WO1997000320A1 - Modified polypeptide, dna encoding the same, transformant, and medicinal composition containing the polypeptide - Google Patents

Abstract

A polypeptide having the amino acid sequence wherein Asn at the 18-position of an amino acid sequence consisting of the amino acids at the 1- to 77-positions of the amino acid sequence of natural human MACIF active protein has been replaced by Gln. This polypeptide is produced and harvested by incubating cells transformed by an expression vector containing a DNA encoding the polypeptide. The modified polypeptide inhibits cytolysis by the complements of nuclear cells and the release of PGI2 and b-FGF by the complements from HUVEC. Also, it inhibits a hyperacute rejection in a model reflecting a hyperacute rejection of transplantation.

Description

 TECHNICAL FIELD The present invention relates to a modified polypeptide, a DNA encoding the same, a transformant, and a pharmaceutical composition containing the polypeptide.

 The present invention relates to a medicament, in particular, a modified polypeptide having human MAC IF activity, an anti-inflammatory action and an action of suppressing hyperacute rejection at the time of transplantation, a DNA encoding the modified polypeptide, and a DN The present invention relates to a recombinant expression vector containing A, a transformant transformed with the recombinant expression vector, a method for producing the modified polypeptide, and a pharmaceutical composition containing the modified polypeptide.

 Here, the human MAC IF activity controls the final stage of a series of activations of the complement system, and forms autologous cells or cells through the formation of a membrane damage complex (hereinafter referred to as MAC). It refers to the activity of inhibiting the damage to self-tissues, or the activity of controlling the complement system, which has the effect of inhibiting the release of cysteine-in, blood coagulation and fibrinolytic factors from cells. Therefore, the modified polypeptide of the present invention is a regulator of the complement system and can be used as a pharmaceutical or the like. Background art

The present inventors purely isolated a natural complement inhibitory protein, a natural human MAC inhibitor (MAC Inhibit Factor: hereinafter referred to as MAC IF), from human normal erythrocyte membrane. The content was disclosed in Japanese Patent Application Laid-Open No. 2-157298. At the same time, (1) the activity of the first half of the complement system in that the natural type human MAC IF inhibits the activation of the second half of the complement system, ie, the inhibition of erythrocyte hemolysis through human MAC formation. (2) the N-terminal amino acid sequence of this natural human MAC IF is eu-Gin-Cys-Tyr -Asn-Cys-Pro-Asn-Pro-Thr (SEQ ID NO: 2 in the sequence listing); and (3) the natural human MAC I The glycoprotein has a glycosylphosphatidylinositol anchor (G1ycosylphosphatidylinosito 1 anchor: hereinafter referred to as GPI anchor) at the C-terminal side of F, and is a glycoprotein with a molecular weight of 18,000 ± 1,000. I made it.

 Subsequently, the present inventors isolated a gene encoding human MA CIF from a human monocyte cDNA library using a mixture of oligonucleotides corresponding to the N-terminal amino acid sequence as a probe, and obtained the gene and GP A gene encoding a soluble human MAC IF-active protein lacking the I anchor and a portion of the C terminal, an expression vector ligated with these genes, and a transformed cell were prepared. An IF active protein was prepared, and its contents were disclosed in Japanese Patent Application Laid-Open No. 3-20198.

 In Trans ρ 1 antati 0 n, (1 995), 59, 1 177, a model that reflects hyperacute rejection of xenograft transplantation, in which human serum is returned to the mouse heart, is used (hyperacuterejection). In the heart of a mouse expressing a membrane-bound human MAC IF derived from a transgenic animal (Transgenic animal), activation of human complement suppressed MAC formation, and No. 6-506604 discloses that the damage reaction of porcine endothelial cells by human complement was suppressed by expressing a membrane-bound human MAC IF using a gene transfer technique.

On the other hand, in Transplan tati on Proceedings, (1995), 27 (1), 328, a similar experiment was performed using porcine endothelial cells expressing the membrane-bound human MAC IF protein. However, it is described that the membrane-bound human MAC IF protein is not effective in suppressing the damage reaction of porcine endothelial cells by human complement. Therefore, there is a clear view as to whether hyperacute rejection at the transplant site is suppressed when transplanting heterologous cells or organs expressing the membrane-bound MAC IF protein. There was no. In addition, the use of a more versatile method of adding human MAC IF and soluble MAC IF at the time of transplantation, instead of expressing it in the transplanted cell's organ, suppresses the hyperacute rejection at the transplant site Be done It was unknown whether or not. Disclosure of the invention

 The present inventors have conducted intensive studies on MAC IF modified polypeptides which are useful as pharmaceuticals and have even more excellent activity.As a result, the modified polypeptide represented by SEQ ID NO: 1 in the following sequence listing was unexpectedly found in nucleated cells. Inhibiting the lysis of human umbilical vein vascular endothelial cells (HUVEC) by complement or the release of arachidonic acid metabolite PG12 and inflammatory mediator b-FGF; The present inventors have found that hyperacute rejection is suppressed by a model that reflects transplant hyperacute rejection, and completed the present invention.

 That is, the present invention relates to a modified polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and being in a soluble form.

 In addition, the present invention provides a DNA encoding the modified polypeptide, a recombinant expression vector containing the DNA, a transformant characterized by being transformed with the recombinant vector, and a modified product thereof. It also relates to a pharmaceutical composition comprising the polypeptide.

 Furthermore, the present invention also relates to a method for producing the modified polypeptide, which comprises culturing the transformant, producing the modified polypeptide, and collecting the modified polypeptide.

The modified polypeptide of the present invention lacks the amino acid sequence at the C-terminal side of native human MAC IF, which is a GPI anchor binding signal, and has no GPI anchor. In addition, asparagine (Asn) at position 18 which is the sugar chain binding site of the polypeptide consisting of amino acid sequences 1 to 77 of the amino acid sequence of the native human MAC IF-active tampon protein _c having the amino acid sequence was replaced with glutamine (G in) the

The activity of native human MAC IF with GPI anchor in the presence of serum is more severe than that of soluble MACIF without GPI anchor, and soluble MACIF is considered desirable as a drug. Can be

As described above, regarding this MAC IF, for example, the natural on the HUVEC cell membrane That type MAC IF has a protective effect on cell lysis by complement and that soluble MAC IF without GPI anchor has an inhibitory effect on the lysis of guinea pig erythrocytes (nucleated cells). Is suggested.

 However, it is still unknown at present whether 1) the mechanism of complement activation in guinea pig erythrocytes is the same as that of human umbilical vein endothelial cells, and 2) the complement membrane. Various regulatory factors are known in addition to MAC IF, but their distribution varies depending on the cell type.3) The sensitivity to complement depends on the cell type, especially erythrocytes and nucleated cells, which are non-nucleated cells. It was unclear whether soluble MAC IF could inhibit the lysis of nucleated cells by complement even if the soluble MAC IF inhibited the hemolytic system of guinea pig erythrocytes. ) MAC IF on the membrane shows CD2 ligand activity, but recombinant soluble MAC IF does not show this activity. 5) MAC IF and soluble MAC IF are added from outside. It is not known to inhibit arakidonic acid cascade and to inhibit the release of inflammatory mediator b-FGF, and 6) cell lysis by MAC IF when present on the cell membrane. Even if it shows an inhibitory effect, MAC IF and soluble MAC

Since it is not known whether or not an inhibitory effect is exhibited when IF is added from the outside, the modified protein of the present invention, in which various activities are confirmed as described later, is an extremely useful protein. It is positioned as being. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the sequences of primers used in the preparation of each of the MAC IF (70, N / Q), MAC IF (77, N / Q), and MAC IF (77) genes in Examples 2 to 4. FIG.

 FIG. 2 is an explanatory view schematically showing a procedure for preparing the MAC IF (70, N / Q) gene prepared in Example 2.

FIG. 3 is an explanatory view schematically showing the structure of a MAC IF (77, N / Q) EX expression plasmid prepared in Example 3. FIG. 4 shows the nucleotide sequence of the gene prepared in Example 3 and the amino acid deduced from the nucleotide sequence for use in expressing the polypeptide of the present invention, MAC IF (77, N / Q). It is explanatory drawing which shows an arrangement.

 FIG. 5 is a diagram schematically showing the results of SDS-PAGE of MAC IF (77, NXQ) performed in Example 6.

 FIG. 6-1 shows the results of cell lysis by urinary MAC IF or MAC IF (77, N / Q), which is a modified polypeptide of the present invention.

 FIG. 6-2 is a graph showing the results of the cytolytic effect of urinary MAC IF or MAC IF (77, N / Q), which is a modified polypeptide of the present invention.

 FIG. 7-1 is a diagram showing the results of cell lysis using LDH activity as an index when MAC IF (77, N / Q), which is a modified polypeptide of the present invention, is not added. FIG. 7-2 shows the results of the b-FGF release inhibitory effect of MAC IF (77, N / Q), which is a modified polypeptide of the present invention.

 FIG. 8 is a graph showing the results of the effect of cytotoxicity on the basis of LDH activity by MAC IF (77, N / Q), which is a modified polypeptide of the present invention.

Figure 9 is a graph showing the results of, HBSS containing human C 9, of HBSS containing human C 8 and human C 9 PG I ₂ release (following Examples 8 (3 1) See for HBSS) HBSS It is.

FIG. 10 is a graph showing the results of suppression of PGI ₂ production by MAC IF (77, N / Q), which is a modified polypeptide of the present invention.

 FIG. 11 shows changes in the perfusion pressure of the guinea pig heart with Krebs buffer containing 3% human plasma (NHP).

 Fig. 11-1-2 shows that the modified polypeptide of the present invention, MAC IF (77, N / Q), was added to a Creps buffer containing 3% human plasma at 10 jt / gZn 1 and 50 g / 1. FIG. 9 is a diagram showing the effect of suppressing the increase in the convection pressure in the case of performing the above.

Fig. 12 shows the temporal changes in the left ventricular pressure and the left ventricular end-systolic pressure of the guinea pig when the control was washed with Krebs buffer containing 3% NHP. FIG. 13 is a graph showing the effect of the modified polypeptide of the present invention, MAC IF (77, N / Q), on guinea pig myocardial dysfunction based on human complement activity. FIG. 14 is a graph showing the effect of inhibiting the release of creatine phosphokinase by MAC IF (77, N / Q), which is a modified polypeptide of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

 As described, the modified polypeptide of the present invention is a polypeptide characterized by having the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing described below, and comprises the first amino acid sequence of a natural human MAC AC IF active protein. It has an amino acid sequence in which the 18th asparagine (Asn) of the polypeptide consisting of the amino acid sequence of the amino acids Nos. 77 is substituted with glutamine (Gln).

 The modified polypeptide of the present invention can be produced, for example, by a gene recombination technique according to the method described below.

 The DNA of the present invention having the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing can be synthesized by, for example, a genetic recombination method or a chemical synthesis method, or a combination thereof as appropriate. it can.

 In the case of using a chemical synthesis method, for example, a nucleic acid is prepared according to a conventional method such as a phosphoramidite method [Hunkap i 11 er, M. eta 1., Naturo, —310, 105-111 (1 984)) Can be chemically synthesized.

When the gene recombination method is used, for example, as described in the above-mentioned Japanese Patent Application Laid-Open No. 3-201985, a naturally-occurring human library can be obtained from a cDNA library prepared from mRNA obtained from human MAC IF-producing cells. A clone containing cDNA encoding MAC IF is isolated, and cDNA encoding native human MAC IF is isolated from the isolated clone. By using this cDNA as a template and performing polymerase "chain" reaction (hereinafter referred to as PCR) with a primer designed to introduce the desired amino acid substitution, natural human MAC can be obtained. In the nucleotide sequence of the cDNA encoding IF, the codon corresponding to asparagine, the 18th amino acid, is replaced by the codon corresponding to glutamine. A partially modified DNA fragment can be obtained. As a template to be used at this time, it is also possible to use the above-mentioned cDNA library itself, or cDNA obtained by synthesizing reverse transcriptase from RNA prepared from MAC IF-expressing cells. The DNA fragment obtained by PCR was replaced with the corresponding portion of cDNA encoding natural human MAC IF, and the template was designed to amplify the desired soluble MAC IF cDNA again as a template. By performing PCR using a primer, the DNA of the present invention can be obtained. Alternatively, the DNA of the present invention can be obtained by replacing the DNA fragment obtained by the PCR with a corresponding portion of a previously prepared cDNA fragment encoding a soluble MAC IF. . Specific design of primers, selection of restriction enzyme sites available for replacement, and the like will be described in detail in Examples below.

 The thus obtained full-length portion of the coding region of the DNA encoding the modified polypeptide having human MAC IF activity of the present invention can be expressed using eukaryotes and prokaryotes as hosts. Expression vectors to be incorporated into these host cells are appropriately constructed according to the host cells.

 Prokaryotic hosts include E. coli strains such as E. coli K12 strain 294 (ATCC 314 46), E. coli B, E. coli 1776 (ATCC 31537), E. coli C600 and E. coli W3110 (F-, λ-, prototrophy, ATCC 27375). ), Bacillus strains, for example, Bacillus subtilis, Salmonella typhimuriium or Serratia marcescens, and other intestinal bacteria other than Escherichia coli, and strains of the genus Pseudomonas. Is mentioned.

When one of the above microorganisms is used as a host, one of the vectors is a promoter and an SD base sequence upstream of the gene of the present invention so that the gene of the present invention can be expressed [Shine, J. et al. of Science of USA, 71, 1342 to 1346 (1974)], and an expression plasmid to which ATG necessary for initiation of protein synthesis has been added can be used. In general, PUC18 and PUC19 are often used as vectors for 97/00320 E. coli strains and the like. The promoter, for example tryptophan promoter, p _L promoter evening one, lac promoter, lpp promoter mono-, -. Comfortable evening can be used mer lactamase promoter, or the like.

 Examples of the marker gene include an ampicillin resistance gene and a tetracycline resistance gene.

 As eukaryotic microorganisms, yeasts are generally used, and among them, yeast belonging to the genus Saccharomyces can be advantageously used. As an expression vector for eukaryotic microorganisms such as the yeast, for example, YRp7 can be used.

 Examples of the promoter of an expression vector for yeast expression include 3-phosphoglyceretokinase or enolase, glyceraldehyde-3-phosphodehydrogenase, and hexonase.

 As the marker gene, a trpl gene or the like can be used.

 As the origin of replication, termination codon, and other DNA sequences for controlling transcription and translation in yeast cells, ordinary known DNA sequences suitable for yeast cells can be used.

 When cultured cells of higher animals are used as hosts, red-haired monkey kidney cells, mosquito larvae cells, African green monkey kidney cells, mouse embryo maintenance blast cells, Chinese hamster ovary cells, and their dihydrofolate reductase-deficient strains [ Ur 1 aub, G., et al., Proceeding ngs of the Nationa l A cademy of Science of the USA, 77, 421 6-4220 (1980)), human cervical epithelial cells, Human fetal kidney cells, moth ovary cells, human myeloma cells, mouse fibroblasts, or the like can be used.

 The vector generally has a functional sequence for expressing the DNA of the present invention in a host cell, such as a replication origin, a promoter to be located upstream of the DNA of the present invention, a ribosome binding site, a polyadenylation site, and a transcription termination sequence. It contains.

Promoters include, for example, adenovirus 2 major late promoter, SV40 early promoter, SV40 late promoter, and promoters from eukaryotic genes. (E.g., the estrogen-induced chicken egg albumin gene, the in vitro enzyme, the glucocorticoid-induced tyrosine aminotransferase gene, the thymidine kinase gene, the major early and late adenovirus genes, the phosphoglycerate kinase gene, or Factor factor gene).

 As an origin of replication, adenovirus, SV40, ゥ papilloma virus (BPV), vesicular stomatitis virus (VSV), or a derivative thereof derived from a vector can be used.

 In addition, as the marker gene at this time, for example, a neomycin resistance gene, a methotrexate-resistant dihydro leaf nucleic acid reductase (DHFR) gene, or the like can be used.

 As an expression vector for higher animal cells, it is effective to use a vector such as pEF-BOS, pSR or the like because of high expression efficiency.

 As described above, the host, the vector, and the components thereof that can be used for expressing the DNA of the present invention have been exemplified, but the present invention is not limited to these examples.

 The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention obtained above into a desired host according to a conventional method.

 The expression vector plasmid is prepared from a host (eg, E. coli HB101 strain) used for gene construction by a general method such as an alkaline lysis method. The method of transforming a host using the prepared vectorplasmid is the calcium phosphate method when using mammalian cells as a host [van der Eb, AJ etal., Method sin Enzymo logy, 65 , 826-839 (1980), Academic Pres s.].

The transformant of the present invention obtained as described above is cultured according to a conventional method, and the modified polypeptide of the present invention having biological activity is produced and accumulated by the culture. The medium used for the culture can be appropriately selected from those commonly used depending on the host used. For example, in the case of the above CHO cells, MEM-spun medium may be used, if necessary, such as fetal calf serum (FCS). Those to which blood components have been added can be used. The expression site of the modified polypeptide of the present invention produced by the transformant may vary depending on the amino acid sequence encoded by the synthesized DNA, the type of vector to be used, the type of host, and a combination thereof. Or can be produced in the culture supernatant of the cells. The modified polypeptide of the present invention produced from various transformants can be subjected to various separation operations utilizing its physical and chemical properties (for example, Biochemical Data Book II, edited by the Biochemical Society of Japan, p. 1175, No. 1). Edition, 1st printing, 1980, Tokyo Chemical Co., Ltd., etc.).

 Examples of the method include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieving chromatography (gel filtration), adsorption chromatography, ion conversion chromatography, affinity chromatography, and high-speed chromatography. Examples thereof include various liquid chromatography such as liquid chromatography (HPLC) and the like, a folding method, a combination thereof, and the like. More specifically, the method differs depending on the expression site of the modified polypeptide.

 The modified polypeptide of the present invention produced in the culture supernatant is purified by the following method.

 First, a target substance is partially purified from the culture supernatant in advance. This partial purification is performed, for example, by a treatment using a salt-forming agent such as ammonium sulfate, sodium sulfate, sodium phosphate, or the like, and an ultrafiltration treatment using Z or a transparent membrane, a flat membrane, a hollow fiber membrane, or the like. Be done. The operations and conditions for each of these processes may be the same as those usually used in this type of method. Then, the crude product obtained above is subjected to adsorption chromatography, affinity chromatography, gel filtration, ion exchange chromatography, reverse phase chromatography, etc., and by a combination of these operations, A fraction showing the activity of human MA CIF can be obtained, and the target substance can be isolated as an equivalent substance.

Recombinant protein produced in cells is treated with an appropriate detergent (eg, NP-40, Triton X-100, octylglycoside) to break the cell membrane and release the recombinant protein into solution. After that, purification can be performed in the same manner as described above. The modified polypeptide of the present invention, DNA encoding the polypeptide, an expression vector containing the DNA, a transformant containing the expression vector, and a method for producing the modified polypeptide have been described above. . Industrial applicability

 Since the modified polypeptide of the present invention is of human origin, it has no antigenicity to humans, is low toxic, and prevents damage to autologous cells and tissues through MAC formation, which is the final stage of the complement activation reaction It can be usefully used as a therapeutic agent for diseases in which complement control components are deleted or reduced.

More specifically, as described in detail in Examples below, the modified polypeptide of the present invention has the following effects: 1) inhibiting lysis of nucleated cells (HUVEC) by complement; Inhibits the release of PG I _{2, a} metabolite of arachidonic acid, and b-FGF, an inflammatory mediator, by the complement from humans (non-1 etha 1 effect), and 3) a model that reflects transplant hyperacute rejection. Hyperacute rejection was suppressed.

 Therefore, the modified polypeptide of the present invention is a useful compound that is expected to have the following diseases or clinical applications.

 That is, the fact that the modified polypeptide of the present invention suppressed lethal effect of nucleated cells (HUVECs) by complement indicates that the modified polypeptide of the present invention caused reperfusion injury after ischemia. It is expected that it can be used as a preventive drug or as a preventive drug for vasculitis associated with autoimmune diseases and the like and cytotoxicity due to complement.

In addition, complement activation signals complement directly to cells, causing Ca ²⁺ influx and releasing various inflammatory media such as arachidonic acid metabolites and protein factors out of the cell. It is known that (Immun 0 and Res. 993; 12: 244—). As arachidonic acid metabolites, arachidonic acid, PGE ₂ , PGI ₂ , TxA ₂ , LTB ₂ and others are reported to be released from epithelial cells, leukocyte cells, vascular endothelial cells, etc. upon stimulation of complement. Have been. B-FGF, PDGF, vWF, etc. are released from vascular endothelial cells by stimulation of complement. Is reported to be. These reactions are called non-lethal effects of complement, and the mechanism of cell lysis by complement, which is known to inhibit MAC IF and soluble MAC IF, is known. Is thought to be caused by a different mechanism. In fact, it is observed that these phenomena occur without lysing the cells. In inflammatory diseases, arachidonic acid metabolites such as PGE ₂ , TxA ₂ , LTB ₄ and b-FGF are known to induce or exacerbate inflammation, and no n-let of complement It is expected that the inflammatory mediators released by haleffect will also cause or exacerbate inflammation.

 The fact that the modified polypeptide of the present invention suppressed the release of PG I 2, a metabolite of rachidonic acid and b-FGF, an inflammatory mediator, by complement from HUVEC means that the modified polypeptide of the present invention It is considered to be useful as an agent for preventing and treating inflammatory diseases such as nephritis, atherosclerosis, and chronic inflammation such as rheumatoid arthritis, which are said to be involved in the non-1 et ha 1 effect of these complements. Furthermore, the fact that the modified polypeptide of the present invention showed an effect of inhibiting complement-induced decline in cardiac function in the guinea pig heart Z human plasma port system was demonstrated by a technically difficult transgenic animal (Transgenic animal). l) It is thought that hyperacute rejection can be suppressed by adding the modified polypeptide of the present invention at the time of organ transplantation without requiring preparation, and is extremely useful clinically.

 At the time of organ transplantation, the transplanted organ temporarily becomes ischemic, and after the transplantation, the blood returns. It is known that cell injury occurs during reperfusion after ischemia, and that MAC IF on the cell membrane disappears during reperfusion injury after ischemia (Labor at or y Inve stigati) on 6, 608-616 (1992)). Therefore, the modified polypeptide of the present invention is considered to be useful not only in xenotransplantation but also in suppressing hyperacute rejection in transplantation between humans.

Although the dosage form of the modified polypeptide of the present invention varies depending on the type and condition of the disease and the condition of the patient, it may be administered orally as a systemic administration route or as an injection, nasal formulation, suppository, implantable preparation, etc. It is administered parenterally. In addition, as a local administration route, 2 Administration, implantable preparations for affected area, etc. are used.

 In preparing these preparations, compositions suitable for the respective dosage forms are formulated. For preparation as an injection, the modified polypeptide of the present invention is dissolved in, for example, a phosphate buffered saline / dextrose aqueous solution, and if necessary, a stabilizing agent, a dispersing agent, etc. are added, and an injection ampoule is prepared. Or freeze-dried in a vial bottle. When administered, distilled water for injection can be dissolved in physiological saline before use.

 The dose of the modified polypeptide of the present invention is usually 10 to 500 mg, preferably 1 to 50 mg per day for an adult, and it may be reduced once to several times a day. Administer separately.

Example

 Hereinafter, the present invention will be described in more detail with reference to Examples and Formulation Examples. It goes without saying that the present invention is not limited by these examples.

Example 1: Synthesis and purification of primer

 (1) Primer design

Based on the nucleotide sequence of the human MA CIF gene cloned from human monocytes (SEQ ID NO: 4 in the sequence listing), six types of primers shown in FIG. 1 were designed. Oligonucleotide M CI having the sequence of SEQ ID NO: 5 in the Sequence Listing, oligonucleotide 5 ′ M lu having the sequence of SEQ ID NO: 8 in the Sequence Listing, and oligonucleotide MA CI having the sequence of SEQ ID NO: 9 in the Sequence Listing In the nucleotide sequence of FNX, the nucleotide sequence underlined in FIG. 1 corresponds to the nucleotide sequence of the human MA CIF gene. Oligonucleotide KM 1 contains the nucleotide sequence from No. 1 to No. 21 of the nucleotide sequence of SEQ ID NO: 4 in the Sequence Listing, and oligonucleotide 5′M1uI is the nucleotide sequence of SEQ ID NO: 4 in the Sequence Listing. The oligonucleotide MA CIF NX contains the nucleotide sequence from No. 136 to No. 165, and the oligonucleotide MA CIF NX contains the nucleotide sequence from No. 1 to No. 21 of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing. Oligonucleotide M3 having the sequence of SEQ ID NO: 6 in the Sequence Listing, oligonucleotide N / Q having the sequence of SEQ ID NO: 7 in the Sequence Listing, and oligonucleotide 77 having the sequence of SEQ ID NO: 10 in the Sequence Listing In the base sequence of XX, the base sequence shown in double underline in Fig. 1. 97 320 corresponds to the base sequence of the complementary chain of the human MAC IF gene. Oligonucleotide M3 contains a nucleotide sequence complementary to the nucleotide sequence from No. 285 to No. 265 of the nucleotide sequence of SEQ ID NO: 4 in the Sequence Listing, and oligonucleotide N / Q is a nucleotide sequence of SEQ ID NO: 4 in the Sequence Listing. Oligonucleotide 77XX contains the nucleotide sequence complementary to the nucleotide sequence from No.156 to No.115 in the nucleotide sequence, and the nucleotide sequence from No.306 to No.287 in the nucleotide sequence of SEQ ID NO: 4 in the sequence listing. And a base sequence complementary to the base sequence of The base sequence shown in italic letters and broken lines in FIG. 1 is a site recognized by the restriction enzyme described immediately below.

 (2) Primer synthesis

 Each primer designed in the above (1) was synthesized using a DNAZRNA synthesizer (Model 394 DNA / RNA synthesizer: Applied Biosystems). The cycle was performed at 40 nM CE, and the END procedure was performed at END CESS.

 (3) Purification of primer

 After heat-treating the reaction solution produced in the above (2) at 56 ° C. overnight, the oligonucleotide purification cartridge (Oli gonuc l e o ti de Purifi ca ti on Car tri dge: Applied Biosystem) Each primer was purified according to the instructions using the following primers and used for the following polymerase-chain reaction (hereinafter referred to as PCR). However, the oligonucleotide MAC I FNX and the oligonucleotide 77XX were purified using a separate oligonucleotide purification cartridge (Oli gopurifi cation Cartridge: Cruach em) according to the instructions. .

Example 2: Preparation of MAC IF (70, NZQ) gene (Fig. 2)

 (1) Amplification of DNA fragment 70a by PCR

Plasmid p GEM 352-3 (plasmid pGEM 352-3) in which cDNA of human native MAC IF is inserted into plasmid p GEM 4 as a template, and 100 nlV each of oligonucleotide KM 1 and oligonucleotide NZQ as primers — 3 is described in Examples (1) to (5) of JP-A-3-201 985. 10 mM Tris—HCl (pH 8.3), 5 OmM KC, 1.5 mM MgCl ₂ , 0.001% (weight Z volume) gelatin, 0.2 mM (51 ^) DNA polymerase (Takara Shuzo) 2. Place a tube containing 100 反 応 1 of the reaction solution consisting of 5 units in a DNA amplifier (DNA Thermocoupler 480i: PERK I). EL MER), heat at 94 ° C for 2 minutes, and then repeat the cycle of heating at 94 ° C for 1 minute, 55 ^eC for 1 minute, and 72 ° C for 1 minute 30 times Finally, a DNA fragment (hereinafter, referred to as DNA fragment 70a) was amplified by performing a PCR reaction in which the temperature was kept at 72 ° C. for 7 minutes In the following Examples, unless otherwise specified, the above conditions were used. Performed PCR.

 (2) Amplification of DNA fragment 70b by PCR

 PCR was performed under the same conditions as in (1) above, except that oligonucleotide 5 ′ M 1 υI and oligonucleotide 3 were used as primers to amplify a DNA fragment (hereinafter referred to as DNA fragment 7 Ob). .

 (3) Ligation of DNA fragment 70a and DNA fragment 70b

 The DNA fragment 70a amplified by PCR in the above (1) is cleaved with restriction enzymes Not I and MlυI, and the DNA fragment 70b amplified by PCR in the above (2) is digested with the restriction enzymes MlυI and The plasmid was digested with EcoRI and the plasmid vector pB1υescript II KS + (STRATA GENE) was digested with restriction enzymes NotI and EcoRI. These DNA fragments were separated by agarose gel electrophoresis, and a DNA fragment 70a 'of about 0.15 kb, a DNA fragment 70b' of about 0.15 kb, and a vector DNA fragment of about 3.0 kb An agarose gel containing was cut out. Extraction and purification of the DNA fragment from the agarose gel pieces were performed using a gel extraction kit (QIAEX> Gel Extrac tion, Kit: QI AGEN). The purified DNA fragments are mixed at a molar ratio of 1: 1: 1 and ligated with a ligation kit (Ligation Kit: Takara Shuzo) to transform Escherichia coli JM109 competent cells (Takara Shuzo). And ampicillin sodium

(Wako Pure Chemical Industries) It was applied to an L-broth plate containing 50 gZml. DNA fragment Ligation and transformation of Escherichia coli were performed according to the attached instructions. An appropriate number of transformants were selected from the transformants that appeared, and plasmid DNA was prepared from each transformant using a plasmid preparation kit (QI AGEN PLASM IDM IN IKIT: QI AG EN). . The obtained plasmid DNA is digested with restriction enzymes Not I, EcoRI and Z or MluI, and then subjected to agarose gel electrophoresis, whereby DNA fragment 70a 'and DNA fragment 70b' are separated. A plurality of plasmids having the linked gene [hereinafter referred to as MAC IF (70, NQ) gene] were selected.

 (4) Determination of DNA base sequence

 Using the plasmid DNA prepared by the above-mentioned plasmid preparation kit (QIAGEN PLASM IDMIN KIT: QIAGEN), the DNA base sequence of the plasmid selected in the above (3) was determined. A DNA sequencer is performed using a sequencing kit (Prims rea dy Reacti on Dy eDeoxy Terminator Cyclic Sequenci ng Kit: Ap p1 ied B iosys ΐ em) to perform a DNA sequencer. (ABI). As a result of the analysis, a recombinant plasmid having the nucleotide sequence from No. 1 to No. 285 of SEQ ID NO: 3 in the sequence listing was selected, and pB1υe-MAC IF (70, N / Q ). The reaction conditions and the like are described in the kit for sequencing (PrimsreadyReactionDyeDeoxyTerminatorCyclic Sequencing Kit) or the DNA Sequencer (ABI). All instructions were followed.

Example 3: Preparation of MAC IF (77, N / Q) expression plasmid (Fig. 3)

 (1) Amplification of DNA fragment MAC I F (77, N / Q) EX by PCR

Using pB1υe-MAC IF (70, N / Q) obtained in Example 2 (4) as a template and oligonucleotide MAC I FNX and oligonucleotide 77XX as primers, Example 2 (1 PCR was performed under the same conditions as in the above) to obtain a DNA fragment of about 0.36 kb. These DNA fragments contain the MAC IF (77, N / Q) gene and a restriction enzyme XbaI recognition site and CD at the 5 'end. It contains a 25 bp 5 ′ untranslated region derived from 4 and a DNA fragment MAC IF (77, N / Q) EX into which a restriction enzyme XbaI recognition site has been introduced at the 3 ′ end. The MAC IF (77, N / Q) gene refers to a DNA having the nucleotide sequence of Nos. 1 to 312 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing and encoding the polypeptide of the present invention. It is. The amplification of the desired 0.36 kb DNA fragment was confirmed by 2% agarose gel electrophoresis.

 (2) Sub-cloning of PCR products

 The DNA fragment amplified by PCR in the above (1) was subcloned into a PCR vector (invitrogen) using a cloning kit (TAC10ningTMKit: invitrogen). That is, the PCR reaction solution 11 obtained in the above (1) was added to sterile water 5〃10 X ligation buffer 121, PCRII vector solution (25 ng / u1) 11, T4

After adding DNA ligase 11 and stirring, the mixture was kept at 14 to 15 ° C for 4 hours or more. Subsequently, Escherichia coli JM109 competent cell (Takara Shuzo) was transformed using the above reaction solution according to the attached instructions, and applied to a selection plate. As a selection plate, agar medium containing ampicillin sodium (Wako Pure Chemical Industries, Ltd.) 501111 and 2% 5-bromo-4 monoclonal 3-indolyl-S-D-galactoside (X — Gal) (Wako Pure Chemical Industries) 501 and 0.1 M isopropyl-111-thio-^-D-galactone (IPTG) (Sigma) 20-1 were used. The selection plate was incubated at 37 ° C overnight, white colonies that appeared were selected, and plasmid DNA was prepared using a plasmid preparation kit (QIAGEN PLASMID MINI KIT: QIAGEN). The obtained plasmid DNA was digested with restriction enzyme XbaI and subjected to agarose gel electrophoresis to select a plurality of recombinant plasmids having an inserted fragment of about 0.36 kb.

 (3) Determination of DNA base sequence

Using the plasmid DNA prepared in the above (2), the DNA base sequence of the plasmid selected in the above (2) was determined. Sequence kit (Pri sm r eady Rea cti on DyeDeoxy Termi na t or A sequence reaction was performed using a Cyclic Sequence Kit (App 1 ied Biosystem) and analyzed with a DNA sequencer (ABI). As a result of the analysis, a recombinant plasmid having the nucleotide sequence of SEQ ID NO: 3 in the sequence listing was selected and named pMAC IF (77, N / Q) EX. The reaction conditions and the like are described in the instruction kit attached to the above-mentioned sequence kit (Prism Ready React on Dy Deoxy Terminator Cyclic Sequencing Kit) or the above-mentioned DNA Sequencer (ABI). I followed all. FIG. 4 shows the base sequence of the translation portion and the stop codon of the MAC IF (77, N / Q) EX gene and its deduced amino acid sequence. The bases or amino acids underlined in FIG. 4 are different from the base sequence or amino acid sequence of the natural type human MAC IF active protein.

 (4) Insertion of DNA fragment MAC I F (77, N / Q) EX into expression plasmid (Fig. 3)

 The plasmid pMACIF (77, N / Q) EX obtained in the above (3) was digested with the restriction enzyme XbaI. In addition, the expression plasmid for animal cells, pEF—BOS CM i s u s h ima et al., Nucl e i c Ac i d s R e s e a r ch h, 18,

5332, (1990)], a plasmid pEBD in which a methotrexate-resistant dihydroleaf nucleic acid reductase (hereinafter referred to as DHFR) expression unit is inserted at the A at 11 site, and a restriction enzyme Xba The fragment was digested with I and dephosphorylated with alkaline phosphatase (Takara Shuzo). Each plasmid is subjected to 1% agarose gel electrophoresis and contains a fragment of the agarose gel containing the desired fragment, that is, a DNA fragment MAC IF (77, NZQ) EX of approximately 0.35 kb with Xba I cohesive ends at both ends. A piece of agarose gel and a piece of agarose gel containing a linear plasmid pEBD fragment having XbaI cohesive ends at both ends were cut out. Extraction and purification of the target DNA fragment from the agarose gel was performed using a gel extraction kit (QIAEX> Gel Extraction <Kit: QIAGEN). The purified DNA fragments are mixed at a molar ratio of 1: 1 and ligated with a Ligation kit (Takara Shuzo) to transform Escherichia coli JM109. (Wako Pure Chemical Industries) was applied to an L-broth plate containing 50 g / m1. Ligation of DNA fragments and transformation of Escherichia coli were performed according to the attached instructions. An appropriate number of transformants were selected from the transformants that appeared, and a plasmid preparation kit (QI AGEN

Plasmid DNA was prepared from each transformant using PLASM IDMIN KIT (QIAGEN). The obtained plasmid DNA is digested with restriction enzymes Not I and EcoRI, and then subjected to agarose gel electrophoresis to obtain DNA fragment MAC IF (77, N / Q) EX plasmid pEBD human A plasmid inserted in the forward direction relative to the elongation factor 1 (EF-1) promoter was selected and named pEBD-MAC IF (77, N / Q) EX. The structure of pEB D—MAC IF (77, N / Q) EX is schematically shown in FIG. As shown in Fig. 3, when the MAC IF (77, N / Q) gene was inserted in the forward direction with respect to the EF-1 promoter, cleavage by the restriction enzymes NotI and EcoRI was performed. A DNA fragment of about 1 kb is generated. On the other hand, when the MAC IF (77, N / Q) gene was inserted in the opposite direction to the EF-1 promoter, a DNA fragment of about 1 kb was obtained by digestion with the restriction enzymes Not I and EcoRI. Does not occur. Example 4: Preparation of MAC IF (77) expression plasmid

 In the method described in Example 3 (1), the DNA fragment MAC IF (77) was prepared according to the method described in Example 3 (1) to (4) except that pGEM35 2-3 was used as a template. A plasmid (hereinafter referred to as pEBD-MAC IF (77) EX) was prepared in which EX was inserted in the forward direction with respect to the human EF-1 promoter of plasmid p EBD overnight.

Example 5: Preparation of CH〇 cells expressing MAC IF (77) or MAC IF (77, N / Q)

 (1) Transfection of CH0 cells

The transfection of CH0 cells was carried out using a ribofectin solution (LIPOFEC TIN ™ Reagent: Gibeo—BRL). Plasmid pEBD—MAC IF (77, N / Q) £ or £ 80—1 ^ 80 IF (77) EX 2 g each, OPT—MEM medium (Gibeo—BRL) 10 This was mixed with 0 n 1 to obtain solution A. The lipofectin solution 201 and OPT-MEM001 were mixed to prepare solution B. The solution A and the solution B were mixed gently, and allowed to stand at room temperature for 15 minutes. Then, 1.8 ml of MEM medium (+) medium (Gibco 0-BRL) was added. This solution, MEM monument (+) CHO and washed with medium Tsumugi胞(DHFR-) gently added to (cell number 1. 3 X 1 0 ⁵ cells / 6 cm dish), Te 37, 5% carbon dioxide condition After culturing for 1 day, the medium was replaced with a MEM medium (+) containing 10% fetal calf serum (hereinafter referred to as FBS), and further cultured for 2 days. The 1Z1 0 amount of the total cell number were transferred to 25 cm ² flask, while exchanging [ME Ma (I) medium (G ibeo- BRL Co.) 5 m l containing 1 0% dialyzed FBS] 2-3 days in culture medium The transfused CH0 cells were selected by culturing at 37 ° C and 5% carbon dioxide.

 (2) Breeding CHO cells expressing MAC IF (77) or MAC IF (77, N / Q)

 Transfer the transfected CHO cell population to Tribcine-EDTA solution

(Gibco—BRL), limiting dilution and plating on a 96-well plate. As a medium, a MEM (-) medium containing 10% FBS permeated with a phosphate buffered saline (hereinafter, referred to as PBS) was used. Select a well containing one cell, and when the cells have grown and become almost confluent,

(77) or a plurality of clones with high expression of MAC IF (77, N / Q) were selected. The selected clones were treated with a suitable concentration of methotrexate (hereinafter referred to as MTX).

Further culturing was performed in a 1 ^ medium (1) medium containing 10% dialysis 83 to induce an increase in the expression level due to gene amplification. The MTX concentration was started at 1 OnM and finally increased to 1 M, and the CH〇 cell line with the highest expression level was selected.

Example 6: Purification of MAC IF (77) and MAC IF (77, N / Q)

 (1) Culture of CHO cells expressing MAC IF (77) or MAC IF (77, N / Q)

To T medium (Wako Pure Chemical) 1 7. 8 3 g / l , glucose 64 g / l, PEG 20 000 lg / l, gentamicin 1 0 mg / l, and NaHCO ₃ Add 2 gZl, and add a final concentration of 1.25 mg of insulin, 2.25 mg of transpurin, 2.38 mg of ethanolamine, 0.38 mg of ethanolamine, and sodium selenite 1.08 // gZl of RD-1 medium (Kyokuto Pharmaceutical Co., Ltd.) , 0.001% Tween 80, 500 nM MTX, and 2% FBS. Cells were seeded at approximately 3 X 10 ⁵ cells / m 1 to 50 Om 1 spinner one flask was aerated medium 50 Om K rotational speed 60 r pm, the N _{2/0 2} gas from the top at 15-25 Z min While culturing for 4-5 days.

 (2) Purification and quantification

 An anti-human MAC IF mouse monoclonal antibody (Hatanaka et al., CI ini ca l Immunology and Immunopathology, 69, 52-59, (1993)) was activated by activated Sepharose 4B (Pharma cia) according to the attached instructions to prepare an anti-MAC IF antibody column. The culture supernatant of CH0 cells producing MAC IF (77) or MAC IF (77, NXQ) is applied to the anti-MAC IF antibody column to adsorb each product, and then Tris buffer containing 15 OmM sodium chloride is applied. The solution was thoroughly washed with a liquid (pH 7.4) and a Tris buffer (PH 7.4) containing 500 mM sodium chloride. MAC IF (77) or MAC IF (77, N / Q) adsorbed on the anti-MACIF antibody column was eluted with a 3 M aqueous solution of potassium thiocyanate, dialyzed against PBS, and subjected to buffer exchange.

 The concentration of the purified protein was assayed using a protein assay kit (Micro BCA protein Assay Kit: Pierce), and the operation was in accordance with the instructions. In addition, the following protein concentration tests were all performed using the above protein assay kit (Micro BCA protein Assay Kit).

 (3) Analysis of MAC IF (77, N / Q)

The purified MAC IF (77, N / Q) was subjected to polyacrylamide gel electrophoresis (hereinafter, referred to as SDS-PAGE) (Daiichi Pure Chemicals) in the presence of sodium dodecyl sulfate by a conventional method, and analyzed. The results are shown in FIG. Figure 5 shows the SD 1 is a drawing schematically showing an electrophoretic image of S-PAGE. Lane 1 shows a migration image of Protein Molecular Weight Marker 1 (Daiichi Pure Chemicals), and Lane 2 shows a migration image of purified MAC IF (77, N / Q) of 50 ng. MAC IF (77, N / Q) formed a single node at a molecular weight of around 7 kDa.

 After applying 500 ng of MAC IF (77, N / Q) to SDS-PAGE, 16 OmA. 1 hour using a transfer device (ISS-semi-dry electroblotting 'Daiichi Pure Chemicals Co., Ltd.) By applying a current, the electrophoresed protein was electrically transferred to a PVDF transfer membrane (Immobilon PVDF transfer membrane: Daiichi Kagaku). This Immobilon PVDF transfer membrane is used as a blocking solution (Block Ace: Dainippon Pharmaceutical) 1

After immersion in 0 ml at room temperature for 4 hours and blocking, 1 〃g / m 1 biotinylated ゥ 15 OmM NaCI / O. 05% Tween 20 / containing a heron anti-MAC IF antibody (IgG) The reaction was carried out in 10 ml of 0.25% BSA / 2 OmM Tris (pH 7.4) buffer (T / B / TBS) at 4 ° C. for 12 hours. Immobilon PVD transfer membrane was added to 150 mM NaC 1 / 0.0 5% Tween 20/2 OmM Tr

After washing with 1 s (pH 7.4) buffer (TZTBS) 2 Oml and diluting it 1 000-fold, streptavidin-piotin-horseradish peroxidase complex (Streptoavidin-biotin- HRPO complex) : Amersham) in TZBZT BS buffer containing 10 ml at room temperature for 1 hour. After washing with 20 ml of TZTBS, the binding of the biotinylated ゥ heron anti-MAC IF antibody was detected using a HRPO detection kit (ECL kit: Amersham) according to the attached instructions. As a result, the band detected by SDS-PAGE was stained. In addition, the biosynthesis of Escherichia anti-MAC IF antibody (IgG) was performed using ImmunPure NHS-CC-Biotin (Pierec) according to the attached instructions.

Example 7: Comparison of MAC IF (77) and MAC IF (77, N / Q) activities Dessauer et al. Ί £ [De ssauer, A. eta 1., Acta Patholofica Mi cr ob iol og ics S candinavia Secti on C Supplement 284, 92, 75-8 1 (1984)] to prepare a human C5b6 complex from human C5 (Cosmo Bio) and 6 (Cosmo Bio). Human C 5 b 6 complex hemolysis rate was diluted to about 50%, in a mixture of guinea pig erythrocytes (2. 5 X 10 ⁸ cells Zm 1) and human serum (final dilution 1 60-fold), MAC IF (77) and MA CIF (77, N / Q) solutions in PBS were added at various concentrations to make a total volume of 1001, and reacted at 37 ° C for 30 minutes. A sample to which no sample solution was added was reacted at the same time as a control. After the reaction, centrifugation was performed at 2000 × g for 3 minutes, and the absorbance (414 nm) of the supernatant was measured. The absorbance (414 nm) of the sample solution was A414 (s), and the absorbance of the control (414 nm) was A414 (c). The absorbance (414 nm) when an EDTA solution was added instead of the sample solution and reacted. To A414 (B). The hemolytic inhibitory activity (%) of the sample is calculated by the formula:

 CA 1 (c) -A41 (s)] / (A414 (c)

 -A 14 (B)) x 100

Was calculated. IC ₅ of hemolysis inhibitory activity of the MAC IF (77, N / Q ). Is about 3 μg / ml in the MAC IF (77), whereas the MAC IF

(77, N / Q) was 1 gZml, and the modified polypeptide of the present invention showed a superior effect.

Example 8: Effect of MAC IF (77, N / Q) on inhibition of cell lysis by complement of nucleated cells

 (1) Preparation of urine MAC IF

 Urine MAC IF has a structure cleaved in the middle of the anchor of natural MAC IF (Archne sof Biochemistry and Biophysics, 31 1 (1), 117-126, (1 994) ) It is known as a protein that has the effect of inhibiting erythrocyte hemolysis in guinea pigs.

 Urine MAC IF was prepared from human urine by the method of Sugita, Y. et al. (Immuno 1 og y, (1994) _8_2_: 34-41).

 (2) Preparation and preparation of a heron anti-HUVEC polyclonal antibody

ゥ For the production of heron anti-HUVEC polyclonal antibody, refer to Broo imans R. According to the method of A. et al. (Eur. J. Immunol., (1992) _2_2_: 31 35-3140).

 (2-1) Immunity of HUVEC

5x 1 0 ⁵ pieces of human umbilical vein endothelial cells (HUVEC: Kurabo, Japan) was suspended in 1 m〗's complete adjuvant (Wako Pure Chemical Industries, Ltd., Japan), Usagi

(Japanese white species: SLC, Japan). Two weeks later, a solution of the same number of HUVECs suspended in 1 ml of incomplete adjuvant (Wako Pure Chemical, Japan) was injected subcutaneously into the same egret three times at two-week intervals. With the above operations, HUVEC immunization was completed.

 (2-2) Preparation of a heron anti-HUVEC polyclonal antibody

 Ten days after completion of the immunization, blood was collected from a heron immunized with HUVEC to obtain a serum fraction (hereinafter, antiserum) according to a conventional method. From the obtained antiserum, an immunoglobulin G fraction was prepared according to a conventional method, and used as a perch anti-HUVEC polyclonal antibody.

 (3) Human complement lysis system of HUVEC

 (3-1) HUVEC PI-PLC processing

1 Ueru per 2. 5 X 10 ³ cells of 96-well microphone port test plate HUVEC and coated with fibronectin (Becton 'Dickinson, USA) planting example, in 5% C0 ₂ presence 4 days at 37 ° C for Cultured. H BSS (1 38m sodium chloride each Ueru 180〃 1, 5 mM potassium chloride, 0. 3 mM-phosphate disodium hydrogen · 12H ₂ 0, 0. 3mM potassium phosphate, 1. 3 mM calcium chloride, 0. 5 mM chloride magnesium · 6Η ₂ 〇, 0. 4 mM sulfuric acid magnesium _{· 7 H 2 0, 5. 6m} D- glucose, 1 0mM HEPE S pH7. after washing with 2), 250 m units / ml Phosphatidyl inosit-Phosp holipase C ( Add HBSS 1001 containing Boehringer Mannheim (Germany) (hereinafter PI-PLC) and 5% inactivated fetal serum (Gibco, USA) (FBS) and add 37. Incubate for 1 hour at C

Each well was washed with 1 HBSS.

(3-2) HUVEC cell injury by human complement Add 50 n1 of HBSS containing 62.5 jug rn 1 of anti-HUVEC polyclonal antibody and 5% inactivated FBS to each well treated in the previous section, and incubate at 4 ° C for 1 hour. Each well was washed with 180 1 HBSS. Add 50% of HBSS containing 50% human serum (hereinafter referred to as final reaction solution I) to these wells, incubate at 37 ° C for exactly 30 minutes, and perform final reaction from each well. Liquid I was collected, and the activity of Lactate Dehydrogenase (hereinafter abbreviated as LDH) in the liquid was measured by the method described in (4). The final reaction solution I supplemented with 20 mM EDTA was used as a control that did not activate complement.

 (4) LDH activity measurement

 Transfer the final reaction solution I 40 〃 1 recovered from each of the tubes in (3) to a 96 ゥ ヱ microwell plate (Nunc) and dissolve it in 0.4 mg ml with 20 mM Tris-HCl pH 9.5-nicotinamide adenine dinucleotide reduced form

(Sigma) (hereinafter abbreviated as NADH) was added in a concentration of 500 mg / m 1 at a concentration of 2.5 mg / m 1 in 100% Tris-hydrochloric acid pH 9.5, and 501 was added. Immediately, the change with time of the absorbance at a wavelength of 330 nm over 150 seconds was measured every 9 seconds using a THERM max (Molecular Devices) microplate reader, and Vmax (mOD / min) was calculated from the decrease rate of the absorbance. (Sample Vmax). The serum-derived LDH activity was determined by adding 2 OmM EDTA to the final reaction solution I (50% human serum) to inactivate complement (background Vmax), while the LDH activity was determined by the total dissolution of HUVEC. The value was obtained when 2% NP-40 was added to the final reaction solution I to lyse the cells (Total Vmax). For the LDH activity from HUVEC by complement, the value (complement Vmax) of the sample to which MAC IF (77, N / Q) was not added was used.

 The lysis rate of HUVEC cells in the reaction sample was shown by formula A, and the inhibitory activity of MAC IF (77, N / Q) and urine MAC IF by formula B. Formula A (HUVEC dissolution rate (%))

100 X (Sample Vmax—Bar / Kung Vmax) / (TotalVmax-Pack Gran Vmax) 32

Formula B (Inhibitory effect (%))

 100 X (complement Vmax—sample Vmax) / (complement Vmax—cogran Vmax)

(5) Inhibitory effect of MAC IF (77, N / Q) and urinary MAC IF on cell lysis by human complement of HUVEC

MAC IF (77, N / Q) or urine MAC IF was added to the final reaction solution I of (3), and its inhibitory effect was examined. Human serum is added to PI-PLC-treated HUVEC cells treated with anti-HUVEC polyclonal antibody at a final concentration of 50%, which is a condition closer to the human body, and reacted at 37 ° C for 30 minutes. It was dissolved (Fig. 6-1). The naturally occurring soluble urinary MAC IF did not show any inhibitory effect on the lysis of HUVEC cells by this human serum at concentrations up to 250 / ng / 1. On the other hand, the MAC IF (77, N / Q) of the present invention very strongly inhibits this cell lysis in a concentration-dependent manner, and its concentration (ED ₅ ) showing 50% inhibition is 10%.

(Fig. 6-2).

Example 9: MAC IF (77, N / Q) inhibits PGI ₂ and bFGF release by complement from HUVEC

 (1) Non-lethal release of bFGF from HUVEC by complement activation using human serum

 (1-1) HUVEC PI-PLC processing

 The same operation as in Example 8 (3-1) was performed.

 (1-2) HUVEC cell injury by human complement

To each well treated as described in the previous section, add 125 1 / g / m1 anti-HUVEC polyclonal antibody (Example 8 (2)) and HBSS containing 5% inactivated FBS, and add 4 ° C. After incubation for 1 hour in each well, each well was washed with 1801 HBSS. Add HBSS containing 12.5% human serum (hereinafter referred to as final reaction solution II) 60 1 to these wells, incubate at 37 for exactly 10 minutes, and then add each well. The final reaction solution II was recovered from the solution, and the Lactate Dehydrogenase The activity was measured by the method described in Example 8 (4), and the concentration of basic fibrob last growth factor (hereinafter abbreviated as FGF) was measured by the method described below.

 (2) b—FGF concentration measurement

Using the recovered final reaction solution I501 obtained in the operation of (1), the b-FGF concentration in the supernatant was quantified by ELISA. The ELISA was performed using Quantikine ^™ HumanFGF basic Immunoassay (R & D systems) according to the attached protocol.

 (3) Non-lethal production of PG I 2 from HUV EC by a complement activation reaction using purified human complement components

 Example 8 Fibronectin was coated by the method described in (3-1).

HUVEC cultured in a 6-well microtest plate until further growth were treated with PI-PLC. To each well was added 501 of HBSS containing 125 μg / ml anti-HUVEC polyclonal antibody (Example 8 (2)) and 5% inactivated FBS, and the mixture was incubated at 4 ° C. for 1 hour. Thereafter, each well was washed with 180 11 of HBS S, and 50 μl of HBSS containing 35% C8-deficient human serum (Quiddel) was added.

One was added and incubated at 37 for 15 minutes. It is thought that C5b7 has been formed on the cell membrane surface of HU VEC by the reactions so far (J.B.C., 2_64 (15) .9053-9060, (1989)). Wash each well with 180 11 HBSS, 0-640 ug / m1 MAC IF (77, NXQ), 0 or 8〃gZm1 human C8 (Quydel) and 0 Or 8 gZm 1 human C 9

HBSS (hereinafter referred to as final reaction solution II) containing (Quiddel) was added in 601, and incubated at 37 ° C for exactly 10 minutes. To recover the final reaction solution IE, and LDH activity in the same solution by the method of Example 8 (4), is a hydrolyzate of prostacyclin (hereinafter be referred as _{PG I 2) 6- keto- prostaglandin Fla} ( hereinafter 6-keto-PGFla) The concentration was measured by the method of (4).

 (4) Measurement of 6-keto-PGFla concentration

PG I ₂ released into the final reaction solution III is immediately hydrolyzed and stable 6-Convert to keto-PGFla. Therefore, the amount of PGI 2 released from HUVEC was measured by measuring 6-keto-PGFla in the reaction solution. Using the recovered final reaction solution 501 obtained above, the concentration of 6-keto-PGFla in the same solution was quantified by ELISA. ELISA was performed using 6-keto Prostaglandin Fla Enzyme Immunoassay Kit (Cayman Chemical) according to the attached protocol.

 (5) Results

 (5-1) MAC I F for release of b—FGF by non-lethal complement activation

(77, N / Q) inhibitory effect

 When the final reaction solution U of (1) was reacted without adding MAC IF (77, N / Q) to the final reaction solution U, the LDH activity in the final reaction solution II was determined by the method of Example 8 (4). The concentration was measured by the method of (2). Under these conditions, the cell lysis rate of HUVEC detected using LDH activity as an index was 4.1 ± 5.7 (%), and HUVEC lysis hardly occurred (FIG. 7-1). On the other hand, the b-FGF concentration increased from 10.8 ± 0.7 (pg / ml) of the control to 31.0 ± 4.7 (pg / ml). This demonstrated that b-FGF was released by non-lethal activation of complement by the method (1).

 Even if MAC IF (77, N / Q) was added to the final reaction solution II of (1) so that the final concentration was 14, 70, or 350 μgZm1, it was still effective for the LDH activity in the recovered final reaction solution II. There were no significant changes (Figure 7-1). However, the release of b—FGF

(77, N / Q) decreased in a concentration-dependent manner, and at MAC I F (77, N / Q) concentrations of 70 gZml or more, the value when inhibition of complement activation was inhibited by 2 OmM EDTA (1

0.8 pgZml) or less (Figure 7-2). This indicates that the soluble MAC IF (77, N / Q) of the present invention can completely suppress the release of b-FGF from HUVEC by non-lethal complement activation.

 (6) Production of PG12 by non-lethal complement activation

When the final reaction solution (3) of (3) was HBSS, HBSS containing human C8, and HBSS containing human C8 and human C9, the LDH activity in each recovered final reaction solution III was determined in Example 8 ( The PGI ₂ activity was measured by the method of (4) by the method of (4). Under the above conditions, C5b7, C5b8, and C5b9 are considered to be formed on the cell membrane surface of HUVEC, respectively (JBC 264 (15). 9053-9060. 1989). In any case, almost no LDH activity was detected in the final reaction mixture ΠΙ, and even when the reaction was performed with HBSS containing human C8 and human C9, the LDH activity in the final reaction mixture 5. was 5. It was 5 ± 1.6 (%), showing almost no cytotoxic activity (FIG. 9). On the other hand, when the final reaction solution M was HBSS and the HBSS containing human C8 was used, the 6-keto-PGFla concentration in the recovered final reaction solution IE was 6.8 ± 4.8 (pg / ml), respectively. 8.6 ± 4.8 (pgZml), and PG12 was hardly produced.However, when 1Π of the final reaction solution was HBSS containing human C8 and human C9, The 6-keto-PGFla concentration was 120.5 ± 21. (Pg / ml), and PGI ₂ release was dramatically increased (FIG. 9). That is, (3) the method according C5b9 of: Ri by the formation of the (MAC membrane attack complex), HUVEC in little damaged, a phenomenon of releasing the PG I ₂ was observed.

For the case where MAC IF (77, N / Q) was added to the final reaction solution (of (3) so that the final concentration was 2.5, 10, 40, 160, 64 O ^ g Zm1, The LDH activity in each recovered final reaction solution DI was measured by the method of Example 8 (4), and the PGI ₂ activity was measured by the method of (4). As expected, LDH activity did not increase even when MAC IF (77, N / Q) was added, confirming that the MAC IF (77, N / Q) protein itself had no cytotoxic activity. (Figure 8). On the other hand, PGI 2 release from HUVEC was suppressed in a concentration-dependent manner with MAC IF (77, N / Q). That is, MAC IF (77, N / Q) inhibited PGI 2 release from HUVE by almost 90% at a concentration of 160 / g / m 1 or more, and its IC ₅ . Was about 20 z / m 1 (Fig. 10). This indicates that the soluble MAC IF of the present invention (77, N / Q), PG I 2 release from HUVECs caused by the formation of C5b9 (MAC: membrane attack complex) by non-lethal complement activation Can be completely suppressed.

Example 10: Effect of MAC IF (77, N / Q) on Guinea Pig Heart Z Human Plasma Perfusion Injury (1) Preparation of human plasma

 Blood was collected from one human volunteer using citrate as an anticoagulant, and the cell component was centrifuged at 3,000 rpm for 15 minutes to obtain human plasma. The obtained human plasma was prepared using a crease buffer (U7 mM sodium chloride, ½M chlorinated chloride, 1.1 ml potassium dihydrogen phosphate, 25 mM sodium bicarbonate, 5 mM glucose, 1.2 mM magnesium chloride, 2.6 mM calcium chloride, 5 mM L-glutamate, 2nA! Pyruvate, 10unit / ml insulin, 14unit / ml heparin, 0.25% BSA) were dialyzed sufficiently and used in the experiment.

 (2) Langendorff experiment

An experiment to examine the inhibitory effect of MAC IF (77, N / Q) on human plasma injury in guinea pig heart was performed in a routine Langendorff experimental system using isolated guinea pig hearts. The method was described by Home ister, JW (Circu 1 ati on Research, (1992), 71: 303-3 19). A Hatley male guinea pig weighing about 300 g was anesthetized with ether, and the heart was removed. Immediately after the heart was transferred to Krebs buffer and the blood in the heart was sufficiently washed out, a force neuron was inserted in a retrograde direction from the aorta toward the aortic valve, and the heart was attached to the Langendorff port flow device and diverted downward.漼流solution warmed to _{37 ° C, 95% 0 2} - was oxygenated with 5% C0 ₂ gas mixture. The port flow was a constant flow port flow of 7 m 1 / min.

 In order to measure left ventricular function, a small rubber balloon was attached to the tip, and a pressure-measuring force-filler filled with water was inserted into the left ventricle vertically along the longitudinal axis of the heart from the incision in the left atrium. End ventricular end diastolic pressure and left ventricular development pressure were measured. At the same time, the heart rate was recorded from the left ventricle generated E pulse wave. The E of the balloon was adjusted to be about 1 Omm Hg during diastole. The heart paged the ventricular muscle directly through the electrodes, keeping the heart rate constant at 273 beats per minute. The 漼 flow pressure was measured via the aortic force neuron.

Guinea pig hearts that were exposed to perfusion with the Langendorff experimental apparatus were preliminarily perfused with Krebs buffer for about 30 minutes. After that, the perfusate was replaced with Krebs buffer containing 3% human plasma, and the perfusion was continued for 60 minutes, and the human plasma was converted to guinea pig cardiac function and And the effects on heart failure were observed. The subjects were examined under the condition that the harbor flow was continued for 60 minutes with Krebs buffer solution containing no plasma component. The effect of MAC IF (77, N / Q) in this experimental system was added to Crepes buffer containing 3% human plasma to give final concentrations of 10 1 u Zm 1 and 50〃 g / m 1 Port flow liquid was examined by port flow using the same operation.

 In order to examine cardiomyocyte damage, the perfusate discharged from the right atrium is collected over time, and the creatine phosphokinase activity in the permeate is tested using the Monotest CK-MB Kit (Beiringer Mannheim). It was quantified according to the instruction of NO300691).

 (4) Inhibitory effect of MAC IF (77, N / Q) on guinea pig heart in human plasma perfusion injury system

 (4-1) Coronany perl us ion pressure (CPP)

 FIG. 11 shows the time-dependent changes in the air pressure in each experimental group.

 When Krebs buffer (NHP) containing 3% human plasma flows into the port, the flow pressure increases extremely from 20 minutes after the start of port flow and reaches 88.1 ± 7 after 60 minutes. It showed a high value of 9 mmHg (Fig. 11-1).

 On the other hand, MAC IF (77, N / Q) was added to 3% human plasma at a final concentration of 10 g / m1 and 50 ugm1 in a Krepp's buffer experiment. (77, N / Q) significantly suppressed the rise of port flow pressure in a concentration-dependent manner, and the flow pressure of the group to which 50 ^ gZml0MAC IF (77, N / Q) was added 60 minutes after the start of flow was 62 It decreased to ± 10.7 mmHg. This indicates that the MAC IF (77, N / Q) of the present invention has an extremely excellent port flow pressure rise suppressing effect. (Fig. 11-2).

 (4-2) Left ventricular pressure

 Changes in the left ventricular pressure over time in each experimental group are shown in FIGS. 12 and 13. In the figure, the left ventricular developed pressure (LVDP) and the left ventricular end diastolic pressure (LVEDP) are shown.

In control experiments with Krebs buffer, the LVDP and LVEDP values The initial values were 62.5 ± 3.84 and 10.0 ± 1.14 mmHg, respectively, and were constant within the study time (Fig. 12).

 In the port flow using 3% human plasma-added Krebs buffer, an extreme increase in LVEDP value was observed from 20 minutes after the start of flow, and a high value of 38.5 ± 4.76 mmHg was shown after 60 minutes. . On the other hand, the MAC IF (77, N / Q) of the present invention significantly inhibited this guinea pig cardiac dysfunction in a concentration-dependent manner, and the concentration of MAC IF (77, NZQ) at a final concentration of 50 g / m1 was increased. At 60 minutes after the start, the LVEDP value was improved to 13.4 ± 4.11, almost the value of the control group. These results indicate that the MAC IF (77, NZQ) of the present invention has an extremely excellent effect on suppressing guinea pig cardiac dysfunction caused by human plasma (FIG. 13).

 (4-3) Creatine phosphokinase release

 Creatine phosphokinase is an enzyme present in cardiomyocytes and is widely used as a marker enzyme for studying cardiomyocyte damage because it is released into blood vessels when cardiomyocyte damage occurs. I have. Therefore, in the present experimental system, the damage of the muscle cells was examined by measuring the enzyme activity in the perfusion fluid discharged from the left atrium. The results of the study are shown in Figure 14.

In the port flow of 3% human plasma-added Krebs buffer, the activity of the enzyme released into the port flow significantly increased from 30 minutes after the port flow started, and 31.8 ± 60 minutes after the port flow started. It showed extremely high value of 4.19 (international unit: IU). This result indicates that human plasma causes extremely severe damage of cardiomyocytes. Investigation of the inhibitory effect of MAC IF (77, N / Q) on guinea pig cardiomyocyte injury caused by human plasma showed that MAC IF (77, N / Q) showed the concentration of the enzyme in the port fluid in a concentration-dependent manner. In the group to which MAC IF (77, N / Q) was added at a final concentration of 50 fji g / m 1, the outflow of the enzyme was 60.70 ± 2.33 IU 60 minutes after the start of the perfusion. There, 3% human plasma _c the result of almost completely inhibiting the outflow of the enzyme by Minatoryu is, MAC IF (77, N / Q) is extremely excellent in suppressing injury in guinea pigs cardiomyocytes by human plasma Minatoryu It has an effect.

Prescription example: A solution prepared by dissolving 5 g of the modified boreptide of the present invention in 100 ml of physiological saline is subjected to aseptic filtration, and then dispensed into vials in a volume of 2 ml. Then, it is freeze-dried to obtain an injectable preparation containing 100 mg of the modified protein of the present invention in one vial.

 Sequence listing

SEQ ID NO: 1

Array length: Ί 7

Sequence type: amino acid

 Topology: linear

Sequence type: protein

Array

 Leu Gin Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr

 1 5 10 15

 Ala Val Gin Cys Ser Ser Asp Phe Asp Ala Cys Leu He Thr Lys

 20 25 30

 Ala Gly Leu Gin Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys

 35 40 45

 Asn Phe Asn Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr

 50 55 60

 Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe Asn Glu Gin Leu

 65 70 75

 Glu Asn SEQ ID NO: 2

Array length: 1 0

Sequence type: amino acid

 Topology: straight chain

Sequence type: protein

Array Leu Gin Cys Tyr Asn Cys Pro Asn Pro Thr 1 5 10 SEQ ID NO: 3

Array length: 3 1 2

Sequence type: nucleic acid

Number of chains: double strand

 Topology: linear

Sequence type: other nucleic acid modified DNA

s ε

Pomani: © Parent: mourn: ^ m

9 OA

usy "10

UI9 "1 usv 3Md usy s naq dsy s 2IS OVl VVl IVV WO 1130V3 VV93VV 111 OVV 101013 OVO OVV

 99 09 99

 s s sAo J JM1 na niusy ^ 3JV ^^ LZ 9VV 391391 OVl 3V103V VIO OVO IVV WO 09V Oil 30333V V3V

 09

 1¾Λ dsv usy 3Md usv SAQ S! H si sA dJi s s usy i

^ ZZ 310 OVO OVV 311 IVV 3011V3 OVO 111 OVV 001191 OVV OVV IV 丄

 9S OS 2

 ui9 ηθ Xio BIV sX JMl d \] s BIV dsv 9Md dsy J3S

081 010 m Vll 000 100 VVV 33V 1 丄 V 3131010303V9111 IVO 131

 OZ 91 01

 J9S s uio eight BIV JMl sAq sAQ dsv ¾IV JMl OJd usy OJJ s

SSI VOX 101 WO 310330 VOV VVV 301 OVO 13010V V033VV 133101

 3 I 9- 01- usv i s uio naq J3s SIH XlO s SIH SAQ a d I¾ BIV naq

06 OVV OVl 391 OVO 013 OOV 1V3190 VOX 1V3301311310130010

 91- 02- 93- Π91 Π3ΐ nan A aqj na \ ^ J9S AIO λΐθ "10 311 ^ ΐθ

^ 310313013010000311013310131090 V90 WO 31V VOO 01V 9I0 / 96 «ir / IDd 0Z £ 00 / Z.6 OW 9 e

001

 OJd S IH na J3S dJj, BIV BIV BIV A8S VVl 330 1V3 113 30V 001 330 V30 V30

 96 06 38

 nai 3Md OJd Jiu Λ Π91 ΠΘ naq J¾ sXq nio S na J9S 09S 013 111 m 13V 010 013 013 110 119 VOV VVV OVO V31 Vll 031

 08 SA OZ.

 Iqi AlO usv nio naq uio "19 usy 9qd usv sAQ na dsv sX 51S VOV 090 100 IVV VV9 113 OVO WO OVV 111 OVV 101 913 3V9 OVV

 99 09 59

 sAq sAQ s jXi JAI j¾ n9 ni3 usv "10 V naq 3JV LZ OVV 301 301 3V1 3V1 03V VIO 0V9 IVV WO 99V Oil 300 33V V3V

 09 ^ 0

 Ι¾Λ dsv usy 9Md usv SAQ S IH nio aqj sA cU 丄 SAQ sAq usy -ΐλχ

310 3V0 OVV 311 IVV 301 IVO 0V9 111 OVV 901 101 OVV OVV IVl

9S 08 93

 uio "31 ^ IO BIV sA Jiji d \\ na s dsv 3Md dsv S

081 010 m Vll 000 109 VVV 33V 丄 丄 V 313 101 939 OVO 111 IVO 131

 OZ 91 01

 J3s SAQ usy ¾IV J 1 sxi SAQ dsy ¾IV JM1 OJd usy OJd s

2δΙ V01 101 IVV 319 300 VOV VVV 391 3V0 100 1DV V33 OVV 133 191

 9 ΐ 9- 01- usv J i s uio J9S S IH A19 s S IH SXQ aqd

06 OVV 3V1 301 OVO 913 30V IVO 109 V31 1V3 391 311 310 130 013

 51- 02--

ΙΒΛ naq na yi na eight S 13 ^ 19 "10 ^ I I ^ IO

^ 010 313 013 010 000 311 013 019 101 000 V09 WO OIV VOO OIV vNa

09I0 / 96df / 13d 0 00/6 OAV SEQ ID NO: 5

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Primer DNA

Array

 GCGGCCGCCA TGGGAATCCA AGGAGGGTCT 30 SEQ ID NO: 6

Array length: 3 4

Sequence type: nucleic acid

Number of chains: single

 Topology: linear

Sequence type: Other nucleic acid Primer DNA

Array

 GCGAATTCTA TTAGTTACAC AGGTCCTTCT TGCA 34 SEQ ID NO: Ί

Array length: 4 2

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Primer DNA

Array

GAGACACGCG TCAAAATCAG ATGAACATTG GACGGCTGTT TT 42 SEQ ID NO: 8

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Primer DNA

Array

 TCTGATTTTG ACGCGTGTCT CATTACCAAA 30 SEQ ID NO: 9

Array length: 6 0

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acid Primer DNA

Array

 TCTAGAGCGG CCGCTCCCCA TCCGCTCAAG CAGGCCACCA TGGGAATCCA AGGAGGGTCT 60 SEQ ID NO: 10

Array length: 3 8

Sequence type: nucleic acid

Number of chains: single strand

 Topology: straight chain

Sequence type: Other nucleic acid Primer DNA

Array

 TCTAGACTCG AGCTATTAAT TTTCAAGCTG TTCGTTAA 38

Claims

The scope of the claims

1. A modified polypeptide having the amino acid sequence of the following formula I (SEQ ID NO: 1 in the sequence listing).

 Formula I

 Leu Gin Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr

 1 5 10 15

 Ala Val Gin Cys Ser Ser Asp Phe Asp Ala Cys Leu lie Thr Lys

 20 25 30

 Ala Gly Leu Gin Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys

 35 40 45

 Asn Phe Asn Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr

 50 55 60

 Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe Asn Glu Gin Leu

 65 70 75

 Glu Asn

 2. A DNA encoding the modified polypeptide of claim 1.

 3. A recombinant expression vector comprising the DNA according to claim 2.

 4. A transformant transformed by the recombinant vector according to claim 3.

 5. The modified polypeptide according to claim 1, wherein the transformant according to claim 4 is cultured to produce the modified polypeptide described in claim 1, and this peptide is collected. Manufacturing method of peptide.

 6. A pharmaceutical composition comprising the modified polypeptide described in claim 1.

 7. A pharmaceutical composition which is an anti-inflammatory agent based on inhibition of the complement system, which comprises the modified boreptide according to claim 1.

8. The pharmaceutical composition according to claim 6, which is an agent for suppressing rejection during organ transplantation.

9. The pharmaceutical composition according to claim 6, which is a non-lethal effect (complement) inhibitor of complement.