ. 555764 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (39) Gene-specific: TTACCTGACCAAGGAGGAGTGC (SEQIDNO: 39) AATCCGCTGCATTCCTGCTGGTG (SEQIDNO: 40) CAGTCACTGGGCCTTGCCGT (SEQIDNO: 41) The generated cDNA sequence is shown in Figure 4E, plus its translation product. At nucleotide levels, the sequence presented differs only slightly from the consensus EST sequence (Figure 4D). The translation of the sequence generates a coding sequence that contains the ATG position of the promoter on the architecture, the signal peptide and the mature placenta diconicin sequence and the transmembrane portion. The translated PCR product sequence was lost due to the choice of 3 'primers in the PCR amplification, so the last 12 amino acid sequences in the cytoplasmic part were lost. The selection of 3 * PCR primers (designed based on the R74593 sequence) is also responsible for introducing artificial S to F mutations at the 211 amino acid position of the translated PCR-derived sequence. The signal peptide derived from the translation of the PCR fragment is somewhat different from the EST-induced sequence. In order to obtain the full-length placenta diconicin cDNA, the PCR-derived product (Figure 4E) was gel purified and used to isolate non-PCR based full-length pure lines representing the diconicin sequence. PCR-derived cDNA sequences were labeled with 32P_CTP using High Prime (Boehringer Mannheim) and used to detect placental cDNA libraries (Stratagene, Unizap ™ libraries), which used colony hybridization technology. About 2 × 10 6 plaques were subjected to 3 rounds of screening and plaque purification. The two pure lines were analyzed by restriction enzymes and based on a comparison with the size of the EST-induced sequence (see above), and they were indeed full length (~ 1.5 bases). Sequencing one of these pure lines by the dioxygen method yielded the oligonucleotide sequence shown in Figure 4F. The translation product obtained from this sequence can produce a protein with a structural promoter of methionine, a signal peptide, and a mature diconine sequence. Mature Placenta Double Corningin sequence and by EST -42- (Please read the note on the back Αβρ to fill out this page) Binding · Binding-Lr I 1LI. This paper size applies Chinese National Standard (CNS) Α4 specification (210 × 297 (Mm) 555764 Consumption cooperation by employees of the Central Bureau of Standards, Ministry of Economic Affairs, Du printed A7 B7 __V. Description of the invention (40) The mature protein sequences generated by the translation of the consistent sequence are the same, although the signal peptide sequence length and sequence are different. Unlike PCR-driven products, cDNA derived from colony hybridization contains the complete outer, transmembrane, cytoplasmic, and stop codons in the framework. In fact, the pure line extends all the way to the tail of poly-A. The starter methionine is followed by a hydrophobic signal peptide, which is the same as the signal peptide encoded in the PCR-derived pure line. Next we expressed and purified placenta succinicin, a soluble fragment of succinicin (1.170), in Sf9 cells (Example 9) and found to be a functional protease inhibitor (Example 10). Furthermore, we isolated a soluble fragment of placenta diconine from the human placenta, which is also an active protease inhibitor (Example 7). Natural proteins and proteins expressed in Sf9 cells are based on the discovery of PTH-amino acids in N-terminal sequencing, which may be glycosylated on asparagine at position 30 (Examples 7 and 9) . Based on the above observations, it appears that the full-length placenta sirekonin has the ability to exist on the cell surface as a transmembrane protein and as a soluble protein. Other membrane-entering proteins containing a Kunitz moiety are known to be proteolytically modified to produce soluble and membrane-bound mixtures. These include type II amyloids such as Amyloid Precursor Protein (APP 751 (Esch F., et al., (1990) Science, 248: 1122-1124) and APP 770 (Wang R., et al. (1991), J Bioi chem, 266: 16960-16964). Contact activation is a process in which the damaged blood vessel surface and the coagulation cascade are exposed and activated. Vascular growth involves blood fibers on the endothelial surface The process of local activation of proteolytic enzymes. The specificity of placenta diconine and its ability to fix i cell surface can suggest the physiology of perforated placenta diconicin • 43-quasi (CNS) M specifications (please read first Note on the back β fill out this page) _ Threading 丨 ^ Γ. 555764 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 _B7___ V. Description of the invention (41) > Functions include contact activation and angiogenesis regulation. Seeking the amino acid sequences of placenta diconine (7-64), diconiline (102-159), and full-length placenta diconine (Figure 4F), using the PIR (Vers · 46.0) of FastA of the Genetics Computer Group program and PatchX (Vers · 46 · 0) protein Library, and protein database of GeneSeq (Vers. 20.0) patent sequence. Using Genetics Computer Group program TFastA (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444-2448), GenBank (Vers. 92.0, supplementing the latest information to 1/26/96) and EMBL (modified Vers. 45.0) nucleotide database and GeneSeq (Vers. 20.0) nucleotide sequence database with patented sequences. These identical protein sequences are sought. The EST and STS subsets of GenBank and EMBL are not included in this research group. The best fits from this study include their full length and those derived from the pure lines R74593 and R35464 that we analyzed The protein sequence of 5 8 amino acids is only about 50% homogeneous. The separation of human dicominin is as described above. The equivalent of dicominin determined by the translated sequence of dicominin (Figure 3) is equivalent to dicominin (Figure 3). 7-64) and diconine (102-159) synthetic peptides can be refolded (Examples 2 and 1) to produce active kallikrein inhibitor proteins (Examples 4 and 3, respectively). We took advantage of this accident Design a purification process based on Isolation of natural placenta dicominin from human tissues. In the purification process, we used kallikrein-sepharose affinity chromatography as the first step to isolate highly purified natural potent kallikrein inhibitors. The isolated natural human diconicin and the predicted sequence from the identical nucleic acid sequence (Figure 3) amino acid residues +1 to +50 (Example 7) have the same -44- '__ _ _ paper size It user standard (CNS) A4 specification (210X 297 mm) (please read the notes on the back 1¾ to fill out this page). Binding and stitching L ___ Γ 555764 5. Description of the invention (42) N-terminal (50 amine groups) Sequence of acid residues). This is the first time that a novel natural kallikrein inhibitor can be isolated from a human placenta. The known-Kunitz section is listed below. Residues that can be contacted with the target protease are particularly emphasized (bold / underlined). These special residues are called positions Xaa1-10, see in particular the description of the flag Xaa below: X 龟 龟 2

1) IHDFCLVSKVV 2) Y2EYCTANAVT 3) -HSFCAFKADD 4) -PDFCFLEEDP 5) -PSWCLTPADR 6) -AEICLLPLDY 7) -PSFCYSPKDB 8)-KAVCSQEAMT 9) RPDFCLEPPYT 10) ---- CQLGYSA 11) VAACNLPIVRAET) 13) K --- CKLPKDB 14) -PNVCAFPMEK

3 456789 GRCRASMPRW GPCRASFPRW GPCKXIHKRF GICRGYITRY GLCKANEMRF GPCRALLLRY GIiCSANVTRY GPCRAVHPRT GPCKARIIRY GPCMCafTSRY GPCRAPIQLW GPCHAHZSRW GTCRDFILKW GPCQTYHTRW

WYNVTDGSCQ YFDVERNSCN FFNIFTRQCE FYNNQTKQCE YYNSVIGKCR YYRYRTQSCR YFNPRYRTCD TFDLSKGKCV FYNAKAGLCQ FYNGTSMACE AFDAVKGKCV YFDVTEGKCA YYDPNTKSCA FFNFETGECS 1 1 111 0 1 234 1 5

LFVYGOCDGN SNNYLTKEEC NFIYOOCRGN KNSYRSEEAC EFIYOOCEGM QNRFBSLEEC BTLEEC TSKQEC QFLYGWCBWN ANNFYTWEAC AFTYTOCOGH DNNFVSREDC RFITOOCGGH Rl · ίNFESEDYC TFVY (3CX: RAK RNNFKSAEDC TFQYQXMGW GNNFVTEKEC LFPYGGCQGM GNKFYSEKEC PFFYOGCG5GH RNNFDTEEYC RFWYGGCOGH ENKFGSQKEC LFAYGGCOGil SNNFLRKEKC RFKYOOCLGN MNNF1 PFKYSOCOGN ENNF ^

LKKCAT.V MLRCFRQ KKMCTRD KNICEDO LRACKKO DDACVm KRACAKA MAVCKAH MRTCGGA LQTC REYCGVP MAVCGSA EKVC EKFCKFT Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs where the serial number 1) is diconine (7-64) (SEQ ID NO: 4); 2) is dicominin (102-159) (SEQ ID NO: 6); sequence 3) is a precursor of tissue factor pathway inhibitor 1, (SEq ID NO: 18); sequence 4) is before tissue factor pathway inhibitor Body 1, (SEQ ID NO: 19); sequence 5) is a precursor of a tissue factor pathway inhibitor, (SEQ ID NO: 20); sequence 6) is a precursor of a tissue factor pathway inhibitor 2, (SEQ ID NO: 21) ); Sequence 7) is Tissue Factor Pathway Inhibitor Precursor 2, (SEQ ID NO: 22); Sequence 8) is 45 This paper size applies to China National Standards (CNS) VIII (210 ^^ 97 mm) 555764 A7 B7 V. Description of the invention (43) The precursor protein of the class Dian powder is homogeneous (SEQ ID NO: 23); sequence 9) is aprotinin (SEQ ID NO: 24); sequence 10) is indirect trypsin Inhibitor precursor (SEQ ID NO: 25); sequence 11) is a meta-α_trypsin inhibitor precursor (SEQ ID NO: 26); sequence 12) is an amyloid precursor protein (SEQ ID NO: 27); sequence 13) is a collagen from -3 (VI) precursor (SEQ ID NO: 28); and 14) is HKI-B9 (SEQ ID NO : 29), it can be seen that 'placenta diconine (7-64) and (102-159) each have the same number of cysteine residues (6) and space, such as the serine protease inhibitor Kunitz Seen among members. The precise bonding of cysteine residues to form three intra-chain disulfide bonds is known 'and is unchanged across all previously known Kunitz family members (Laskowski, M et al., 1980, Ann. Rev. Biochem. 49: 593-626). According to this known bond type and placenta diconicin (7-64) and (102_159) folded into active protease inhibitors, a large number of reductions are consistent with the fact that three intra-chain disulfide bonds are formed (Examples 2 and 1 ), There is a high probability that the formation of disulfide bonds within the placental bisconine Kimitz portion occurs between the following cysteine residues: C11 and C61; C20 and C44; C36 and C57; C106 and C156; C115 and C139; C131 and C152. Furthermore, this type of disulfide bond is highly likely in larger placenta diconine containing two Kunitz moieties, because this protein type is also an active serine protease inhibitor, and because of the natural placental double Corning's 50-cycle N-terminal sequencing (Example 7) generates a sequence that is silent at the expected position of the cysteine residue. The placenta diconine, the isolated part or other variants thereof, can be produced by standard solid-phase peptide synthesis, using t-Boc chemistry such as Merrifield RB · and Barany G ·, in: The peptides, Analysis, Synthesis, Biology ， -46- This paper size applies to Chinese National Standard (CNS) A4 specification (2 丨 0 > < 297 public directors) (please read the notice on the back first and fill in this page) Printed 555764 A7 B7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (44) 2, Gross E. et al · Eds · Academic Press (1980) Chapter 1; or use F-moc chemistry, such as Carpino LA And Han G. (1970) J. Amer Chem Soc., 92, 5748-5749, and explained in Example 2. In addition, the expression of the placenta diconicin variant DNA can be used to generate recombinant placental diconicin variants. The present invention is also related to DNA constructs which encode variants of the placenta diconine protein. These structures can be prepared synthetically, WBeaucageS.L. And Caruthers M.Η., (1981) Tetrahedron Lett, 22, ρρ1859-1862 »Matteucci MD and Caruthers MH? (1981), J. Am. Chem. Soc. 103 , p 3185, or prepared from a genomic body or cDNA, which is obtained by screening a genomic body or a cDNA library with a cDNA probe designed to hybridize with placenta diconine in a coding DNA sequence. The genomic or cDNA sequence can be modified at more than one position to obtain a cDNA encoding any amino acid substitution or deletion described in this disclosure. The present invention is also a related expression vector, which contains the placenta diconicin encoding the present invention, and is used to produce a recombinant placental diconicin variant after being separated or other variants. The cDNA should be ligated to a suitable promoter sequence, which can show transcriptional activity in the host cell of choice, and has suitable terminator and polyadenylation signals. The cDNA encoding the placenta diconicin variant can be fused to a 5 · signal peptide, which can generate a protein encoded by the cDNA and perform secretion. The signal peptide can be identified by the host organism. In the case of mammalian host cells, the signal peptide may also be a natural signal peptide present in the full-length placenta diconine. The method for preparing this carrier for expressing placenta diconicin is well known in the art and is described in, for example, Sambrood et al., Molecular Cloning: A laboratory Manual, Cold Spring Harbor, New York, (1989) 〇-47 · (Please read the notes on the back to fill in this page first) • Binding _ Thread-This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 555764 Printed by A7 _B7 of the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Explanation of the invention (45) The present invention also relates to a transformed cell containing a DNA construct, and the construction system can encode the placenta dikonin in the present invention, and isolate a part or other variants to generate a recombinant placental dikonin. Various combinations of expression vectors and host organisms can be used to produce placenta succintocin variants. Suitable host cells include baculovirus-infected Sf9 insect cells, mammalian cells such as BHK, CHO, Hela and CM27, bacteria such as coliform bacteria, and yeasts such as Saccharomyces cerevisiae. Mammalian, insect, and microbial expression systems required to achieve placenta dual-coninin expression; usage is well known in the art and described in, for example: Ausubel F. M et al.? Current Protocols in Molecular Biology, John Wiley & Sons (1995), Chapter 16. For placental diconicin fragments containing a single Kunitz inhibitor moiety, such as diconicin (7-64) and (102-159), yeast and E. coli are preferred, and yeast systems are the best. Typically, yeast performance can be performed as described in U.S. Pat. No. 5,164,482 (for aprotinin variants), and can be applied to placenta diconine (102-159) of Example 5 of this patent. E. coli expression can be performed using the method described in U.S. Patent No. 5,032,573. Mammalian and yeast system usage is best for larger placenta diconine with two inhibitors, such as variant diconine (7-159). DNA encoding a placental diconicin variant having a natural amino sequence amino acid substitution can be prepared from a recombinant protein using the method of Kunkel τ · A · (1986) Proc · Natl · Acad · Sci USA 82: 488-492. In short, the DNA to be mutated is cloned into a single-stranded phage vector, such as Ml3. The oligonucleotide that occupies the region to be altered and encodes a substitution is hybridized to single-stranded DNA and double-stranded using standard molecular biotechnology. This DNA was then transformed into a suitable bacterial host and confirmed by dideoxynucleotide sequencing. The correct DNA is re-selected to the expression quality -48- This paper size applies the Chinese National Standard (CNS) M specification (21〇 > < 297 Gongchu) (Please read the notes on the back first and fill in this page) Line 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. In addition, the target DNA is mutated, sequenced, and embedded in a suitable expression plastid using standard PCR techniques. The following specific examples are presented by way of illustration, but this does not limit certain aspects of the invention and preferred specific examples. Example 1 Preparation of Synthetic Placenta Dikonin (102-159) Materials and Methods / Reagents. Fluorescent substrate Tos-Gly-Pro-Lys · AMC purchased from Bachem BioScience Inc (King of Prussia, PA) 0 PNGB, Pro_Phe_Arg_AMC, Ala-Ala-Pro-Met-AMC, bovine trypsin (type III), human Plasma kallikrein, and human plasmin were purchased from Sigma (St. Louis, MO). Recombinant aprotinin (Trasylol®) was obtained from Bayer AG (Wuppertal, Germany). Pre-filled Gin Wang resin was obtained from Novabiochem (La Jolla, CA). Thioanisole, ethanedithiol, and tertiary butyl methyl ether were obtained from Aldrich (Milwaulkee, WI). 0. Functional placenta diconine (7-64) and (102-159) are quantitatively present in the refolded sample The content of trypsin inhibitory activity in each purification stage was measured by GPK-AMC. Bovine trypsin (200 picomoles) at 37 ° C with diconine (7-64) or (102-159) from each purification stage in buffer solution A (50 mM Hepes, pH 7.5, 0.1 M NaCl , 2 mM CaCl2 and 0.01% triton X_100) were incubated for 5 minutes. Add GPK-AMC (final 20 // M), and measure the fluorescence (ex = 370 nm, em = 432 nm) in a Perkin_Elmer LS-50B fluorometer, and determine the coumarin content produced in 2 minutes. According to the equation, the percent inhibition of each test sample is estimated, where R0 is the increased labor rate in the presence of the inhibitor, and Ri is the rate determined in the absence of the added sample. -49- This paper size applies Chinese national standards (CNS) A4 specification (21〇X 297 mm) (Please read the notes on the back and fill in this page first) _ Thread 555764 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of Invention (47) Rate . One unit of inhibitor activity is defined as the amount required to reach 50% inhibition in the assay under the conditions. Inhibition% = 100 X [1-R. /! ^] (1) Synthesis. Placenta bis-conine (102-159) was synthesized on an Applied Biosystems 420A peptide synthesizer, where NMP-HBTU Fmoc chemistry was used. The peptide was synthesized on a pre-filled Gin resin with 8 times the appropriate amount of amino acid (for each coupling). Dissociation and deprotection in 84.6% trifluoroacetic acid (TFA), 4.4% thioanisole, 2.2% ethanedithiol, 4.4% liquid phenol, and 4.4% H20 at room temperature for 2 hours effect. The crude peptide was precipitated, centrifuged and washed twice with tert-butyl methyl ether. The peptide was purified on a Dynamax 60A C18 reverse phase HPLC column using a TFA / acetonitrile gradient. The final preparation (61.0 mg) yields the correct amino acid composition and molecular weight. It is known from the electron spray mass spectrum () ^ 111 + = 683 6.1; estimated 値 = 683 5.5), and the predicted sequence is YEYECTANAV TGPCRASFPR WYFDVERNSC NNFIYGGCRG NKNSYRSEEA CMLRCFRQ (SEQ ID NO: 6) was purified. Refolding of placenta diconine (102-159) was performed according to the method of Tam et al (J. Am. Chem. Soc. 1991, 113: 6657-62). A portion of the purified peptide (15.2 mg) was dissolved in 4.0 ml of 0.1 M Tris, pH 6.0 and 8 M urea. A solution containing 23% DMSO, 0.1 M Tris, pH 6.0 was added dropwise to obtain a final concentration of 0.5 mg / ml peptide in 20% DMSO, 0.1 M Tris, pH 6.0, and 1 M urea. The oxidation of the disulfide bond is completed. After it was diluted 1:10 in a buffer solution containing 50 mM Tris, pH 8.0 and 0.1 M NaCl, the solution was stirred at 50 ° C for 24 hours. The material was purified using a kallikrein affinity column, which was adhered from 30 mg of bovine pancreatic kallikrein (Bayer AG) to 3.5 ml of CNBr-activated Sepharose (Pharmacia), produced according to the manufacturer's instructions -50- Applicable to Chinese National Standard (CNS) A4 specification (21〇 < 297 mm) (Please read the notes on the back and fill in this page first) • Installation and wiring 555764 A7 B7 5. Description of the invention (48). The refolded material was added to the affinity column at a flow rate of 1 ml / min and washed with 50 mM Tris, pH 8.0 and 0.1 M NaCl until the 280 nm absorbance of the wash solution was no longer measured. The column was dissolved with 3 volumes of 0.2 M acetic acid, pH 4.0 and 1.7. Pool the active fractions (see below) and adjust the pH of the solution to 2.5. The material was added directly to a Vydac C18 reverse-phase column (5 μm, 0.46 X 25 cm), which had been equilibrated with 22.5% acetonitrile at 0.1% TFA. Separation was achieved using a linear gradient of 22.5 to 40% acetonitrile in 0.1% TFA over 40 minutes at 1.0 ml / min. The active fractions were pooled, lyophilized, redissolved in 0.1% TFA, and stored at -20 ° C until needed. result. Using 20% DMSO as the oxidant, as described above, the synthetic placenta diconicin (102-159) was refolded and purified according to the following 2-step purification strategy to generate an active trypsin inhibitor (Table 1 below) . (Please read the precautions on the back-: Write this page) Printed by the Consumers Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs Printed Table 1 Purification Table of Separated Placenta Twin Corningin (102-159) Milligram unit c SpA Yield step (ml) Milliliter (U) (U / mg) 8.0M urea 4.0 3.75a 15.0 0 0 20% DMSO 32.0 0.47a 15.0 16,162 1,078 100 Kallikrein 9.8 0.009b 0.09 15,700 170,000 97 Pro Afterburner C18 3.0 0.013ab 0.04 11,964 300,000 74 a protein is determined by AAA. b The protein is determined by OD280nm, and the elongation coefficient determined by the purified protein (1.7X104 liters Mohr 4 cm) is used. c Unit 1 is defined as the amount of material required to inhibit 50% trypsin activity in standard analysis. Line --- -51-This paper size applies to China National Standard (CNS) Α4 size (210X 297 mm) 555764 A7

Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economics. The crude refolded material was analyzed on a solidified column of bovine pancreatic kallikrein by column chromatography. It selectively separated 6.0% of the protein and had 97% trypsin inhibition. C18 reversed phase chromatography can be used to generate 2 times further purification, and the overall recovery is 74%. In RPHLC, the reduced and refolded placenta diconicin (102-159) exhibited dissolution times of 263 and 20 minutes, respectively. The spectroscopic analysis of the purified material showed a molecular weight of 68298.8, which lost 6 mass units from the starting material. This illustrates the complete formation of the three disulfide bonds predicted from the peptide sequence. The Multiphor II electrophoresis system (Pharmacia) was used in accordance with the manufacturer's recommendations, plus pi standards, and the preformed Ampholine® PAGplate (pH 3 · 5 to 9.5) was used to determine the purified, refolded synthetic placenta diconine (1 〇2_ 159), and the focal length reaches 1.5 hours, find its isoelectric point. After staining, the moving distance from the cathode edge of the gel to different protein bands was detected. A standard curve is generated by plotting the distance of the standard against the equivalent pl, s to determine the pi 値 of each unknown. With this technique, the pi 値 of placenta diconine (102-159) was determined to be 8 · 3 ' consistent with that expected from the amino acid sequence. This is lower than the 10.5 number developed by aprotinin p I 値 (Tenstad et al., 1994, Acta Physiol Scand. 152: 33_50). Example 2 Preparation of Synthetic Placenta Dikonin (7_64) Synthesized as described in Placenta Di Cornin (1 02- 1 59), and then folding and purifying the placenta Di Cornin (7-64), but with the following changes: In refolding, the synthetic peptide was stirred in a 20% DMSO solution at 25t for 30 hours; C18 RP-HPLC purification was achieved with a linear gradient of 25 to 45% acetonitrile in 0.1% TFA, 40-52- Standards are applicable to China National Standard (CNS) A4 specifications (210 ^ 297 mm) (Please read the precautions on the back II: Write this page) Order-line · Printed by the Central Consumers Bureau of the Ministry of Economic Affairs Consumer Cooperatives 555764 A7 B7 5 2. Description of the invention (50) minutes (1 ml / min). The active fraction from the first C18 process was refilled into the column and fractionated with a linear gradient of 20 to 40% acetonitrile in 0.1% TFA (60 minutes, 1 ml / min). result. The final purified reduced peptide showed MH + = 6563, which is consistent with the following sequence: IHDFCLVSKV VOICRASMPR WWYNVTDGSC QLFVYGGCDG NSNNYLTKEE CLKKCATV (SEQ ID NO: 4) Refolding and purification can generate a functional Kunitz portion, which is an active trypsin inhibitor Agent (Table 2 below).

Table 2A Purification of synthetic placenta diconicin (7-64) % DMSO 64.0 0.31 20.0 68,699 3,435 100 Kali affinity pH 4.0 11.7 0.10 1.16 43,333 36, 110 62 Kali affinity pH 1.7 9.0 0.64 5.8 4972 857 7.2 C18-1 4.6 0.14 0.06 21,905 350,143 31.9 C18-2 1.0 0.08 0.02 7,937 466,882 11.5 Purified The refolded protein showed MH + = 6558, which is 5 ± 1 mass unit, which is less than the reduced peptide. This proves that refolding can cause the formation of at least one suitable disulfide bond. The method of determining the pi 値 of placenta twin-corninin (102-159) is determined by the method of determining the pi 値 of placenta-di-koninin (102-159). Placenta Shuang Corningsu (7 · 64) presents pi 値 -53- This paper size is applicable to China National Standard (CNS) Α4 size (210X 297 mm) (Please read the note on the back to write this page) -

, 1T-line 555764 A7 B7 V. Description of the invention (51) Test radon (pl = 7.9) is higher. The refolded placenta diconine (7-64) moves to the anodized edge (pH 9.5) of the gel, and the correct pi 决定 cannot be determined under these conditions. Continuous preparation of synthetic placenta dikonin (7-64) Because the synthetic placenta dikonin (7-64) cannot be completely deprotected before purification and refolding, a protein that must be completely deprotected is used to repeat the process. fold. Basically synthesize, refold, and purify placenta dikonin (7-64) as described in placenta dikonin (102-159), but with the following changes: in refolding, a synthetic peptide (0.27 mg / ml) Presented as a solution in 20% DMSO, stirred at 25 ° C for 30 hours; a linear gradient of 22.5 to 50% acetonitrile in 0.1% TFA was used to achieve C18RP_HPLC purification in 40 minutes (1 ml / min). result. The final purified reduced peptide showed MH + = 6567.5, which fits the following sequence: IHDFCLLVSKV VOICRASMPRW WYNVTDGSC QLFVYGGCDG NSNNYLTKEE CLKKCATV (SEQ ID NO: 4) Refolding and purification can generate a functional Kunitz moiety, which can be an active trypsin inhibitor (Table 2B below). (Please read the note on the back first and write this page)-Printed by the Central Bureau of Standards of the Ministry of Economic Affairs, Printed by the Consumer Cooperatives of Table 2B Synthetic Purification Table of Placenta Twin Corningin (7-64) Table 2B Purified Vel mg / mg Yield steps per milliliter (ml) Milliliter (U) (U / mg) 8.0M urea 4.9 2.1 10.5 0 0-20% DMSO 39.0 0.27 10.5 236,000 22,500 100 Kail affinity (PH 2) 14.5 0.3 0.43 120,000 279,070 50.9 Cl 8 inverse Phase 0.2 1.2 0.24 70,676 294,483 30.0 -54- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (52) Purified The refolded protein showed MH + = 6561.2, which is 6.3 mass units, which is less than the reduced peptide. This indicates that refolding can lead to the formation of the expected three disulfide bonds. The pi 値 of placental doxorubicin (7-64) was determined by the method of determining placenta doxycycline (102-159) pl 値. Refolded placenta diconine (7-64) showed a pi 値 of 8.85, slightly higher than the predicted 预测 (pl = 7.9). Example 3 In-tube specific proteases of functional placenta diconicin fragment (102-159). As described previously, bovine trypsin, human plasmin, and bovine pancreatic kallikrein were quantified using para-nitrophenyl para-guanidinobenzoate by active-site titration. (Chase, T., and Shaw, E., (1970) Methods Enzmol., 19: 20-27). Human kallikrein was quantified by active site titration, using bovine aprotinin as a standard and PFR_AMC as the substrate, assuming the formation of a 1: 1 complex. Under the conditions used by each enzyme, the Km of GPK-AMC using trypsin and fibrinase is 29 "] ^ 1 and 7 2 6 a M; PFR-AMC uses human plasma kallikrein and bovine pancreas The Km of visceral kallikrein was 457 " M and 81.5 "M; AAPR-AMC with Km 値 of elastase was 1600 βM. Human tissue kallikrein (Bayer, Germany) was quantified by active-site titration, and para-nitrophenyl para-guanidinobenzoic acid HC1 salt (Chase, T., and Shaw, E., (1970) Methods Enzmol · 19: 20-27) 〇Inhibition kinetics: To detect the inhibitory effect of placenta dikonin (102-159) or aprotinin on trypsin, 50 pM trypsin and placenta dikonin (10-159) (0-2 nM) or aprotinin (0 · 3 nM) was incubated in buffer solution a for a total volume of 1.0¾ liters. After 5 minutes at 37QC, add 15 microliters of 2 mM GPK- -55- This paper size applies Chinese national standard ((: milk) 8 4 size (210 乂 297 mm) _ (Please read the notes on the back first ml fill in this page). Binding and Threading 555764 Employees ’cooperation with the Central Standards Bureau of the Ministry of Economic Affairs, printed A7 B7__V. Invention Description (53) AMC, and track changes in fluorescence. Fibrinolytic enzyme (50 pM ) And placenta biscominin (102-159) (0-10 nM) or aprotinin (〇-4 nM) in 50 mM Tris-HCl (pH 7.5), 0.1 M NaC and 0.02% triton x The buffer solution of -100 determines the inhibitory effect of placenta diconine (102-159) and aprotinin on human plasmin. After 5 minutes at 37 ° C, 25 microliters of 20 mM GPK-AMC was added. And track changes in fluorescence. Use kallikrein (2.5 nM) and placenta dicominin (102-159) (0-3 nM) or aprotinin (0-45 nM) at 50 mM Tris- HCl (pH 8.0), 50 mM NaCl, and 0.02% triton χ · 100 determine the inhibition of human plasma kallikrein by placenta diconine (102-159) or aprotinin. 5 minutes at 37 ° C Afterwards, add 15 μl of 20 mM P FR-AMC, and track changes in fluorescence. In a similar manner, kallikrein (92 pM), placenta diconine (102_159) (0_1 · 6 nM) and aprotinin (0-14 pM) and 100 & quot The quality of the final concentration of Μ determines the inhibitory effect of placenta diconicin (102-159) and aprotinin on bovine pancreatic kallikrein. Enzfitter software (Biosoft, Cambridge, UK) was used to analyze the program using nonlinear regression Determine the apparent inhibitory constant K Guang: According to the equilibrium formula for tightly bound inhibitors, the kinetic data of each experiment:% / ¥. = 1-India.-1. + 1 ^-[(£. + 1. + 1 ^) 2-4 £. 1.] 1/2) / 2 £. (2) where Vi / V. Is the enzyme activity (inhibited versus uninhibited rate) of the fraction, and E. And I. The total concentration of enzymes and inhibitors. Ki 値 is obtained by correcting the mass effect according to the following formula: K ^ KiVci + tSo] / ^) (3) (Boudier, C., and Bieth, JG, (1989) Biochim Biophys Acta.? 995: 36-41) About Placenta Shuang Corningin (102-159) and Aprotinin on Human Neutrophils -56- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) (Fill in this page)-Installed, 11 lines 555764 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

A7 B7 V. Description of the invention (54) Inhibitory effect of protease, elastase (19 nM) is co-cultivated with placenta diconicin (102-159) (15 nM) or aprotinin (0-7.5 " M) In a buffer solution containing 0.1 M Tris-HCl (pH 8.0) and 0.05% TritonX-100. At 37 ° 0 for 5 minutes, AAPM-AMC (500 " M or 1000 " M) was added and the fluorescence was measured at 2 minutes. Ki 决定 was determined from the Dixon mapping of 1 / V pair [I] at two different substrate concentrations (Dixon et al., 1979). Combine the human kallikrein of 0.35 nM with placenta dikonin (7-64) (0-40 nM) or placental dikonin (102-159) (0-2.5 nM) or aprotinin (0- 0.5 nM) was incubated in 1 ml reaction volume containing 50 mM Tris-HCl buffer solution pH 9.0, 50 mM NaCl, and 0.1% triton X-100 to detect aprotinin, placenta diconicin fragment (7-64 ) Or placenta diconine (102-159) inhibits kallikrein in human tissues. After 5 minutes at 37 ° C, 5 μl of 2 mM PFR-AMC was added to a final concentration of 10 // μM, and changes in fluorescence were followed. Under the conditions used, the PFR-AMC Km 肽 of human tissue kallikrein is 0.5 # M. Incubate 0.87 nM human factor Xa and an increasing inhibitor in a buffer solution containing 20 mM Tirs (pH 7.5), 0.1 M NaCl, and 0.1% BSA to detect synthetic placenta diconine (102-159) Inhibitory effect of recombinant placenta diconine and aprotinin on human factor Xa (American Diagnostica, Inc, Greenwich, CT). After 5 minutes at 37 ° C, 30 μl of 20 mM LGR-AMC (Sigma) was added and the change in fluorescence was tracked. Human urine can be detected by incubating urokinase (2.7 nanograms) and inhibitor in a total volume of 1 ml containing 5010] ^ 111 ^ -HCl (pH 8.0), 50 mM NaCl and 0.1% Triton x-100. Kinase (Sigma) is an inhibitory effect of Kunitz inhibitor. After 5 minutes at 371, 35 microliters of 20 mM GGR · AMC (Sigma) was added and the fluorescence changes were tracked. FXIa (0.1 nM) and 0-800 nM -57- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the notes on the back first and fill in this page) Binding and Threading 555764

7 B V. Description of the invention (55) Placenta diconicin (7-64), 0-140 nM placental diconicin (102-159) or 0 · 40 uM aprotinin cultured at 50 mM Hepes pH 7.5, 100 mM NaCl, 2 mM CaCl2, 0.01% Triton x_100 and 1% BSA in a 1 ml volume buffer solution were used to measure the inhibitory effect of factor XIa (from Enzyme Research Labs, Southbend, IN). After 5 minutes at 37 ° C, 10 microliters of 40 mM Boc-Glu (OBzl) -Ala-Arg-AMC (Bachem Biosciences, King of Prussia, PA) was added, and the changes were followed. result. A direct comparison of the placenta succinicin (102-159) and aprotinin inhibition profiles was made by using various proteases to detect their inhibition constants under the same conditions. Ki 値 is listed in Table 3 below. Table 3 (Please read the notes on the back to fill in this page) m. Shuang Corningsu (102-159) inhibits various proteases. Ki 印 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. Shuangkangxin (102-159) Ki (nM) inhibitor peptide Ki (nM) (_ Km (mM) trypsin (48.5 pM) 0.4 0.8 CPK-AMC (0.03 mM) 0.022 chymotrypsin (5 nM) 0.24 0.86 AAPF-pNA (0.08 mM) 0.027 Bovine visceral kallikrein (92.0 pM) 0.4 0.02 PFR-AMC (0.1 mM) 0.08 human blood cable kinase (2.5 nM) 0.3 19.0 l * FR-A) VlC (0.3 mM) 0.46 Another type of fibrinolytic agent (50 pM) 1.8 1.3 GpK-AMC (0.5 mM) 0.73 Human neutrophil elastase (19 nM) 323.U 8500.0 AAPM-AMC (1.0 / / M) 1.6 factor Xlla > 300.0 12,000.0 PFR-AMC (0.2 " M) 0.35 human tissue fine release enzyme (0.35 nM) 0.13 0.004 PFR-AMC (10 " M) 0.0057 factor Xa (0.87nM) 274 NI at Μ LGR-AMC (0.6 mM) ND. Urokinase 11000 4500 GGR-AMC (0.7 mM) ND Factor Xia (O.lnM) 15 288 E (OBz) AR-AMC (0.4 mM) 0.46 -58- line _ This paper size applies to Chinese National Standards (CNS ) A4 specification (21 × X 297 mm) 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 __ B7___ V. Description of the invention (56) η.? Under the conditions of application, placenta dual-conine (102-159 ) And aprotinin inhibit bovine trypsin and human plasmin to a comparable extent. Aprotinin inhibits elastase with Ki 値 of 8.5 // M. Placenta siretin (102 · 159) to 323 nM Ki 値 inhibits elastase. The placenta diconine (102-159) has a 20-fold higher Ki 値 inhibitory effect on bovine pancreatic kallikrein than the aprotinin. In contrast, the placenta diconine (102-159) ) Is an inhibitor of human plasma kallikrein that is more potent than aprotinin, and its affinity is 56 times higher. Because it acts as a kallikrein inhibitor, placenta dikonin (102-159) is 50 times stronger than Trasylol®, so in order to maintain a patient dose effective for inhibitors in KIU, the required amount of human placenta diconine, or its fragment (ie placenta diconine (102-159)) is lower than Trasylol ®. This reduces the cost per dose of the drug and reduces the possibility of adverse renal effects during re-exposure of medicine and patients. Furthermore, the protein is derived from the human body and is therefore less immunogenic in humans than bovine-derived aprotinin. This result can greatly reduce the risk of adverse immune events if the drug and patient are re-exposed. Example 4 In-tube specificity of the functional placenta diconicin (7 · 64) Using the materials and methods of the above example, the in-tube specificity of the functional human placental diconicin (7-64) was determined. Results: The following table shows the efficacy of placenta diconine (7-64) as an in vitro inhibitor of various serine protease enzymes. The data shown is a comparison of the inhibitory data obtained with placenta bisconine (102-159) or aprotinin (Trasylol®). -59- Applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) for "Zhang's scale" " '----- (Please read the notes on the back first and fill in this page)

1T line 555764 A7 B7 V. Description of the invention (57) Table 4A Diconine (7-64) inhibits Ki of various proteases Table 4A Protease diconine (7-64) Aprotinin diconine (102-159 ) (Concentration) Ki (nM) Ki (nM) Ki (nM) Trypsin (48.5 pM) 0.17 0.8 0.4 Bovine pancreatic kallikrein (92.0 pM) 0.4 0.02 0.4 Human blood beam kallikrein (2.5 nM) 2.4 19.0 0.3 Human plasmin (50 pM) 3.1 1.3 1.8 Bovine chymotrypsin (5ηΜ) 0.6 0.9 0.2 Factor Xlla > 300 12000 > 300 elastase > 100 8500 323 (Please read the (Notes to fill out this page)

The 1T results showed that the amino acid sequence encoding the placenta diconine (7-64) can be refolded to obtain an active serine protease inhibitor, which can effectively antagonize at least 4 types of trypsin serine proteases. Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. The following table 4B also shows the effectiveness of refolded placenta diconine (7-64) as an in-vitro inhibitor of serine proteases. Refolded placenta diconine (7-64) is prepared from proteins that have been determined to be fully deprotected prior to purification and refolding. The data shown are compared with the results obtained using placental biconictin (102-159) or aprotinin (Trasylol®) inhibition.

Table 4B Refolded di-conicin (7-64) inhibits K i 値 -60 of various proteases- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) System 5. Description of the invention (58) Table 4B Protease diconine (7-64) Aprotinin diconine (102-159) (Concentration) Ki (nM) Ki (nM) Ki (nM) Trypsin (50 pM ) 0.2 0.8 0.3 Human plasma kallikrein (0.2 nM) 0.7 19.0 0.7 Human plasmin (50 pM) 3.7 1.3 1.8 Factor Xlla not performed 12,000 4,500 Factor XIa (O.lnM) 200 288 15 Human tissue Kal. 2.3 0.004 0.13 Surprisingly, placenta dikonin (7-64) is stronger than aprotinin in inhibiting human plasma kallikrein, and it is at least similar in acting as a plasmin inhibitor. Effect. These data show that placenta diconine (7-64) is at least as effective as inhibiting peptidase (using in vitro assays), and we can expect better or similar efficacy in vivo. Example 5 Expression of placental diconicinin in yeast (102.159). A synthetic oligonucleotide was used to generate a DNA sequence encoding placental diconicin 102-159 (SEQ ID NO. · 6). The final DNA product contains (5 'to 3 ·) 15 nucleotides, which is fused from the yeast α-pairing factor polypeptide sequence to the in-architectural cDNA sequence (encoding placenta dicominin (102-159)). Stop codon. Once cloned into the yeast expression vector pS604, the cDNA can direct the expression of a fusion protein containing an N-terminal yeast pairing factor polypeptide fused to the placenta diconine (102-159) 5 8 amino acid sequences. Handle and modify the fusion at the dissociation position of the α-pairing factor and the joint ΚΕ-2 at the Kunitz part -61-This paper size is applicable to China National Standard (CNS) A4 (210X 297 mm) (Please read the note on the back first (Fill in this page) -Consumer cooperation of employees of the Ministry of Economics and the Central Bureau of Standards, printed 555764 A7 B7, 5. Description of invention (59) ~ protein, to release the Kunitz part at its natural N-terminus. Fencheng ’s following 5 stocks ’nuclear acid’ which contains > an alternative Hindlll position:

GAA GGG GTA AGC TTG GAT AAA AGA TAT GAA GAA TAC TGC ACC GCC

AAC GCA GTC ACT GGG CCT TGC CGT GCA TCC TTC CCA CGC TGG TAC TTT GAC GTG GAG AGG (SEQ ID NO: 42) B amHI position and 'stop codon:

CGC GGA TCC CTA CTG GCG GAA GCA GCG GAG CAT GCA GGC CTC CTC

AGA GCG GTA GCT GTT CTT ATT GCC CCG GCA GCC TCC ATAGAT GAA

GTT ATT GCA GGA GTT CCT CTC CAC GTC AAA GTA CCA GCG (SEQ ID NO: 43) Glucosinolic acid is dissolved in 10 mM Tris buffer solution pH 8 · 0, which contains 1 mM EDTA, plus 12 μg of each oligo After mixing, make it reach 0.25M NaCl. During hybridization, the oligonucleotide was boiled for 5 minutes to denature, and then allowed to cool from 65 ° C to room temperature for 2 hours. Klenow fragments were used to extend the overlap and then hydrolyzed with Hindlll and BamHI. The resulting hydrolyzed double-stranded fragment was cloned into puci9 and its sequence was confirmed. The pure line containing the correct sequence fragment was hydrolyzed with BamHI / Hindlll, which released the dicominin containing the following ten sequence fragments:

GAA GGG GTA AGC TTG GAT AAA AGA TAT GAA GAA TAC TGC ACC GCC

AAC GCA GTC ACT GGG CCT TGC CGT GCA TCC TTC CCA CGC TGG TAC

TTT GAC GTC GAG AGG AAC TCC TGC AAT AAC TTC ATC TAT GGA GGC

TGC CGG GGC AAT AAG AAC AGC TAC CGC TCT GAG GAG GCC TGC ATG CTC CGC TGC TTC CGC CAG TAG GGA TCC (SEQ ID .: 44) which was then gel purified and linked to pS604 which had been cleaved by BamHI / Hindlll. The mixture was ligated for extraction by gas / gas imitation and purified on an S-200 minispin column. The connected product is directly transformed to yeast strains SC101 and WHL341, and coated -62- This paper size is applicable to China National Standard (CNS) A4 specifications (210X297 mm) l · ---.------ batch 1 (Please read the note on the back Hr to fill out this page first) Order line 555764 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 _V. Description of the invention (60) On the ura screening plate. Twelve colonies from each strain were drawn in a straight line over the ura shedding disc. Single colonies were inoculated into 2 ml of ura DO medium and grown at 30 ° C overnight. Cells were agglomerated at 14000 x g for 2 minutes, and the supernatant was evaluated for the content of placenta biconictin (102-159). Detecting the performance of placenta diconicin (102-159) in transformed yeast. First, evaluate the ability of the supernatant (50 microliters per analysis) to inhibit trypsin activity in the test tube, using the analysis method described in Example 1. (1 ml analytical volume). Unused medium and pure yeast lines showing inactive aprotinin variants were used as negative control groups. Yeast pure lines that can express natural aprotinin were used as a positive control group and used for comparison. The second method to quantify the performance of placenta diconicin (102-159) is to use polyclonal antibodies (pAbs) that antagonize synthetic peptides to track the accumulation of recombinant peptides using Western blots. These studies were performed only with recombinants derived from the SC101 strain, as they can produce greater inhibitory activity than recombinants derived from WHL341. In order to produce pAb, two 6-8 week old New Zealand white female rabbits (Hazelton Research Labs, Denver Pa) were purified and reduced synthetic placental diconicin (102-159) at 250 micrograms on day 0, Immunized in complete Freund's adjuvant, and then chased if cases with incomplete Freund's adjuvant with 125 μg of the same antigen on days 14, 35, 56 and 77. The antisera used in this study were collected after a third addition in a developmental procedure. Multiple antibodies were purified from antisera on protein A. Colonies 2.4 and 2.5 from yeast SC101 transformation (Figure 8) and the aprotinin control group were cultured overnight in 3 (50 ml of TC DO ura DO medium. Cells were agglomerated and the supernatant was made using Centriprep 3 ( Amicon, Beverly, MA) concentrated -63-This paper size is applicable to China National Standard (CNS) a4 size (210X 297mm) (Please read the note on the back first: fill in this page) Pack-、?! Line LT Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 555764 A7 B7 V. Description of the invention (61) The shrinking device is concentrated 100 times. Each sample (30 microliters) uses the manufacturer's steps at 10-20% Tricine buffer gel (Novex , San Diego, CA) SDD-PAGE. Repeated repair gels were unfolded with a silver-stained kit (Integrated Separation Systems, Nantic, MA), transferred to a nitrocellulose membrane, and biosynthesized succintocin (102_159 ) Of purified multiple antibody development. Goat anti-rabbit antibody conjugated with alkaline phosphatase was used as the secondary antibody, according to the manufacturer's instructions (Kirkegaard and Perry, Gaithersburg, MD) ° Purification of placenta from SC101 transformants Corning (102-159) from SCI 01 2.4 strains 1 liter The fermented gravy of the nutrient was harvested by centrifugation (4,000 gX30 minutes), and then filled into a 1.0 ml anhydrous curd trypsin-sepharose column (Takara Biochemical Inc., CA), which had previously been buffered with 50 mM Hepes. pH 7.5, containing 0.1 M NaCl, 2 mM CaCl2 and 0.1% (v / v) triton X-l00 equilibrated. The column was washed with the same buffer solution containing ΐ · M NaCl until A280nM dropped to 0, at this time the tube The column was dissolved with oi Μ formic acid, pH 2.5. The separated fractions were pooled and packed into a ci8 column (Vydac, 5 microns, 4.6 X 250 mm) which had previously been equilibrated with 0.1% TFA and 20-80% Dissolve in a linear gradient of acetonitrile / 0.1% TFA for 50 minutes. Pool fractions containing placenta diconine (102-159) and rechromatograph on C18. Apply a linear gradient of 22.5 to 50% acetonitrile / 0.1% TFA. Results. Fig. 8 shows the percentage of trypsin activity inhibited by 12 colonies derived from the transformation of each of SciOi and WHL341 strains. The results show that the trypsin inhibitor placenta diconine (102-159) All the colonies of the 12 strains of yeast SC101 transformed had a significant amount of trypsin inhibitory activity Force, the comparison with the negative control group, did not enter the ability to inhibit trypsin. So live _ -64 paper size (CNS) A4 specification (21〇χ297 public envy) '' (Please read the notes on the back first and fill in this page) Installation · Line 555764 A7 B7 Employee Consumer Cooperatives, Central Standards Bureau, Ministry of Economic Affairs Printed 5. Description of the invention (62) Sexuality is related to the performance of specific inhibitors in placental strobilin (102-159) transformed cells. Yeast WHL341 samples contain very small amounts of trypsin inhibitory activity. This is related to the slow growth observed by the strain under the conditions applied. Figure 9 shows SDS-P AGE and Western blot analysis of yeast SC101 supernatant. Supernatants derived from recombinant yeasts 2.4 and 25 expressing placenta diconicin (102-159), and supernatants derived from yeasts expressing peptidases, and their silver-stained SDS-PAGE A protein band at about 6 kDa can be generated, which is equivalent to the expected size of each recombinant Kunitz inhibitor portion. Western blot analysis shows that 6 kDa expressed by 2-4 and 2.5 strains needs to be capable of reacting with the pAb of defamable placenta succintin (102_159). In the aprotinin control group, the same 6 kDa band did not react with the same antibody, demonstrating the specificity of the placenta diconicin variant (102-159) antibody. Silver-stained sdS-PAGE of the final preparation of the placenta diconicin C-terminal portion was highly purified (Figure 10). In the final formulation, the overall recovery of trypsin inhibitory activity derived from the gravy was 31%. Sequencing of the purified inhibitor at the n-terminus showed that 40% of the protein could be correctly processed and modified to produce the correct placental dicominin (ι02 · ι 59) N-terminus, while approximately 60% contained a part of the yeast 々 · pairing factor. The purified substance contained an active serine protease inhibitor, which showed Ki 35 in the in vitro inhibition of plasma kallikrein by 0.35 nM. In summary, the protease inhibitor activity and accumulation of synthetic dicomynin (102-159) immunochemically relevant proteins in fermented gravy, and the isolation of placenta dicominin (10) from one of the transformants. 2.159) Proof of the performance of placenta succinicin in the recombinant yeast strain described herein, showing for the first time that yeast can be used to produce placenta succinicin fragments. -65- This paper size is in accordance with Chinese National Standard (CNS) Α4 size (21GX ^ 97 mm).

1T line 555764 A7 B7 V. Description of the invention (63) Preparation of additional constructs in an effort to enhance the performance level of the Kunitz part contained in placenta diconine 102-159 and increase the yield of the correct N-terminal protein . We hypothesized that the N-terminal residue (YEEY--) of placenta diconicin 102-159 can present a dissociation position that can only be confirmed by the yeast KEX-2 protease defect, which can enzymatically remove the yeast a-factor before Area. Therefore, we prepared the yeast expression constructs to produce placenta succinicin 102-159 (the N-terminus of ΕΕΥ · _), 101 · 159 (the N-terminus of ΝΥΕΕΥ ··), and 98-159 (DMFNYEEY ·· ) The P 'subpositions around the dissociation position of KEX · 2 are changed. In an attempt to enhance the level of recombinant protein performance, we used yeast preferred codons rather than mammalian preferred codons for the preparation of certain constructs described below. The structure was prepared basically as described above (as the placenta diconine 102-159, defined as the # 1 conformation) with the following changes: construct # 2 placenta diconine 103-159, yeast codon usage 5 · Conscious stock oligonucleotide GAAGGGGTAA GCTTGGATAA AAGAGAAGAA TACTGTACTG CTAATGCTGT TACTGGTCCA TGTAGAGCTT CTTTTCCAAG ATGGTACTTT GATGTTGAAA GA (SEQ ID NO: 55) and 3 'anti-conscious stock oligonucleotide ACTGGATCCT CATTATTCGAA AACATCTCACATCACACA CATACA NO: 56) Operate the construct # 3 as the above-mentioned expression construct (upper construct # 1) to express the placenta diconine 102-159. Placental diconine 101-159, yeast codon usage -66- Dimensions are applicable to China National Standard (CNS) A4 specifications (210X297 mm) (Please read the notes on the back first and fill out this page)

P, τ Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 _B7__ V. Invention Description (M) 5 · Awareness Shares TACTTTGATG TTGAAAGA (SEQ ID NO: 57) and 3 'anti-conscious oligonucleotide as same as construct # 2, operate as a performance construct (upper construct # 1) to express placenta diconine 102-159 . Construct # 4 Placenta diconicin 98-159, yeast codon usage 5 »Conscious oligonucleotide GAAGGGGTAA GCTTGGATAA AAGAGATATG TTTAATTACG AAGAATACTG TACTGCTAAT GCTGTTACTG GTCCATGTAG AGCTTCTTTT CCAAGATGGT ACTTTGATGTTGAAAGA (SEQ ID NO: 58) and 2 # Identical anti-conscious strand oligonucleotides were operated as described in the expression construct (Constructor # 1, see above). Yeast strain SC101 (MAT from, ura 3-52, sue 2) was transformed by a plastid containing each of the above-mentioned eDNA, and the protein was expressed by the method described above for the production of placenta diconine 102-159 with human codon usage. About 250 ml of each yeast culture can be recovered, and the supernatant from the centrifugation (5 minutes X 3000 RPM) was separately purified by the above 1 ml kallikrein-sepharose column. The relative amounts of trypsin inhibitory activity, the quality of the recovered purified protein, and the N-terminal sequence of the purified protein were determined in the lysate, and are listed in Table 7. Table 7 Relative production levels of different proteins containing the C-terminal Kunitzin part of placenta twin-cornin (please read the notes on the back first and fill in this page) • Binding Line -67- This paper size is applicable to Chinese National Standards (CNS ) a4 specification (210X 297 mm) 555764 A7 B7 printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, description of the invention (65) Table 7 N-terminal sequencing in the lysate of the construct suggested the phasor sequence pair concentration of the inhibitor (pmol) # 2 103-159 Untested and No No No Performance # 3 101-159 25% Inhibition No No Low Performance U 98-159 93% Inhibition 910 DMFNYE- Good Performance Correct Product # 1 102-159 82% Inhibition 480 AKEEGV- Performance of incorrectly processed protein with active results shows that placenta diconicin fragments of different lengths, which contain a C-terminal Kunitz portion, have large differences in their ability to express functionally secreted proteins. The structures of the performance fragments 101-159 and 103_159 produced little or low enzyme activity in the supernatant before purification, and each purified fraction was sequenced at 0.05 N-terminal to generate a measurable amount of inhibition. Agent. On the other hand, placenta biconicin 102-159 or 98-159 is shown to produce significant amounts of protease activity before purification. However, the N-terminal sequencing showed that the purified protein recovered from 102-1596 performance was again greatly incorrectly modified, and the N-terminal and most of the previous proteins it achieved were within the previous sequence of the yeast pairing factor. The processing modification somewhere is consistent. However, the purified protein recovered from placenta succinicin 98-159 can be fully processed and modified in the correct position to produce the correct N-terminus. In addition, compared with the recovery of placenta stiltinin 102-159, almost twice as much protein was recovered. Therefore, the placenta diconicin 98-159 represents a preferred segment length of the C_ terminal Kunitz portion of the placenta diconicin from the brewer's yeast α-paired mother-child pre-primary sequence / KEX-2 processing modification system. -68- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) (please read the note on the back first! Filled in this page)-Installation · 1T-Line 555764 A7 ____B7___ 5. Description of the invention (66) Example 6 Different steps of yeast performance A 58 amino acid peptide derived from the R74593 translation product was also cloned from the R87894-R74593 PCR product to the TA vector TM (Invitrogen, San Diego, CA) after DNA sequencing , Or PCR amplification from human placental cDNA. The amplified DNA product contains 19 nucleotides from the yeast α-pairing factor leader sequence, which pairs with the R74593 sequence and guides the synthesis of YEEY-CFRQ (58 residues), thus making the translation product structurally constitute a Fusion protein from the -pairing factor / Kunitz part. The protein sequence also contains a kex 2 dissociation, which releases the Kunitz moiety at its natural N-terminus. 5. Awareness strand oligonucleotide, which contains the position of Hindlll for breeding, contains the following sequence: GCCAAGCTTG GATAAAAGAT ATGAAGAAT ACTGCACCGC CAACGCA (SEQ ID NO: 30) 3 • The anticonsciousness oligonucleotide contains an available for breeding BamHI position, and a stop codon, and has the following sequence: GGGGATCCTC ACTGCTGGCG GAAGCAGCGG AGCAT (SEQ ID NO: 31) cDNA sequence of 206 nucleotides, can be cloned into yeast expression vector, with the following sequence: CCAAGCTTGG ATAAAAGATA TGAAGAATAC TGCACCGCCA ACGCAGTCAC TGGGCCTTGC CGTGCATCCT TCCCACGCTG GTACTTTGAC GTGGAGAGGA ACTCCTGCAA TAACTTCATC TATGGAGGCT GCCGGGGCAA TAAGAACAGC TACCGCTCTG AGGAGGCCTG CATGCTCCGC TGCTTCCGCC AGCASEQGAGDATAT Standard (CNS) A4 specification (210X297 mm) (Please read the note on the back 9 to fill —: write this page) Printed by the Consumers 'Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 555764 Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Printed A7 B7 V. Description Ming (67) cloned into the yeast expression vector pMT 15 (see US Patent 5,164,482, it has been the case entirety by reference), which has been hydrolyzed by first Hindlll and BamHI. The resulting plastid vector was transformed into yeast SC106 strain using the method of U.S. Patent No. 5,164,482. Isolate and culture URA 3 + yeast transformants under defensive conditions. Depending on the amount of trypsin inhibitory activity accumulated in the culture supernatant over time, the yield of the recombinant placenta diconicin variant can be determined using the in-tube analysis method described above. The fermented gravy was centrifuged at 9000 rpm for 30 minutes. The supernatant was filtered through 0.4 and then through a 0.2 micron filter, diluted to a conductivity of 7.5 ms, and adjusted to pH 3 with citric acid. The samples were reabsorbed in batches to 200 ml of S-86 卩 11 & 1 * 086, and flowed quickly (? 11 & 1111 & (^ &) in 5〇111] \ [citrate steel 卩 113, and stirred to 60 The gel was washed sequentially with 2 liters of 50 mM sodium citrate, pH 3.0; 50 mM Tris-HCl pH 9.0; 20 mM HEPES pH 6.0. The washed gel was transferred to a suitable column, and Dissolve in a linear gradient of 0 to 1 M sodium chloride at 20 mM HEPES pH 6.0. Resolve fractions containing trypsin inhibitory activity in test tubes are pooled and a) solidified anhydrous trypsin column chromatography (basically As described in Example 2); b) solidified bovine kallikrein column chromatography; or c) a combination of traditional chromatography steps, including gel filtration and / or anion-exchange chromatography. Example 7 Isolation and identification of natural human placenta from the placenta. The dicominin protein was purified from intact frozen placenta (Analytical Biological Services, Inc, Wilmington, DE) to be homogeneous in performance. The placenta (740 g) was thawed to room temperature, cut into 0.5-10 cm slices, placed on ice and washed with 600 ml of PBS buffer solution. Wash and decante, and place 240ml placenta sheet -70- This paper size is applicable to Chinese National Standard (CNS) A4 size (210X297mm) (Please read the notes on the back first and fill in this page). Standard Bureau employee consumer cooperative printed A7 B7 V. Invention Description (68) Entered into the Waring Mixer. After adding 300 ml of a buffer solution containing 0.1 M Tirs (pH 8.0) and 0.1 M NaCl, the mixture was mixed at high speed for 2 minutes, decanted into a 750.0 ml centrifuge tube, and placed on ice. This step is repeated until all materials have been processed. The mixed slurry was centrifuged at 4500 X g for 60 minutes at 4 ° C. The supernatant was filtered through gauze, and placenta dikonin was purified using a kallikrein affinity column. The preparation was covalently attached to 70 mg of bovine pancreatic kallikrein (Bayer AG) to 5.0 ml of CNBr-activated Sepharose (Pharmacia ) Follow the manufacturer's instructions. The material was added to the affinity column at a flow rate of 2.0 ml / min, and washed with 0.1 M Tris (pH 8.0) and 0.1 M NaCl until the absorbance of the washing solution at 280 nm was no longer measured. The column was further washed with 0.1 M Tris (pH 8.0), 0.5 M NaCl, and then dissolved with 3 volumes of 0.2 M acetic acid, pH 4.0. Fractions containing kallikrein and trypsin inhibitory activity (see below) were pooled, frozen and lyophilized. Placenta succintin was further purified by gel filtration chromatography on a Superdex 75 10/30 (Pharmacia) column, which was adhered to a Beckman System Gold HPLC system. Briefly, the column was equilibrated in 0.1 M Tris, 0.15 M NaCl and 0.1% Triton X-100 at a flow rate of 0.5 ml / min. The freeze-dried sample was reconstituted in 1.0 ml of 0.1 M Tris pH 8.0 and injected into a gel-filtration column in 200 microliter portions. Fractions were collected (0.5 ml) and analyzed for trypsin and kallikrein inhibitory activity. The active fractions were pooled and the pH of the solution was adjusted to 2.5 with TFA. The material was packed directly into a Vydac C18 reverse-phase column (5 microns, 0.46 X 25 cm), which was first equilibrated in 20% acetonitrile / 0.1% TFA. A linear gradient of 20-80 / 0 acetonitrile / 0.1% TFA was used, 1. Separation was completed at 50 ml / min for 50 minutes. At the beginning, washing was performed with 20% acetonitrile / 0.1% TFA for 20 minutes. Collect fractions (1 ml) and analyze pancreatic eggs -71-This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the notes on the back first and fill in this page)-Pack ·

1T-line 555764 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (69) Inhibitory activities of white enzyme and kallikrein. Fractions containing inhibitory activity were concentrated using a speed · vac concentrator (Savant) and subjected to N-terminal sequence analysis. Functional analysis of placenta diconine: Inhibition of bovine trypsin and human plasma by detecting functional placental kallikrein Kallikrein's ability to achieve their identification. Trypsin inhibitory activity was analyzed in analysis buffer solution (50 mM Hepes, pH 7.5, 0.1 M NaCl, 2.0 mM CaCl2, 0.1% Triton χ-100), at room temperature, in a 9 6-well microtiter plate (Perkin Elmer ), And Gly-Pro-Lys-aminomethylcoumarin was used as the substrate. On a Perkin-Elmer LS_50B fluorometer equipped with a disk reader, the amount of coumarin produced by trypsin was determined by detecting the fluorescence (ex = 370 nm, em = 432 nm). Trypsin (23 µg in 100 µl buffer solution) was mixed with 20 µl of the test sample and incubated at 25 ° C for 10 minutes. The reaction was started by adding 50 microliters of the subject GPK-AMC (33 // M final concentration) to the analysis buffer solution. Detect the fluorescence intensity and determine the% inhibition of each fraction as follows: Inhibition o / flOOxn-Fc / Fd where F. Unknown fluorescence, Fi * only trypsin-control fluorescence. 7.0 nM kallikrein was used in the analysis buffer solution (50 mM Tris, pH 8.0, 50 mM NaCl, 0.1% triton χ · 100) and 66.0 ju M Pro-Phe-Arg- AMC was used as the substrate. Kallikrein inhibitory activity in the test fraction. Determination of Intratube Specificity of Placenta Siaconin The materials and methods described in the previous example were used to determine the in vitro specificity of natural human placenta Siaconin. Placenta diconicin was quantified by trypsin titration at a known concentration in the active position, where CPK-AMC was used as the substrate to track the unbound trypsin. -72- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X 297mm) (please read the notes on the back Plfr first and fill in this page).

1T line-555764 B7 V. Description of the invention (70) Protein sequencing 1ml fraction (C18-29 Delaria) was reduced to 300ml in Speed Vac to reduce the amount of organic solvents. The sample was repacked into a Hewlett-Packard miniature dual-phase reaction column and washed with 1 ml of 2% trifluoroacetic acid. The samples were sequenced on Hewlett-Packard Model G1005A protein sequencing system using Edman degradation. Version 3.0 sequencing methods and all reagents were supplied by Hewlett-Packard. The sequence confirmed 50 cycles with 0 results. Placenta diconicin is purified to apparent homogeneity with sequential kallikrein affinity, gel-filtration, and reverse phase chromatography (see purification table below) ... (Please read the note on the back first. Ipξ: Fill out this page ) Packing Table 5 Purification Table of Natural Placenta Shuang Corningsu U · 179) Table 5 Step Vol (ml) OD 280 (/ ml) OD 280 Unit a ⑼ Unit / OD 280 Placenta Supernatant 1800.0 41.7 75,060 3,000,000 40.0 Miscellaneous Release Enzyme affinity pH 4.0 20.0 0.17 3.36 16,000 4,880 New Zealand release enzyme affinity pH 1.7 10.2 0.45 4.56 12,000 2,630 Superdex 75 15.0 0.0085 0.13 3,191 24,546, 11 Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairsa — Units are defined as can be used in standard analysis Kallikrein and trypsin inhibitory activity are inhibited by 50% of the amount of kallikrein and most of the trypsin inhibitory activity are released from the kallikrein affinity column, pH 4.0. The subsequent gel filtration chromatography (Figure 5) can generate a kallikrein and a high level of inhibitory activity of proteases. A standard curve generated with molecular weight standards under the same conditions was judged to have a molecular weight range of 10 to 40 kDa. Reversed-phase C18 chromatography (Figure 6) can generate 4 inhibitory activities -73- This paper size is applicable to Chinese National Standard (CNS) A4 specifications (210x297 mm) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 555764 A7 B7 Description of the invention (71)-'~ peak' dissolves the strongest one at about 30% acetonitrile. The activity related to the first peak (29th fraction) eluted from C18 showed an amino acid sequence starting from the first amino acid predicted by the placenta diconine (ADRER. ·· ·, SEQ ID NO: 1), and is the same as the sequence detected in 50 cycles of sequencing (amino acid underlined in Figure 3). The cysteine residues within this sequence are silent, as expected by oxidized protein sequencing. Once PTH-pyridylethyl-cysteine is recovered at the 11th and 20th cycles, the cysteine residues at the 11th and 20th amino acid positions of the mature placenta diconine can then be ethylated from sp. Known in protein sequencing. Interestingly, asparagine (Figure 3) at the amino acid residue of sequence 30 is silent, showing that this position appears to be glycosylated. The second 9th fraction generates a major sequence, which is equivalent to the placenta diconicin starting from residue # 1 (2 7 picomoles in cycle 1), plus a small sequence (2 picomoles), which is also Derived from placenta, starting from the 6th place, double-conicin (SIHD). This shows that the final formulation sequenced in fraction 29 is of high purity and seems to be related to the protease inhibitory activity associated with this fraction (Figure 6). Therefore, the final preparation of placenta succintocin obtained by C18 chromatography has high purity according to silver-stained SDS-PAGE analysis (Figure 7). San Diego, CA) showed Mr movement at 24 kDa, and the molecular weight markers for calibration are as follows: insulin (29 kDa); bovine trypsin inhibitor (5.8 kDa); lysozyme (14.7 kDa); lactoglobulin (18.4 kDa); carbonic anhydrase (29 kDa); and milk albumin kDa). The above-mentioned size of placenta diconine SDS-PAGE is consistent with that predicted from the full-length coding sequence (Figure 4F). -74- This paper size is in accordance with Chinese National Standard (CNS) Α4 specification (210X 297 mm) (Please read the notes on the back first and fill in this page).

1T 555764 A7 B7 V. Description of the invention (72) As expected from the above N-terminal sequencing results, the purified protein can be reacted with the antibody that can destroy the placenta diconine (7-64) to produce A band with the same Mr (Figure 12A) as the purified formulation observed on a gel with a silver frame (Figure 7). However, when the same preparation was reacted with a biosynthetic antibody to placenta diconine (102-159), no band equivalent to the full-length protein was observed. Conversely, a fragment co-moved with combinin (102-159) of about 6 kDa was observed. The simplest explanation of these results is that the purified preparation is degraded after purification to generate an N-terminal fragment containing an N-terminal portion and a C-terminal fragment containing a C-terminal portion. Assuming that the antiserum-reactive fragment of placenta diconulin (7-64) lacks the C-terminus of the full-length protein, the size (24 kDa) can suggest a state of hyperglycosylation. Table 6 below shows the in vitro inhibitory effects of placenta diconine on various serine proteases. Comparison of data with aprotinin (Trasylol®). Table 6 (please read the note on the back first-please fill out this page) • Packing · 11 The inhibitory effect of placenta dikonin on various proteases by the Central Consumers Bureau of the Ministry of Economic Affairs. Aprotinin (concentration) Ki (nM) Ki (nM) Yueyi protease (48 · 5 pM) 0.13 0.8 Human plasmin 1.9 1.3 (^ 〇pM) The results showed that it was isolated from a natural source (human placenta) The placenta dikonin is a potent inhibitor of trypsin-like serine proteases. Example 8 -75- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm)-line 555764 Printed by A7 B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (73) Placenta Shuang Corning Su Phenotype multiple tissue samples in human organs and tissues were purchased from Clontech, which contained 2 micrograms of polyA + RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Two different cDNA probes were used: 1) a gel-purified cDNA encoding placenta diconicin (102-159); 2) a 780 bp cDNA derived from PCR (Figure 4E), which was deciphered from TAA pure line with EcoRhJc Released and gel purified. Each probe was labeled with 32P-dCTP, using any guide labeling kit from Boehringer Mannheim Biochemicals (Indiana), and then hybridized to multiple recombinant samples as instructed. Biomax film and 18-hour exposure were used to expand with Umax Scanner and scan with Adobe Photoshop to generate autoradiograms. result. The tissue phenotypes observed with the placenta dual-conicin (102-159) probe (Figure 11A) or a larger probe containing two Kunitz fractions (Figure 11B) were basically the same as expected. Placenta succintin mRNA is most abundant in the pancreas and placenta. Significant levels were also seen in the lung, brain, and kidney, but lower levels in the heart and liver, and mRNA was not detected in skeletal muscle. In all cases, the transcript size was 1.95 仟 bases, which closely matched the predetermined size of placenta diconicin derived from the EST overlapping alignment and the selection of the full-length cDNA in the previous illustration. The extensive tissue distribution of mRNA shows that placenta succintocin is widely expressed. Since the protein also contains a leader sequence, it will have an enlarged exposure to the human immune system as long as it can be considered to be its own protein. Evidence that placenta diconicin mRNA exhibits another extensive tissue distribution can be derived from the fact that certain EST entities (Figure 4B) that are homogeneous to placental diconicin are composed of human adult and child brains, and human retinas, breasts , Ovarian, nose epithelium and placenta. Therefore, it can be concluded that administration to human patients -76- This paper size applies the Chinese National Standard (CNS) A4 specification (21 × 297 mm) (Please read the precautions on the back first! Fill out this page)-Binding · 555764 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of Invention (74) Natural human proteins do not seem to defame the immune response. Interestingly, the phenotypic pattern of placenta diconicin is reminiscent of bovine aprotinin, which is found at high levels in bovine lung and pancreas. In order to further illustrate the performance of placenta dicominin, the RT-PCR of total RNA obtained from the following human cells was decided: unstimulated human umbilical vein endothelial cells (HUVECs), HK-2 (cells derived from renal proximal tubules) Strain), TF-1 (red and white blood cell line) and phorbolester (PMA) -stimulated human peripheral blood white blood cells. The probes used are: CACCTGATCGCGAGACCCC (consciousness unit; SEQ ID NO: 59); CTGGCGGAAGCAGCGGAGCATGC (anti-consciousness unit; SEQ ID NO: 00), which is designed to expand 600 b.p. of placenta diconicin-encoding cDNA fragments. Actin primers were included to expand 800 b.p. actin fragments to allow comparisons to be normalized. Although the 800 b.p. fragment was identified in all columns as ethidium bromide on all agarose gels with the same intensity, the 600 b.p. placenta diconicin fragment did not exist in HUVECS, but was present in large amounts in other cell lines. We concluded that placenta succintocin is not expressed in at least some endothelial cells, but in some white blood cell populations. Example 9 Purification and Characterization of Placenta Dikonin (1-170) Highly Purified from Baculovirus / Sf9 Expressive Lineage Placental dikonin (large placenta 1-170) containing two Kunitz moieties, as follows Expressed in Sf9 cells. The placenta diconicin cDNA was obtained by PCR (Figure 4E), which was contained in the TA vector (see the previous example), and then interpreted with Hindlll and Xbal water to generate a 5 'Xbal position and 3. Hindlll -77- This paper size applies the Chinese National Standard (CNS) Α4 specification (210 × 297 mm) (Please read the note on the back first! Pi: Fill out this page) Binding line 555764 Printed by A7 _B7___ 5. Description of the invention (75) A segment of the position measurement connection. This fragment was purified by gel and cloned into M13mpl9 vector (New England Biolabs, Beverly, MA). Using in-tube mutation method (Kunkel TA, (1985) Proc. Natl. Acad. Sci. USA, 82: 488-492) can generate the PstI position of V at the Xbal position at the 5 'end, but it is located at the starting position encoding ATG The 5 'end of the natural placenta diconicin signal peptide and the mature placental diconicin coding sequence. Oligonucleotide used for mutation has the following sequence: 5f CGC CTC TCG GCT GAC CTG GCC CTG CAG ATG GCG CAC GTG TGC GGG 3, (SEQ ID NO: 61) The following oligonucleotides are similar at the 1 end of cDNA 3 The genetic operation operates a stop codon (TAG) and Bglll / Xmal position: 51 CTG CCC CTT GGC TCA AAG TAG GAA GAT CTT CCC CCC GGG GGG GTG GTT CTG GCG GGG GTG 3, (SEQ ID NO: 62). The stop codon and the sequence encoding the placenta diconicin are located on the structure and cause an immediate termination after the lysine of the amino acid residue No. 170, so the truncated placenta encoding the lack of the putative transmembrane portion is encoded. Corning fragment. The product produced by the hydrolysis of Pst2 and Bglll was isolated and then cloned into the BacPac8 vector to represent the placental diconicin fragment (1-170), which contains two Kunitz parts, but immediately at the N_terminus of the transected membrane fragment. Truncate. The performance of Sf-9 insect cells on dicominin was best when the culture medium was recovered 72 hours after infection, and it was the best when multiple infections occurred one to one. After recovery, the baculovirus cell culture supernatant (2 liters) was adjusted to pH 8.0 by adding Tris-HCl. Shuang Corningin Chromatographic purification was performed using a 5 ml bovine pancreatic kallikrein affinity column described in Example 7, which purified the natural placental Shuang Corningin from the placenta. The dissolved substance was adjusted to pH 2.5 with TFA, and accepted C1 8 reverse phase column chromatography. 78- This paper size is applicable to China National Standard (CNS) A4 size (210X 297 mm) (Please read the note on the back first) ^^ Fill in this page) _ Binding line 555764 A7 B7 V. Description of the invention (76) (1.0 x 25 cm), which has previously been equilibrated in 10% acetonitrile / 0.1% TFA with a flow rate of 1 ml / min. Shuang Corningsu dissolves in a linear gradient of 10-80% acetonitrile / 0.1% ΊΤΑ in 40 minutes. The active fractions were pooled, cold-dried, and redissolved in 50 mM Hepes (pH 7.5), 0.1 M Naa, 2 mM CaCl2, and 0.1% triton X-100, and stored at -20 ° C until needed. . The amino acid analysis was used to determine the concentration of recombinant diconine. result. Recombinant diconicin was purified from a baculovirus cell culture supernatant using a 2-step purification strategy shown below to generate an active trypsin inhibitor (Table 8 below). Table 8 Purification of the transformed culture supernatant of the recombinant sigunicin. Table 8 Purification of Vol OD 280 / ml OD280 Unit specific activity step (ml) Total amidine (U / OD) supernatant 2300.0 9.0 20,700 6,150,000 297 Kal affinity 23.0 0.12 2.76 40,700 14,746 C18 RP-HPLC 0.4 3.84 1.54 11,111 72,150 (Please read the notes on the back and fill out this page first) 94. Equipment · Central Bureau of Standards, Ministry of Economic Affairs, prints crude materials for cured cattle Pancreatic kinase releasing enzyme affinity column chromatography can selectively separate 0.013% protein and 0.67% trypsin inhibitory activity. Most of the trypsin inhibitory activity present in the starting supernatant did not bind to the immobilized kallikrein, and was not related to diconine (results not shown). The C18 reversed phase chromatography can be further purified 5 times, and the recovery rate is 0.2%. The final preparation was highly purified by SDS-PAGE (Fig. 13), showed Mr 21.3 kDa, and was able to react with rabbit anti-placenta diconine 102-159 (not shown) on the immunosucker. N_end sequence (26 cycles) can produce mature -79 · This paper size applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) line 555764 A7 B7 V. Description of invention (77) Central Bureau of Standards, Ministry of Economic Affairs The expected sequence of placenta diconicin printed by the employee consumer cooperative (Figure 4F), starting from residue +1 (ADRER ...) shows that the signal is correctly processed and modified in Sf9 cells. Purified placental diconicin (100 picomoles) from Sf9 cells was calcined at pyridylethyl, hydrolyzed by CNBr, and sequenced with the resulting fragment unresolved. Sequencing 20 cycles can generate the following ends: Sequence quantity placenta double-conicin residue # LRCFrQQENPP-PLG --- 21 Pico Morse 154-168 (SEQ ID NO: 63) ADRERSIHDFCLVSKWGRC 20 Pico Morse i_2〇 (SEQ (ID NO: 64) FNYeEYCTANAVTGPCRASF 16 pico-moles 100-119 (SEQ ID NO: 65) Pr-YV-dGS-QFYG 6 pico-moles 25-43 (SEQ ID NO: 66) Therefore, it can be recovered equivalent to the expected 4 ] Sf-terminus. This confirms that the Sf9-expressing protein contains the entire external sequence of placenta diconine (1-170). N-terminal sequencing (50 cycles) of additional samples of unhydrolyzed placenta diconine (1_170) can result in the absence of any amino acid sequence of PTH-amino acids at 30 cycles (expected to be PTH-days Winter melamine). Similar results were obtained from the sequencing of natural proteins from the human placenta (Example 7), and this was consistent with the glycosylation of this residue as detected by the amino acid sequence surrounding the asparagine residue. Furthermore, cysteine residues in this region are also silent, consistent with their involvement in disulfide bonding. Example 10 Inhibitory Specificity of Purified Placenta Dikoninin Derived from Sf9 Cells The materials and methods described in Examples 3, 4 and 7 were used to determine the in vitro specificity of the recombinant dikoninin. In addition, 0.3 5 nM human tissue kallikrein recombinant dicominin was cultivated in a buffer solution containing 50 mM Tris (pH 9.0), 50 mM NaCl and 0.01% Triton x-100 to detect the dicominin pair. Inhibition of Kallikrein in Human Tissues. After 5 minutes at 37 ° C, 5 μl of 2 mM PFR-AMC was added and the changes in fluorescence were tracked. (Please read the note on the back first. Fill out this page) -S-T »

I -80- This paper size applies Chinese National Standard (CNS) M specification (210X 297 mm) 555764 A7 _ B7 V. Description of the invention (78) > Inhibition of tissue plasminogen activator (tPA) It can be determined as follows: tP A (single-chain version from human melanoma cell culture, Sigma Chemical Co, St Louis, MO) and the inhibitor are pre-incubated at 2011] ^ 1 ^ buffer solution ( ? 117.2) for 2 hours, which contains 151111 NaCl, and 0.02% sodium azide. Next transfer to a reaction system containing the following initial component concentrations to initiate the reaction: tPA (7.5 nM), inhibitor 0-6.6 // M, DIle-Lpro-Larg-p nitroaniline (1 mM) at 28 mM Tris The buffer solution has a pH of 8.5 and contains 0.004% (ν / ν) triton x-100 and 0.005% (v / v) sodium azide. After 2 hours of incubation at 37 ° C, A405 was detected to determine the formation of para-nitroaniline. The following table shows the effectiveness of recombinant dicominin as various serine protease inhibitors in test tubes. The data shown are compared with the inhibitory effect obtained by using recombinant dicominin, or aprotinin. Table 9 Ki 値 Comparison of Inhibitory Effects of Recombinant Placenta Dikonin (1-170) or Aprotinin on Various Proteases 11-^ ------ install ------ order (please read the note on the back first (Fill in this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs, Table 9 Protease recombinant succinicin skin inhibitor (concentration) Ki (nM) Ki (nM) Teng protease (48.5 pM) 0.064 0.8 Human plasma Kal (2.5 nM) 0.18 19.0 Human tissue Kal (0.35 ηM) 0.04 0.004 Bovine pancreas KalClOOpM) 0.12 0.02 Human plasmin (50 pM) 0.23 1.3 Factor Xa (0.87ηΜ) 180 5% inhibition (over 31 " Μ) Factor XIa (O.lnM) 3.0 288 Tissue plasminogen activator < 60 Uninhibited (at 6.6 ju Μ) (7.5 nM) Tissue Vila factor 800 Not inhibited (at 1 ju Μ) Line -81 -This paper size is in accordance with Chinese National Standard (CNS) A4 (21 × 297 mm) 555764 Printed on A7 B7 by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (79) The results show that the recombinant Shuang Corning can be used in Expressed in insect cells to produce active protease inhibitors that effectively antagonize Less than 5 different serine protease inhibitors. Recombinant kallikrein is more potent than aprotinin in antagonizing human plasma kallikrein, trypsin, and plasmin. Surprisingly, the recombinant dicominin was more potent than the synthetic derived dicominin fragments (7-64) and (102_159) in antagonizing the enzymes tested. These data show that recombinant tannins are more potent than aprotinin, which is known from in-tube analysis, and we can expect them to have better in vivo efficacy. In addition to detecting the strength of specific proteases, the ability of placenta dikonin (1-170) to prolong the activated partial thromboplastinase time (APTT) can be evaluated and compared with the activity related to makeup inhibitors . The inhibitor was diluted in 20 mM Tris buffer solution pH 7.2, which contained 150 mM NaCl and 0.02% sodium azide, and added to a quartz tube in the MLA ElectraR 800 Automatic Coagulation Timer (Medical Laboratory Automation, Inc. ·, Pleasantville, N · Υ ·). The instrument is set in APTT mode with 300 seconds activation time and repeat mode. After adding 0.1 ml of plasma (Specialty Assayed Reference Plasma lot 1-6-5185, Helena Laboratories, Beaumont, TX), APTT reagent (Automated APTT-lot 102345, from Organon Teknika Corp., Durhan, NC) and 25 mM CaCl2 automatically It is dispensed to start clotting, and clotting time is automatically tracked again. The results (Fig. 14) show that the doubling time of coagulation requires a final concentration of aprotinin 2 ju M, but the placental bicomycin derived from Sf9 requires only 0.3 # M. These data show that placenta diconine is an effective anticoagulant and can be used as a drug to treat diseases related to the pathological activation of the intrinsic pathway of coagulation. -82- This paper size applies to China National Standard (Cns) A4 (210X297 mm) (Please read the notes on the back first and fill in this page). Binding.

Claims (1)

555764 Patent Application No. 086105368 Chinese Patent Application Replacement (July 1992) S A8 B8 Patent application scope-Mis 1 _ A substantially purified protein having serine protease inhibitory activity, the protein is selected from one of the following amino acid sequences: ADRERSIHDF CLVSXWGRC RASMPRWWYN VTEX3SCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDKSSDK? 100 . NYEEYCTANA VTGPCRASFP RWYFDVZRKS CNN7IYGGCR GNXKSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK (170 / * (SEQ ID NO ·· 52); MAQLCGL RRSRAPLALL GSLLLSGVLA ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKX CATVTSNATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCHASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQ y ENPPLPLGSK VWLAGLFVH VLILFLGASM VYLIRVARKK 200 QERALRTV ^ S SGDDKEQLVK NTYVL 225 (SEQ ID NO: 49);? ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKX CATVTENATG DLATSRNAAD SSVPSAPRRQ DSZDHSSDH 100 NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSSS 150 ACHLRCFRQQ ENPPLPLGSK VWLAGLFVH VXILFLGASM VYLIRVARRN 200 QERALRTV ^ S SGDDKEQLVK NTYVX 225 (SEQ WORK D NO: 71); AGSFLAWL 'GSLLLSGVLA -1 ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKX CATVTENATG DLATSRNAAD SSVPSAPRRQ DSRCQQ SRQQ RCYQQRC 100 NZYFD paper scale applicable Chinese national standard (CNS) A4 size (210X297 mm) 555764 A8 B8 C8 D8 patented range KLR XEAEGVSRLL GSLLLSGVLA XDR £ RSIHDF CLVSKWGRC RASMPRWWYN VTCGSCiLFV YGGCDGNSNN 50 YLTKZECUKX CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 jsTVEEYCTANA VTGPCHASFP RWYFDVERNS CNNFIYGCCR GNXNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK VWLAGLFVH VLILFLGASH VYLIRVARRN 200 QERALRTVWS SGDDKEQLVK NTYVL 225 (SEQ ID NO: 45); MAQLCGL RRSPAFLALL GSLLLSGVLA XDRERSIHDF CLVSKWGRC RASHPRWWYN VTDGSCQLFV VGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSSDHSSDMF 100 NYEEYCTANA VTGPCRAS ?? RWYFDVERNS CNNFIYGGCR GNKNSYRSEE ISO ACMLRCFRQQ ENPPLPLGSK VWLAGLFVM VLILFLGASM VYL station RVARRN 200 QERALRTVWS FGD.. twenty one 3 (SEQ ID NO: 47); XDRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTK ££ CLKX CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCT ^ A VTGPCRIR FRGLCRGRCLRQQLRAGQYRCLRQQ 150 IHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCCGNSNN YLTKEECLKK CATV (SEQ ID NO: 4); 'CLVSKWGRC RASMPRWWYN VTDGSCQLFV VGGCDGNSNN 50 YLTKEECLIOC C (SEQ ID. NO: 5); YE £ YCTANA VTGPCRASFP CNYFIYRWY 5064 61 CTANAVTGPC RASFPRWYFD VERNSCKNFI YCCCRGNKNS YRSEE 150 ACMLRC 156. This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) k Order 555764 8 8 8 8 AB c D patent application scope (SEQ ID'NO ^ 7); IHDF CLVSKWGRC RASHPRWWYN VTDCSCQLP / YGGCCGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ * DSEDHSSDM? L〇〇bffEEYCTANA VTGPCRAFR RCY RWY NO: 3); CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN YLTKE £ CLX: < CATVTZNATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMP NYEEYCTANA VTGPCRASFP RWYFDVZRNS CNN7IYGGCR GNKNSYRSEH ACHLRC (SEQ ID NO: SO); ADRERSIHD CLVSKWGRC RASHPRWWYN VTDGSCQLFV YGGCDGNSNN YLTKZECLKX CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE ACMLRCFRQQ? ENPPLPLGSK VWLAGAVS (SEQ ID NO: 1); trans 50 100 150 156 50 100 150 179 50 50 ADR £ RSIHDF CLVSKWGRC RASHPRWVTfM VTDGSCQLFV YGGCEX3NSNN VLTKEZCLXK CATVTZNATG DLATSRNAAD SSVPSAPRRQ DS * (SEQ ID NO: 8) Numbered according to the natural human placenta diconicin amino acid sequence shown below, where the N-terminal residue resulting from the signal peptide was removed A first residue; CDNA CONA CDNA CONA CDNA CDNA CDNA GCACGAGTTG GTGTCGCAGG GCCGAGAAGG GGGGGCTTTG GAGGCGCTCC GAGACCCCAA atggcgcagc translation MAQL GGAGGTGTAG CGGCGAGGGC: GTCC 'GGCG ATTGCGCGTG CGGCTGGTGG TGTGCGGGCT CGL CCGGGCC GCACCTC CGCGGCTCTG AACGCGCTGA' GAGG AGCAGACCCA .CTGAA GGTCCGGAAA GACCCTCCCG GAGCGTCGGC CGCGTTGAGG GGCTTCCCGC CGTCGCCTGC GCGTCTCGGC GAGGCGGAGC CGGGCGTTTC RRSRAFL GCGAGTC CCACAC1 GGGCC GGCA1 GGCM CGTTGA 50 TCGCGC 100 GACTTCC 150 ACCTGAACGC 200 ACCTGAtCGC 250 TGAGCTGGCC 300 TCGCCCTGCT 350 ALL -II This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 555764 A8 B8 C8 D8 cDNA application translation GGGATCGCTG GSL CTCCTCTCTG LLSG GGGTCCTGGC GGCCGACCGA GAACGCAGCA VLAXDRERSI 400 7 CDNA TCCACGACTT CTGCCTGGTG TCGAAGGTGG SKVV TGGGCAGATG CCGGGCCTCC 450 translation HDFCLVGRCRAS 23 cDNA ATGCCTAGGT GGTTCGTGGCTCT GGTGGCTCA 40 CDNA GTAXGGGGGC TGTGACGGAA ACAGCAATAA TTACCTGACC AAGGAGGAGT 55 .: translation YGGCDGNSNNYLTKE ε C 57 cDNA GCCTCAAGAA ATGTGCCACT GTC ^ CAGAGA ATGCCACGGG TGACCTGGCC 600 translation LKK CAT V 1: £ NATGDLA 73 cDNA ACCAGCAGGA ATGCAGCGGA TTCCTCTGTC CCAAGTGCTC CCAGAAGGCA 650 translation TSRNAADSSVPSAPRRQ 90 CDNA GGATTCTGAA GACCACTCCA GCGATATGTT CAACTATGAA GAAXACTGCA 700 translation D s ε DHSSDMFNYS ε rc τ 107 cDNA CCGCCAACGC AGTCACTGGG CCTTGCCGTG catccttccc ACGCTGGTAC 750 translation ANA VTGPCRASFPRWY 123 cDNA TTTGACGTGG AGAGGAACTC CTGCAATAAC TTCATCTATG GAGGCTGCCG 800 translation FDVERNS CNN FIYGGCR 140 CDNA GGGCAATAAG AACAGCTACC GCTCTGAGGA S £ £ GGCCTGCATG CTCCGCTGCT 850 translation GNK 'NSYR ACM LRCF 157 cDNA TCCGCCAGCA GGAGAATCCT CCCCTGCCCC TTGGCTCAAA GGTGGTGGTT 900 tran ^ latioxx RQQ E NPPLPLGSKVVV 173 cDNA CTGGCGGGGC TGTTCGTGAT GOTGTTGATC CTCTTCCTGG GAGCCTCCAt 950 tr anslation LA Γ; LFVMV Γ, t LFL n ASM 190 loaded with cDNA GGTCTACCTG ATCCGGGTGG CACGGAGGAA CCAGGAGCGT GCCCTGCGCA 1000 translation vr L t RVARRSQERALRT 207 CDNA CCGTCTGGAG CTCCGGGGAG GACGAGGCGCCTGGTGAA GAACACATAt OCCC ACC 225 ΝλΝΑΝΑΝΑΝΑΝΑΝΑκλΜΑ50 CDCDCDCDCDCDCOCOCD15; TGTGAC ^ GGGCTC TGGCAGC TGGCCTC tttttttaaa AXG:: GA GGTCTGTTTC ^ GGGAT GGGTTTGCTT CGCAG tCTGGCAGCA CTTTCCTCCA GGTAGAGTTT ττττοταίΑΤ cacagaagtg TTTAtGGTTT TTTTAAGTAT ^ TGTACA AATAAATTTC CAGCATGTTG TAGAGGGATT TCTGGGAGGT TGGAAATCCT GCCCCGAGTT TCTTTGCTTA ATGTTGGAAT CAtGGTTT TTTTAAGTAT AAACAAAAGT ^ GGAAAGT AAAATGTACA AGTTTAATAA rAAATTTC CAGCATGTTG CTTTCAAAAA GACTCGGATT AGGACGGCTG CTAGGAGGCT GTTTCCTCGC TGTTGAATTC CGTTTCTTTT TTTTTATTAG AAAGGGGCCT TGAGTGATCA CTTCCTGGTC CCTCCTCGCA TGATCGATTT CATTGCCTCC GTTTGTCTGA CATTCTGAAA TCCCCTTTAG 1150 1200 1250 1300 1350 1400 1450 1500 -4- This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm), patent application scope 2. The protein according to item i of the patent application scope, where the glycosylation, or Contains at least one intra-chain cysteine-cysteamine iL sulfur bond 'or: is glycosylated and contains at least one intra-chain cysteine-sulfide-cysteine disulfide bond. 3. A pharmaceutical composition for inhibiting the activity of a serine protease, which comprises a protein according to item 1 or 2 of the scope of the patent application, and a pharmaceutically acceptable carrier. 4 · The pharmaceutical composition according to item 3 of the patent application, which contains the protein according to item 3 of the patent application, plus a pharmaceutically acceptable carrier. 5. An isolated nucleic acid sequence ' which encodes a protein according to item 1 or 2 of the scope of patent application. 6. The isolated nucleic acid sequence according to item 5 of the patent application, and encoding the protein of item 2 of the patent application. 7. A method for inhibiting serine protease activity in vitro, which method comprises contacting a serine protease with an effective amount of at least one protein according to claims 1 or 2 of the patentable scope of the application. 8. The method according to item 7 of the patent, which comprises contacting serine protein with an effective dose of at least one protein according to item 2 of the scope of the patent application. 555764 Zheng 1 Supplement No. 86105368 Patent Application Chinese Schematic Correction Page (December 87) 1 ΛΛ u; η ^. IQO 80604020 ο ο Retention time Figure 5 555764 Patent Application No. 86105368 Chinese Schematic Correction Page (December 87) Figure 6 \ nn OD215 nm% Trypsin Inhibition 〇 SQO 丨% Inhibition of Dermal Release Enzyme g 908Ο7Ο605Ο403Ο2ΟΙΟ I · * I V / _ ^; grab the spectrum φίφί ο Residence time Patent Application No. 86105368, Chinese Schematic Correction Page (December 87) Figure 9 SDS ^ PAGE Western Ink Dot SCM 2 谧 驾 # ιηζ * 3