Genes coding for a protein having human macif activity, expression vectors containing said genes, transformant cells and proteins having human macif activity

Abstract: Genes coding for protein having human MACIF activity, expression vectors containing the genes, cells transformed with the vectors and proteins having human MACIF activity.

Description: This invention relates to genes coding for a protein having human MACIF (membrane attack complex inhibition factor) activity, expression vectors with the genes respectively inserted therein, cells transformed with the expression vectors, and proteins having human MACIF activity. The term "protein having human MACIF activity" includes, within the meaning thereof, a group of proteins which regulate the complement system in the final stage of complement activation and inhibit damaging of human cells and tissues as a result of membrane attack complex formation. The present inventors previously succeeded in isolating a naturally occurring human MACIF, a so-far unknown protein regulating the complement system, in pure form from the human normal erythrocyte membrane (Japanese Patent Application No. 63-310642). They found that the human MACIF inhibits the activation of the late complement components or, in other words, inhibits hemolysis resulting from human MAC formation and, in this respect, the human MACIF is distinguished from and superior to the known complement-regulating substances that inhibit the activation of the early complement components, and that the naturally occurring human MACIF has the following N-terminal amino acid sequence: Furthermore, they found that this naturally occurring human MACIF is a glycoprotein having a molecular weight of 18,000±1,000 (as determined by SDS-polyacrylamide gel electrophoresis) with a phosphatidylinositol anchor (hereinafter abbreviated as "PI-anchor") at the C terminus. For further studying protein having human MACIF activity and for developing the practical use of such protein as a drug, it is essential to obtain such protein in a pure and homogeneous form, and in sufficiently large quantities. For this purpose, application of recombinant DNA technology appears to be the most effective means. The present invention can provide a gene coding for a protein having human MACIF activity. The present invention can also provide a replicable expression vector capable of expressing the gene coding for a protein having human MACIF activity. The present invention can further provide a microorganism or cells transformed with the expression vector. The present invention can still further provide a genetically engineered protein having human MACIF activity. The present invention provides genes respectively coding for polypeptides having human MACIF activity in which an amino acid sequence is represented by the following general formula (I): In the above formula, X and Y are defined as follows: X is H, Met, or the amino acid sequence: Y is OH, or or The present invention further provides expression vectors respectively containing the genes mentioned above, microorganisms or cells transformed with the vectors, and genetically engineered proteins expressed in the microorganisms or cells and having human MACIF activity. Fig. 1 illustrates the gene isolated from human leukocytes and coding for an amino acid sequence including the naturally occurring human MACIF. Fig. 2 illustrates an amino acid sequence deduced from the nucleotide sequence of the gene as shown in Fig. 1. Fig. 3 illustrates the whole structure of an expression vector, pKK223-3, for use in Escherichia coli . Fig. 4 illustrates the whole structure of an expression vector, PVY1, for use in CHO cells. Fig. 4a illustrates a construction scheme for the expression vector pVY1. Fig. 5 illustrates the whole structure of a vector, pTEJ001, for expression in Escherichia coli . Fig. 6 illustrates the results of purification of a human MACIF protein (E86) expressed in Escherichia coli using Q-Sepharose. Fig. 7 illustrates the results of SDS-PAGE analysis of fractions obtained by purification of the human MACIF protein E86 expressed in Escherichia coli using Q-Sepharose. Fig. 8 illustrates the dose dependency of the inhibitory activity in hemolysis of guinea pig erythrocytes of the human MACIF protein E86 expressed in Escherichia coli and the result of neutralization thereof with an anti-human MACIF antibody. Fig. 9 illustrates the dose dependency of the inhibitory activity in hemolysis of guinea pig erythrocytes of the human MACIF protein (C77) expressed in transformant CHO cells and the results of a neutralization test thereof with an anti-human MACIF antibody. Fig. 10 illustrates the dose dependency of the inhibitory activity in hemolysis of guinea pig erythrocytes of the human MACIF protein (C70) expressed in transformant CHO cells. For reference, the gene defined by the DNA sequence shown in Fig. 1 contains a gene sequence coding for the amino acid sequence of that protein which exhibits physiological activities of naturally occurring human MACIF and, in addition, a gene coding for a naturally occurring human MACIF precursor amino acid sequence (inclusive of a secretory signal peptide and a PI anchor-attachment signal amino acid sequence, among others). The secretory signal peptide-encoding gene is a gene coding for a peptide necessary for the protein having human MACIF activity which is produced in host cells to be released from the cell, while the PI anchor-attachment signal amino acid sequence-encoding gene is a gene coding for a hydrophobic amino acid sequence portion necessary for the attachment of the PI anchor to the protein having human MACIF activity. In the naturally occurring human MACIF, the PI anchor is attached to the 76th amino acid (glutamic acid) residue of the amino acid sequence shown, for reference, in Fig. 2. The PI anchor has a skeletal structure composed of phospho-ethanolamine, glycan and phosphatidylinositol (PI). The amino group of phospho-ethanol-amine is attached to the carboxyl group of the C-terminal amino acid residue of peptides, and the fatty acid side chains of the PI is bound to the cell membrane. In this way, the PI-anchor serves as an anchor for binding peptides to the cell membrane. In the amino acid sequence shown in Fig. 2, the sequence from the methionine residue at position -25 to the serine residue at position -1 is the secretory signal sequence and the sequence from the asparagine residue at position 77 to the proline residue at position 103 is the PI anchor-attachment signal sequence. Thus, the amino acid sequence comprising the 1 st to 76th Amino acid residues constitutes a peptide core of naturally occurring human MACIF. This human MACIF may have a sugar chain of various kinds of structures depending on the host cells used for phenotypic expression or depending on culturing conditions of cells used therein. For practical use as a drug having physiological activities of human MACIF, a protein is not required to have all the constituent elements of the above-mentioned human MACIF. It may be lacking in the PI anchor, or it may partially differ in amino acid sequence from human MACIF, provided that it has human MACIF activity. Thus, the proteins according to the present invention are proteins having human MACIF-like physiological activities but differing from naturally occurring human MACIF in that a part of the amino acid sequence of this human MACIF is missing or replaced with some other amino acid sequence, in that some amino acid sequence is added to or inserted in them, in that they have no PI anchor and/or in that they have no carbohydrate chain or differ in the kind of carbohydrate. The genes provided by the present invention are genes coding for human MACIF proteins, specifically a gene coding for the amino acid sequence from the 1st amino acid residue (Leu) to the 70th amino acid acid residue (Asn) of the above-mentioned amino acid sequence of formula (I), a gene coding for the amino acid sequence from the 1st to the 77th amino acid residue (Asn) of the sequence of and a gene coding for the amino formula (I), acid sequence from the 1st to the 86th amino acid residue (Thr) of the sequence of formula (I). The genes according to the invention can be prepared by various methods. For example, a suitable method comprises isolating a clone containing cDNA coding for human MACIF from a cDNA library prepared from mRNA obtained from human MACIF-producing cells and isolating the MACIF cDNA from the thus-isolated clone. Another method comprises chemically synthesizing nucleic acids in the conventional manner, for example by the phosphoamidite method [Hunkapiller, M. et al., Nature, 310 , 105-111 (1984)], based on the genetic information encoding human MACIF as disclosed herein. A combination of the above two methods may be mentioned as a further example. In the following, the method mentioned above that makes use of mRNA is described in further detail. Oligonucleotide probe preparation A oligodeoxyribonucleotide probe is prepared which is complementary to mRNA coding for the N-terminal amino acid sequence of purified, naturally occurring human MACIF isolated from human erythrocytes, for example, by chemical synthesis by the phosphoamidite method using a commercial DNA synthesizer (e.g., Applied Biosystems model 380A DNA synthesizer). cDNA library preparation (1) Preparation of raw material cells: Human cells of any kind in which the human MACIF according to the invention can be expressed may be used as the material for human MACIF mRNA in the practice of the invention. Advantageous as such cells from the high mRNA content viewpoint are human peripheral blood-derived leukocytic cells, human lymphocytic cells and other tissue cells as well as appropriate cell lines established therefrom. Human peripheral blood-derived leukocytes and lymphocytes can be isolated from a normal human-derived buffy coat by density gradient centrifugation [Bøyum, A., Scandinavian Journal of Clinical Laboratory Investigations, 21 , Supplement 97, 77-89 (1968)] using dextran or Ficoll-Hypaque, for instance. Tissue cells can be prepared from a tissue homogenate or the like in the conventional manner. As the established human cell lines, there may be mentioned, for example, human erythroblastic leukemia cell lines (e.g., K562), human B cell leukemia cell lines (e.g., Raji), human T cell leukemia cell lines (e.g., MT-2), human monocytic leukemia cell lines (e.g., U937, HL60) and other tissue cancer cell lines (e.g., Bowes melanoma). The cell lines to be used are not limited to these, however. Such cell lines are readily available from the Salk Institute Cell Bank (California, U.S.A.) and other similar institutions. The cells are cultured in the manner of stationary culture, spinner culture or roller bottle culture, for instance, using an appropriate animal cell culture medium, e.g., commercially available RPMI 1640 medium [Moore, G.E. et al., Journal of American Medical Association, 199 , 519-524 (1967)]. In some instances, stimulation of cells during cultivation can result in intracellular expression of human MACIF mRNA in increased amounts. The use of an immune complex as the stimulant is advantageous. As the stimulant, there may be further mentioned lectins [e.g., concanavalin A (ConA), phytohemagglutinin (PHA)], various antigens, phorbol esters [in particular 12-O-tetradecanoylphorbol-13-acetate (TPA)] and physiological stimulatory factors (e.g., interleukins, interferons, colony stimulating factors), among others. These may be used in combination of two or three of them. (2) mRNA extraction: An RNA fraction containing human MACIF-encoding mRNA can be extracted from cells of any of the kinds mentioned above in the conventional manner. Thus, for instance, the cells are partially or completely disrupted and/or solubilized by means of a guanidine thiocyanate solution or an appropriate detergent (e.g., SDS, NP-40, Triton X-100, deoxycholic acid) or by a physical means such as homogenization or hemolysis. Chromosomal DNA is then subjected, to a certain extent, to the shearing action of a mixer (e.g., Polytron) or a syringe, followed by separation of a nucleic acid fraction from proteins. Particularly useful for this fractionation procedure is the technique of extraction with phenol and chloroform or cesium chloride density gradient ultracentrifugation [Chirgwin, J.M. et al., Biochemisty, 18 , 5294-5299 (1979); Maniatis, T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, (1982)], among others. In the above-mentioned extraction procedure, an RNAase inhibitor, for example, heparin, polyvinyl sulfate, diethyl pyrocarbonate or a vanadium complex, may be used as an additive for preventing RNA degradation due to RNase. Isolation and purification of mRNA from the RNA obtained by the above extraction procedure can be effected, for example, by using an adsorption column of, for example, oligo-dT-cellulose (Collaborative Research) or poly-U-Sepharose (Pharmacia), or in a batchwise manner. The thus-obtained mRNA is a mixture of mRNAs coding for a variety of proteins. Therefore, it may be purified and concentrated with respect to the desired mRNA that corresponds to human MACIF prior to cDNA library preparation. This purification and concentration can be performed as follows. Thus, the mRNA obtained in the above manner is fractionated by sucrose density gradient centrifugation, for instance, and the resulting fractions are tested for the presence of the desired human MACIF mRNA by dot plot hybridization, for instance. (3) cDNA Library preparation: The purified mRNA obtained in the above manner is generally unstable. Therefore, the mRNA is converted (reverse transcribed) to a stable complementary DNA (cDNA) and connected to a microorganism-derived replicon, enabling amplification of the desired gene. The in vitro mRNA conversion can be generally carried out by the Okayama-Berg method [Okayama, H. and Berg. P., Molecular and Cellular Biology, 2 , 161-170 (1982)]. Thus, oligo-dT is used as a primer, which may be free oligo-dT or in the form already attached to a vector primer, and a single-stranded cDNA complementary to the mRNA is synthesized using the mRNA as a template and using reverse transcriptase in the presence of dNTPs (dATP, dGTP, dCTP and dTTP). The next step depends on whether the oligo dT is used in the free form or in the form attached to a vector primer. In the former case, the template mRNA is removed by decomposing it by treatment with an alkali, for instance, and then a double-stranded DNA is synthesized in the presence of reverse transcriptase or DNA polymerase I with the single-stranded DNA as a template. The resultant double-stranded DNA is then treated at both ends thereof with SI nuclease and, after addition of an appropriate linker DNA or a plurality of bases whose combination allows annealing to the respective ends, inserted into an appropriate vector, for example, an EK system plasmid vector (either of the stringent type or of the relaxed type), or a λgt phage vector. In the latter case, the mRNA to serve as a template is allowed to remain as it is, and the opened circular plasmid with the same linkers as mentioned above added thereto is annealed with a linker DNA (frequently a DNA fragment containing a region autonomously replicable in cells and an mRNA transcription promoter region) to give a closed circular form. Then, the mRNA is replaced with a DNA chain in the presence of dNTPs and in the simultaneous presence of RNase H and DNA polymerase I to give a complete plasmid DNA. The cDNA-containing plasmid vector obtained in the above manner can be introduced into a host for transformation thereof. Typical as the host is Escherichia coli . However, the host is not limited to this but may be, for example, Bacillus subtilis or Saccharomyces cerevisiae . The host can be transformed by introducing the DNA mentioned above by various methods commonly used in the art, for example, by collecting cells mainly at the logarithmic growth phase, treating them with calcium chloride for rendering them ready for spontaneous DNA uptake and allowing them to take up the plasmid. In the procedure mentioned above, magnesium chloride or rubidium chloride may be allowed to additionally coexist in the transformation system for further improvement of the transformation efficiency, as is generally known in the art. It is also possible to convert host cells to the spheroplast or protoplast state prior to transformation. Cloning of cDNA A strain carrying the desired human MACIF cDNA can be detected from among the transformants obtained in the above manner by various methods, for example, the methods mentioned below. (1) Screening using a synthetic oligonucleotide probe: In cases where a part of the amino acid sequence of the desired protein is known, as in the case of the present invention, oligonucleotides corresponding to the amino acid sequence portion is synthesized and this is used as a probe (after labeling with ³²P or ³⁵S) for detecting and selecting a positive strain by hybridization with transformant-derived DNAs denatured and immobilized on a nitrocellulose filter. The oligonucleotide may have a base sequence derived based on the codon frequency data or a combination of presumable base sequences. In the latter case, the number of probes can be reduced by incorporating inosine thereinto. (2) Selection using an antibody to human MACIF: The cDNA is inserted in advance into a vector capable of protein expression in transformants, protein production is allowed to proceed therein, and the anti-human MACIF antibody and a second antibody to the antibody are used to detect and select a desired human MACIF producer cell. (3) Screening by producing human MACIF in other animal cells: Transformant strains are cultured for gene amplification. The genes are used to transfect animal cells (using a plasmid capable of autonomously replicating and containing an RNA transcription promoter region or a plasmid capable of integrating into animal cell chromosomes). The proteins encoded by the genes are allowed to be produced and the culture supernatants or cell extracts are assayed for human MACIF activity. Alternatively, a transformant strain carrying the desired cDNA coding for human MACIF is selected from among transformant strains by detecting the human MACIF produced therein by using an antibody to human MACIF. Confirmation of human MACIF cDNA The gene according to the present invention obtained by the use of mRNA as a starting material can be confirmed to be a gene properly coding for human MACIF by using an appropriate translation system. Most commonly used is the method developed by Krieg et al. [Krieg, P.A. et al., Nucleic Acids Research , 12 , 7057-7070 (1984)], which comprises synthesizing a large quantity of mRNA in vitro using a potent promoter and an RNA polymerase specific to the promoter, followed by translation of the mRNA into a protein using a simple translation system. Thus, in the above method, the cDNA of the present invention is inserted into an appropriate plasmid downstream from a potent promoter, such as the SP6 promoter, T7 promoter or T3 promoter, (in the case of the use of mRNA as a starting material, the vector containing these promoters can be previously used in the library pereparation), and the resulting plasmid is purified and then cleaved at an appropriate restriction enzyme cleavage site occurring downstream from the human MACIF cDNA which occurs downstream from such promoter. The resulting double-stranded DNA is transcribed to mRNA in vitro using a polymerase specific to the promoter used, such as SP6 polymerase, T7 polymerase or T3 polymerase, respectively. The thus-transcribed mRNA is then translated into a protein by using a cell-free protein synthesizing system, such as a rabbit reticulocyte lysate or wheat germ lysate, or by the method comprising injecting the mRNA into Xenopus laevis oocytes. That the gene correctly coding for human MACIF has been obtained can be confirmed by assaying for MACIF activity of the translation product protein, or by an immunological method using an antibody specific to human MACIF. Gene sequence determination The nucleotide sequence of the thus-obtained gene of the present invention can be determined, for example, by the dideoxy method using a plasmid vector [Chen, E. Y., DNA , 4 , 165-170 (1985)] or by the 7-DEAZA method [Mizusawa, S. et al., Nucleic Acids Research , 14 , 1319-1324 (1986)]. The thus-obtained gene for naturally occurring human MACIF (derived from human leukocytes) is shown in Fig. 1. While the method of preparing the gene of the present invention as detailedly described above goes via mRNA, the gene coding for human MACIF can also be prepared by chemical synthesis based on the nucleotide sequence disclosed herein. A typical example of the method of chemical synthesis is the phosphoamidite method. Construction of expression vectors (1) Selection of the host-vector system: The coding region of the desired human MACIF gene can be expressed using a eukaryote or prokaryote as the host. The vector to be integrated into such host cells can be constructed in an appropriate manner depending on the host cells. As the prokaryote host, there may be mentioned Escherichia coli strains [e.g., E. coli K12 294 (ATCC 31446), E. coli B, E. coli χ1776 (ATCC 31537), E. coli C600, E. coli W3110 (F-, λ-, prototrophic; ATCC 27325)], Bacillus strains [e.g., B . subtilis], enteric bacteria other than E. coli, for example, Salmonella typhimurium and Serratia marcescens, and Pseudomonas strains. Usable as the vector for such microorganism hosts is an expression vector which contains the gene of the present invention with a promoter and an SD base sequence [Shine, J. et al., Proc. Natl. Acad. Sci. U.S.A., 71 , 1342-1346 (1974)] located upstream of the gene, together with ATG necessary for protein synthesis initiation. Generally, pBR322, pBR327 and the like are vectors suited for use in Escherichia coli and other microbial strains. Usable as the promoter are, for example, the tryptophan promoter, PL promoter, lac promoter, lpp promoter and β-lactamase promoter. As typical examples of the marker gene, there may be mentioned the ampicillin resistance gene and tetracycline resistance gene. Yeasts are generally used as the eukaryotic microorganism. In particular, yeasts belonging to the genus Saccharomyces can be used advantageously. A typical example of the expression vector for use in yeasts and other eukaryotic microorganisms is YRp7. Useful examples of the promoter which the expression vector for expression in yeasts should have are the 3-phosphoglycerate kinase enolase, glyceraldehyde-3-phosphate dehydrogenase or hexokinase promoters. The trpl gene, for example, can be used as the marker gene. The origin of replication, termination codon and other DNA sequences which serve to regulate transcription and translation in yeast cells may be ordinary DNA sequences known to be suited for use in yeast cells. When cultured higher animal cells are used as the host, they may be rhesus monkey kidney cells, mosquito larva cells, African green monkey kidney cells, mouse fetus fibroblasts, Chinese hamster ovary (CHO) cells, a dihydrofolate reductase-deficient strain thereof [Urlaub, G. et al., Proc. Natl. Acad. Sci., U.S.A., 77 , 4216-4220 (1980)], human cervical epithelial cells, human fetus kidney cells, moth ovary cells, human myeloma cells or mouse fibroblasts, for instance. The vector generally contains functional sequences for expression of the DNA of the present invention in host cells, for example, the origin of replication, promoter located upstream from the DNA of the present invention, ribosome binding site, polyadenylation site and transcription termination sequence. Preferred examples of the promoter are the adenovirus 2 major late promoter, SV40 early promoter, SV40 late promoter, eukaryotic gene-derived promoters (e.g., estrogen-inducible chicken egg albumin gene, interferon gene, glucocorticoid-inducible tyrosine aminotransferase gene, thymidine kinase gene, adenovirus major early and late genes, phosphoglycerate kinase gene, α-factor gene). The origin of replication may be derived from adenovirus, SV40, bovine papilloma virus (BPV), vesicular stomatitis virus (VSV), or any of vectors derived from these. The neomycin resistance gene, and methotrexate resistant dihydrofolate reductase (DHFR) gene, among others, can be used as the marker gene in this case. The examples of the host, vector and constituent elements thereof that have been described hereinabove as usable for the expression of the human MACIF cDNA and modified human MACIF protein cDNAS are by no means limitative of the scope, of the present invention. (2) Construction of expression vectors for human MACIF protein: A method of constructing vectors for the expression of human MACIF proteins is described in the following. These are recombinant vectors comprising a DNA sequence derived from the DNA base sequence shown in Fig. 1 by deleting any portion thereof, for example, such that the expression product lacks in a part of its N-terminal or C-terminal amino acid sequence, or by modifying any portion thereof such that the corresponding amino acid sequence be replaced by some other amino acid sequence, or by adding an appropriate sequence such that some amino acid sequence be added to the expression product, together with those constituent elements necessary for expression in a variety of host systems mentioned in the above section (1). More specifically, these expression vectors include a series of recombinant vectors in which a gene coding for a polypeptide defined by the amino acid sequence of formula (I) is effectively connected to and placed under the control of regulatory DNA sequences capable of causing expression of the polypeptide. When bacterial cells, which have no PI anchor synthesizing mechanism, are used as the host for expression of the above-mentioned expression vectors, the expression product is always a polypeptide having no PI anchor. When mammalian cells are used as the host, the PI anchor addition is controlled in a complicated manner. It is generally known that when PI anchor attachment to the C terminus of a polypeptide takes place, elimination of a specific hydrophobic C-terminal polypeptide portion (PI-anchor attachment signal sequence) from a precursor for the polypeptide precedes modification with the PI anchor. In the case of human MACIF shown in Fig. 2, the polypeptide from the 77th amino acid residue (Asn) to the 103rd amino acid residue (Pro) acts as a signal for PI anchor attachment. However, the respective protein genes inserted in the expression vectors for those proteins of the present invention composed of amino acid residues No.1 to No.70,77 and 86 are lacking in the portion of DNA which codes for a part or the whole of the PI anchor attachment signal sequence. It is known that deletion of a gene fragment coding for a part of the hydrophobic attachment signal sequence from a gene coding for a protein precursor to which a PI anchor is to be added results in production of a soluble protein [Berger, J. et al., Journal of Biological Chemistry , 263 , 10016-10021 (1988)]. Therefore, the genes of the present invention can possibly be used as genes for producing soluble human MACIF proteins. Among the vectors for the expression of these proteins, a vector for the expression in Escherichia coli and in mammalian cells of MACIF proteins comprising those amino acid residues up to the 86th, 77th and 70th, respectively, are described in more detail in the following together with methods of their construction and expression. (a) Expression of a protein comprising the 1st to 86th amino acid residues: When Escherichia coli is used as the host, plasmid pGEM352-3 is cleaved with Ava II. Separately, a synthetic DNA of the formula (III): is phosphorylated at the AvaII cleavage site. The cleavage product and phosphorylated synthetic DNA are ligated together using T4 DNA ligase. The ligation product is then cleaved with Pst I and ligated with the synthetic DNA of formula (II): phosphorylated at the Pst I cleavage site, using T4 DNA ligase. The thus-produced ligation product (now EcoRI/Hind III fragment) from the synthetic DNA and DNA fragment is isolated and purified by agarose gel electrophoresis. The thus-prepared Eco RI/ Hind III DNA fragment is ligated with an Eco RI/ Hind III main body fragment of pKK223-3 shown in Fig. 3. A recombinant Escherichia coli strain, which allows expression of a polypeptide comprising the 1st to 86th amino acid residues, can be obtained using this ligation product. Plasmids can then be isolated from transformants by the alkaline method and analyzed using restriction enzymes. In this way, a transformant harboring the desired plasmid with the Eco RI/ Hind III DNA fragment inserted therein can be selected. For polypeptide expression in CHO cells, pGEM352-3 is cleaved with Ava II and then ligated with the synthetic DNA of formula (III), as in the case of expression in Escherichia coli cells. The ligation product is then cleaved with Sac I and a DNA fragment of about 375 base pairs is isolated and purified by agarose gel electrophoresis. This DNA fragment is rendered blunt-ended by treatment with mung bean nuclease. The resulting DNA fragment is inserted into pVY1 cleaved with Bgl II and rendered blunt-ended to give an expression plasmid. Transformation of CHO cells with the expression plasmid gives CHO cells capable of expressing and producing a polypeptide comprising the 1st to the 86th amino acid residues. (b) Expression of proteins comprising the 1st to 77th amino acid residues and the 1st to 70th amino acid residues, respectively: For causing expression of the gene coding for the 1st to 77th amino acid residues or the 1st to 70th amino acid residues in Escherichia coli or CHO cells, essentially the same procedure as mentioned above can be followed except that, in the plasmid preparation step, the synthetic DNA to be joined to pGEM352-3 after cleavage of the latter with Ava II is a synthetic DNA of the formula (IV): when the polypeptide comprising the 1st to the 77th amino acid residues is to be expressed, or a synthetic DNA of the formula (V): when the polypeptide comprising the 1st to the 70th amino acid residues is to be expressed. The subsequent steps, the corresponding genes and recombinant vectors for the expression of the genes can be obtained by proceeding in the same manner as described above in (a). Transformation Introduction of the thus-obtained expression vectors containing the human MACIF protein cDNA into desired host cells, namely transformation of the cells with the vectors, can be effected using these techniques that are used generally. Each expression vector plasmid can be prepared from the host used for gene construction (e.g. E . coli HB101) by a method in general use, for example, alkaline bacteriolysis. The vector plasmid prepared is used to transform the host. The transformation can be effected by the method of Hanahan [Hanahan, D., Journal of Molecular Biology , 166 , 557-580 (1983)], for instance, when bacterial cells are used as the host, or by the calcium phosphate method [van der Eb, A. J. et al., Methods in Enzymology , 65 , 826-839 (1980), Academic Press], for instance, when mammalian cells are used as the host. Cultivation and purification The transformant obtained in the above manner can be grown in the conventional manner and cultivation thereof results in production and accumulation of biologically active human MACIF proteins. The medium for the cultivation can suitably be selected from among various conventional media depending on the host cells employed. For instance, when the above-mentioned CHO cells are used as the host, MEM-α medium if necessary supplemented with a blood component such as fetal calf serum (FCS), may be used. The site of expression for the production of the recombinant human MACIF proteins in the transformant differs depending on the amino acid sequence encoded by the cDNA selected, the kind of vectors, that of the host cells and the combination of these. Thus, the recombinant human MACIF can be produced on the cell membrane, within the cell or in the cell culture supernatant. The human MACIF proteins produced in transformed cells can be isolated and purified therefrom by various separation techniques [e.g., Japanese Biochemical Society (ed.), Biochemical Data Book II, 1st edition, 1st printing, page 1175, Tokyo Kagaku Dojin (1980)] based on the physical and chemical properties thereof. To be concrete, the techniques include, among others, treatment with an ordinary protein-precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), other liquid chromatographic techniques, dialysis, and combinations of these. To be more concrete, the technique to be used may vary depending on the site of expression of the recombinant protein. Recombinant proteins produced in the culture supernatant can be isolated and purified in the following manner. First, the desired substance is partially purified from the culture supernatant in advance. This partial purification is realized, for example, by treatment with an agent for salting out, such as ammonium sulfate, sodium sulfate or sodium phosphate, and/or ultrafiltration treatment using a dialyzing membrane, flat membrane or hollow fiber membrane. The procedure and conditions of each of these treatments may be the same as those generally employed in the art. The roughly purified product obtained in the above manner is subjected to adsorption chromatography, affinity chromatography, gel filtration, ion exchange chromatography, reversed-phase chromatography or the like or a combination of these, and a fraction showing human MACIF activity is recovered. In this manner, the desired substance can be isolated in a pure and homogeneous form. A recombinant protein produced on the cell membrane may have a PI anchor and, therefore, may be bound to the cell membrane via the anchor. Such membrane-bound recombinant protein can be purified by disrupting the cell membrane by treatment with an appropriate detergent (e.g. NP-40, Triton X-100, octylglycoside) and then proceeding in the same manner as described above. Alternatively, a recombinant protein bound to the cell membrane via the PI anchor can be solubilized by an appropriate treatment for PI anchor cleavage. As the means of PI anchor cleavage, there may be mentioned, for example, cleavage with phosphatidylinositol-specific phospholipase C (PI-PLC) and cleavage with phosphatidylinositol-specific phospholipase D (PI-PLD). The recombinant protein solubilized by the above treatment is released into the cell culture supernatant and therefore can be purified in the same manner as described above. A recombinant protein produced in the cell can be purified by disrupting the cell membrane by treatment with an appropriate detergent, as in the case of membrane-bound recombinant proteins, to thereby cause release of the recombinant protein into the solution phase and then proceeding in the same manner as mentioned above. The activity of the thus-purified recombinant human MACIF, or soluble recombinant human MACIF can be identified, for example, by measuring of reactive lysis [Thompson, R.A. et al., Journal of Experimental Medicine , 131 , 629-641 (1970) and Lachmann, P.J. et al., Journal of Experimental Medicine , 131 , 643-657 (1970)] inhibiting activity assay. In the foregoing, the genes according to the present invention, the vectors for the expression of the genes and the transformed microorganisms and cells capable of allowing the expression of the genes as well as the methods for their preparation have been described. As a result of the present invention, genes coding for human MACIF proteins practically useful as drugs have been provided. Thus, it is now possible to produce a pure and homogeneous grade of human MACIF protein in large quantities using recombinant DNA technology. Human MACIF proteins are of the human origin. Therefore, they have no antigenicity and their toxicity is low. They can prevent cells and tissues from being damaged as a result of MAC formation in the last stage of complement activation. Thus, they can be used advantageously as therapeutic agents for various diseases, in particular diseases resulting from the absence or reduced level of a complement-regulating component(s) and all diseases classifiable under the category of type II or type III allergy. Furthermore, not only in disease due to the absence or reduced level of one or more complement-regulating components, typically in paroxysmal nocturnal hemoglobinuria (PNH), but also in various inflammatory or autoimmune diseases accompanied by cell and/or tissue damage, a qualitative or quantitative abnormality in human MACIF may possibly be observed. Therefore, human MACIF, monoclonal or polyclonal antibodies specific to human MACIF, human MACIF protein DNAs and, further, DNAs complementary to the human MACIF protein genes can be used in specific diagnosis of the above-mentioned diseases. The dosage form of human MACIF proteins may vary depending on the diseases, symptoms and patient's conditions. Generally, however, non-oral dosage forms, such as injections, nasal preparations, suppositories and implants, are used for systemic administration, while intraarticular preparations and preparations to be implanted into affected sites, for instance, are used for local administration. In preparing these dosage forms, compositions suited for the respective forms are used. In preparing injections, human MACIF protein is dissolved in phosphate-buffered physiological saline or an aqueous dextrose solution, for instance, and, after addition of a stabilizer, a dispersant and/or the like, as necessary, distributed into ampules or lyophilized in vials. In the latter case, the preparation is reconstituted prior to use by dissolving in distilled water for injection or physiological saline. The daily dose of MACIF for human adults is generally within the range of 100 µg to 5,000 mg, preferably within the range of 1 mg to 500 mg. Such daily dose is administered in a single dose or in divided doses. Dosage form preparation example A solution of 5 g of MACIF in 100 ml of physiological saline is subjected to aseptic filtration and then distributed in 2-ml portions into vials. Lyophilization gives a preparation for injection, each vial containing 100 mg of MACIF. REFERENCE EXAMPLES (1) Determination of N-terminal and C-terminal amino acid sequences of a purified sample of naturally occurring human MACIF: 1) N-terminal amino acid sequence determination A purified sample of naturally occurring human MACIF (18 kilodaltons) was reduced in the conventional manner with 2-mercaptoethanol in the presence of 8 M urea, then S-carboxymethylated with iodoacetic acid, and analyzed for its N-terminal amino acid sequence using a gas phase protein sequencer (model 470A, Applied Biosystems, U.S.A.). The result was as follows: 2) C-terminal amino acid sequence determination: A 0.6 mg portion of the purified sample of naturally occurring human MACIF was reduced in the conventional manner with dithiothreitol in the presence of 6 M guanidine hydrochloride and S-carboxymethylated with iodoacetic acid. The alkylation mixture was dialyzed against distilled water overnight at 4°C and the dialyzate was concentrated to 0.5 ml using a centrifuge-type reduced-pressure concentrator. The whole carboxymethylated human MACIF solution was buffered by addition of 1 M Tris hydrochloride buffer (pH 8.0). Then, a solution of 100 µg of Pronase (Calbiochem) in 20 µl of 50 mM Tris hydrochloride buffer was added to the solution, and the reaction was allowed to proceed at 37°C for 22 hours. A chloroform-methanol (1:1) mixture (0.5 ml) was added to the reaction mixture. After thorough shaking, the whole mixture was centrifuged, whereupon it separated into a transparent upper layer, a cloudy middle layer and a transparent lower layer. A portion of each layer was taken, hydrolyzed with 6 N hydrochloric acid and analyzed in the conventional manner using a Picotag work station equipment (Waters, U.S.A.). Ethanolamine was detected in the middle layer. It was thus revealed that the middle layer contained a Pronase digestion fragment with a PI anchor attached at the C terminus. The middle layer (600 µl) containing the above PI anchor-attached C-terminal fragment was lyophilized, 1.2 ml of a water:n-butanol:l N hydrochloric acid (600:600:3) mixture was added and, after thorough shaking, the mixture was centrifuged. Hydrolysis was performed with 6 N hydrochloric acid in the same manner as described above and analyzed using a Picotag work station equipment. Ethanolamine was detected in the butanol layer (upper layer). An equal volume of n-butanol-saturated 5 mM hydrochloric acid was added to the upper layer, the mixture was shaken and then centrifuged and the upper layer was separated. This extraction procedure with acidic butanol was repeated twice in all. Half of the final n-butanol layer was subjected to amino acid sequence determination using the protein sequencer mentioned above. As a result, the following sequence was found: this covers from the 72nd to the 76th amino acid residue in the sequence shown in Fig. 2. No amino acid residue was detected behind the 76th amino acid residue (Glu). The above facts indicated that erythrocyte-derived, naturally occurring human MACIF has the PI anchor attached to the Glu which is the 76th amino acid residue from the N terminus. (2) Preparation of oligonucleotide probes for detecting cDNA clones coding for human MACIF: Based on the amino acid sequence revealed in the above section (1), 15-mer and 17-mer mixed deoxyoligonucleotide probes complementary to the mRNA regions coding for amino acid residues Nos. +1 to +5 and +4 to +9, respectively, were chemically synthesized by the phosphoamidite method using a model 380A DNA synthesizer (Applied Biosystems) and labeled with ³²P at the 5' end. The 15-mer mixed probe (hereinafter referred to as "M1 probe") had the nucleotide sequences: and while the 17-mer mixed probe (hereinafter referred to as "M4 probe") had the nucleotide sequences (3) Preparation of a human monocyte cDNA library: Recombinant plasmids were constructed by the Okayama-Berg method (vide supra) starting with mRNA derived from immune complex-stimulated human peripheral blood monocytes and the commercially available plasmid vector pGEM4 (Promega). The recombinant plasmids thus constructed had cDNA inserted between the Kpn I cleavage site and Sac I cleavage site of the multicloning sites occurring between the SP6 promoter region and T7 promoter region of pGEM4. The directionality of cDNA was such that the Kpn I side (T7 promoter side) was on the 3' side of mRNA and the Sac I side (SP6 promoter side) was on the 5' side of mRNA. The recombinant plasmid mixture thus obtained was used to transform E. coli HB101 competent cells (Takara Shuzo) and a cDNA library comprising about 400,000 transformants was obtained. (4) Cloning of human MACIF cDNA: For isolating a transformant harboring a plasmid containing cDNA coding for human MACIF, the human monocyte cDNA library obtained as described in section (3) was subjected to colony hybridization using the synthetic oligonucleotide probes prepared as described in section (2) to detect clones hybridizing with both of M1 and M4 probes. A clone containing the longest cDNA (about 2,000 bp) was selected and, for the plasmid pGEM 352-3 isolated therefrom, a partial base sequence of the cDNA portion was determined by the dideoxy method (vide supra) using the plasmid vector and by the 7-DEAZA method (vide supra). The thus-determined cDNA sequence (about 500 bp) from the SP 6 promoter side (5' side of mRNA) of pGEM4 is shown in Fig. 1. In the cDNA sequence of the pGEM352-3 clone, there is a sequence coding for the N-terminal ten amino acid residues of the purified human MACIF described in section (1), with the translation initiation codon ATG at a site corresponding to amino acid residue No. -25 upstream from the N terminus and the translation termination codon TAA at a site corresponding to the +104th amino acid residue downstream from the N terminus. The open reading frame thus formed codes for a protein comprising 128 amino acid residues. It is strongly suggested that the amino acid sequence corresponding to the region of -25 to -1 should be the so-called secretion signal sequence very rich in hydrophobicity. EXAMPLES (1) Expression in Escherichia coli of a gene coding for a human MACIF protein 1) Construction of a human MACIF protein expression vector and expression in Escherichia coli 1-1) Preparation of a vector for expression in Escherichia coli The vector pYEJ001 (Pharmacia; Fig. 5) for expression in Escherichia coli was cleaved with the restriction enzyme Hind III, and the vector main body containing the origin of replication and the promoter for expression of the gene in question was isolated and purified by agarose gel electrophoresis and then treated with alkaline phosphatase for terminal phosphate group elimination. The DNA fragment to be inserted into the vector, inclusive of the SD sequence, human MACIF protein gene and transcription terminator, was prepared in the following manner. 1-2) Preparation of an insert DNA fragment for the expression of a modified human MACIF protein (E86) comprising the peptide sequence up to the 86th amino acid residue pGEM352-3 was cleaved with the restriction enzymes Pst I, Hinc II and Bam HI and a DNA fragment of about 310 base pairs was isolated and purified by agarose gel electrophoresis. This 310 base pair DNA fragment was cleaved with the restriction enzyme Mbo II and a DNA fragment of about 250 base pairs was isolated and purified by agarose gel electrophoresis. Separately, two (B and C) of the four synthetic DNAs of the formulas (VII): were terminally phosphorylated using polynucleotide kinase. A and the phosphorylated B were heated to 90°C and then gradually cooled to 16°C for effecting annealing. The phosphorylated C and unphosphorylated D were treated in the same manner for annealing. The two annealing product DNAs and the previously prepared DNA fragment of about 250 base pairs were ligated together using T4 DNA ligase and a DNA fragment of about 325 base pairs containing the region coding for the modified MACIF protein E86 was isolated and purified by agarose gel electrophoresis. 1-3) Preparation of a vector for the expression of the human MACIF protein in Escherichia coli and transformation therewith: The thus-prepared DNA fragment was terminally phosphorylated using polynucleotide kinase and then ligated with the separately prepared vector main body using T4 DNA ligase. The ligation mixture was used to transform E . coli K12JM109. Plasmids were prepared from the transformants obtained by the alkaline method (vide supra) and analyzed using restriction enzymes (e.g. by cleavage with Pst I), and a plasmid with the gene in question inserted therein in the direction enabling the gene to be expressed was selected out. 1-4) Expression and cultivation: One volume of 2×YT medium (16 g of Bactotryptone, 10 g of yeast extract and 5 g of sodium chloride per liter) supplemented with 100 µg/ml of ampicillin was inoculated with 1/50 volume of a preculture of a recombinant E . coli strain allowing expression of the gene in question. Then, culture was shaken at 37°C and the recombinant E . coli strain was grown until cell density had reached about 5x10⁷/ml, and then isopropyl thiogalactopyranoside was added in a concentration of 2.5 mM. After further 16 hours of cultivation, cells were harvested. The cells collected were suspended in PBS containing 2 mM EDTA, 2 mM EGTA, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride (PMSF) (all purchased from Sigma) and disrupted using a Manton-Gaulin homogenizer. 2) Purification of the protein resulting from the expression of the human MACIF protein gene in Escherichia coli and biological activity assay: 2-1) Purification: The Escherichia coli cell disruption product prepared as described above was centrifuged (12,000 rpm, 30 minutes) and the sediment was stirred overnight at 4°C in the presence of 6 M guanidine hydrochloride for solubilization. The remaining insoluble matter was removed by centrifugation under the same conditions as mentioned above. The expression product protein (E86) contained in the supernatant was reconstituted by treatment in the presence of oxidized-form and reduced-form glutathione (Sigma) according to the method of Winkler and Blaber [Winkler, M.E. and Blaber, M., Biochemistry , 256 , 4041-4045 (1986)]. The reconstitution mixture was thoroughly dialyzed against 10 mM Tris buffer (pH 8.0) and then purified by application to Q-Sepharose (Pharmacia) preequilibrated with the same buffer. 2-2) Activity of the modified human MACIF protein (E86): The activity data obtained failed to show a main peak of activity neutralizable by the antibody (Fig. 6). However, based on the results of SDS-PAGE analysis as shown in Fig. 7, the main band-containing fractions (Nos. 26-30) were purified on an anti-human MACIF antibody column, followed by reactive lysis inhibiting activity measurement. As shown in Fig. 8, the purified expression product protein (E86) showed antihemolytic activity in a dose-dependent manner. Furthermore, the inhibitory activity was completely neutralized by the antibody. The above results indicate that the human MACIF protein expressed in Escherichia coli has the same activity as that of human erythrocyte-derived MACIF and that human MACIF can exhibit reactive lysis inhibiting activity even when it has no carbohydrate chain or no PI anchor. (2) Expression of the gene coding for a human MACIF protein in CHO cells and confirmation of its biological activity: 1) Expression of a human MACIF protein (C86) in CHO cells: 1-1) Construction of a recombinant plasmid for expression in CHO cells: The plasmid pVY1 was used as the vector for expression in CHO cells; and was constructed as follows, as illustrated in Fig. 4a. a) The DNA of the vector pAdD26SV(A) No. 3 [obtained from Dr. H. Handa; made known by the paper: Kaufman, R.J. and Sharp, P.A., Molecular and Cellular Biology , 2 , 1304-1319 (1982)] was first cleaved with Bgl II, followed by phenol-chloroform extraction and ethanol precipitation. The precipitate thus obtained was dissolved in sterile distilled water, rendered blunt-ended in the conventional manner using Klenow enzyme (Boehringer Mannheim), then subjected to phenol-chloroform extraction and ethanol precipitation, and dissolved in sterile distilled water. It was further self-ligated using a DNA ligation kit (Takara Shuzo). The ligation mixture was used to transform competent cells of E . coli HB101. Plasmid DNAs were obtained from tetracycline-resistant transformants. A portion of each of these DNAs was treated with Bgl II and electrophoresed on a 0.7% agarose gel. In this way, a clone having no Bgl II site any more, pAdD26SV(A) No. 3(N), was obtained. The plasmid DNA was then digested with EcoRI, followed by phenol-chloroform extraction and ethanol precipitation. The precipitate was dissolved in sterile distilled water, and the EcoRI cleavage site was rendered blunt-ended using mung bean nuclease (Pharmacia), followed by phenol-chloroform extraction and ethanol precipitation. The precipitate thus obtained was dissolved in sterile distilled water. b) The pKSV10 (Pharmacia) DNA was cleaved in the conventional manner with the restriction enzymes KpnI and Bam HI and then rendered blunt-ended using T4 DNA polymerase (Takara Shuzo) and Klenow enzyme. After electrophoresis on a 0.7% agarose gel, the gel portion containing a fragment about 2.9 kbp in size was separated and the DNA was recovered by electroelution. c) The DNA fragment obtained as described in the above 1) and the DNA fragment obtained as described in the above G) were ligated together using a DNA ligation kit and the ligation mixture was used to transform competent cells of E . coli HB101. Plasmid DNAs were prepared from tetracycline-resistant transformants by a conventional method. A part of each plasmid DNA was digested with Pst I and the digest was subjected to 1.0% agarose gel electrophoresis. In this manner, the plasmid pVY1 giving bands at about 3.6 kbp, about 3.25 kbp and about 1.5 kbp was obtained. Thus, pVY1 was cleaved with Bgl II and submitted to the following recombinant plasmid construction. pGEM352-3 was cleaved with Eco 0109I and Sty I and ligated with a synthetic DNA of the formula (VIII) phosphorylated at the Sty I cleavage site and a synthetic DNA of the formula (IX) phosphorylated at the Eco 0109I cleavage site, using T4 DNA ligase. A DNA fragment of 341 base pairs was then isolated and purified by agarose gel electrophoresis. This DNA fragment was ligated with the BglII-cleaved pVY1 using T4 DNA ligase and the ligation mixture was used to transform E . coli HB101 to give tetracycline-resistant transformants. Plasmids were prepared therefrom by the alkaline bacteriolytic method and analyzed using restriction enzymes (PstI etc.) and a recombinant expression plasmid for the gene in question was selected out. 1-2) Confirmation of gene expression in CHO cells: DHFR-deficient CHO cells (vide supra) were transfected with the expression plasmid constructed in the above manner by the calcium phosphate method. A transformant strain capable of growing in a selective medium [MEM alpha (-), Gibco] in the presence of methotrexate was obtained and used in the subsequent studies. 10⁸ CHO cells transformed in the above manner were cultured and the culture supernatant was subjected to affinity chromatography using an anti-MACIF monoclonal antibody column prepared by binding a purified mouse monoclonal anti-human MACIF antibody to activated Sepharose 4B (Pharmacia, Sweden). The column was then washed thoroughly with phosphate-buffered saline (PBS) containing 0.1% NP40 and with 2 M aqueous solution of sodium chloride containing 0.1% NP40. Fractions bound to this antibody column were eluted with 3 M aqueous solution of sodium thiocyanate. Each eluate fraction was desalted on a PD-10 column (Pharmacia) and evaluated for the presence or absence of antigen by competitive ELISA using a rabbit anti-naturally occurring human MACIF antibody. As a result, it was confirmed that a human MACIF antigen, was present in a "bound" fraction in the above affinity chromatography. This result proves that a human MACIF protein comprising the peptide composed of the 1st to 86th amino acid residues had been successfully expressed in CHO cells in accordance with the invention. 2) Expression of a modified human MACIF protein (C77) in CHO cells and confirmation of its biological activity: 2-1) Construction of a recombinant plasmid for gene expression in CHO cells: The plasmid pVY1 obtained as described in a),b), and c) above used as the vector for gene expression in CHO cells. pVY1 was cleaved with Bgl II and submitted to the following recombinant plasmid construction. pGEM352-3 was cleaved with Eco0109I and Sty I and ligated with a synthetic DNA fragment of the formula (VIII) phosphorylated at the Sty I cleavage site and a synthetic DNA fragment of the formula (XI) phosphorylated at the Eco 0109I cleavage site, using T4 DNA ligase. A DNA fragment of 314 base pairs was then isolated and purified by agarose gel electrophoresis. This DNA fragment was ligated with the Bgl II-cleaved pVY1 using T4 DNA ligase and the ligation mixture was used to transform E . coli HB101 to give tetracycline-resistant transformants. Plasmids were prepared from these by the alkaline bacteriolytic method and analyzed by using restriction enzymes ( Pst I etc.) and a recombinant plasmid capable of expression of the gene in question was selected out. 2-2) Confirmation of gene expression in CHO cells: DHFR-deficient CHO cells (vide supra) were transfected with the expression plasmid constructed in the above manner by the calcium phosphate method and a transformant capable of growing in a selective medium [MEM alpha (-) Gibco] in the presence of methotrexate was isolated and used for further studies. 10⁸ CHO cells transformed by the above method were cultured and the culture supernatant was subjected to affinity chromatography using an anti-MACIF monoclonal antibody column and proceeding as described in (2)-1-2) above. Fractions bound to this antibody column were eluted with 3 M aqueous solution of sodium thiocyanate and eluate fractions were desalted on a PD-10 column (Pharmacia) and assayed for the presence or absence of antigen by competitive ELISA using a rabbit anti-MACIF antibody. The presence of a MACIF antigen was confirmed in a "bound" fraction in the above affinity chromatography. This result indicates that a human MACIF protein comprising the peptide composed of the 1st to 77th amino acid residues had been expressed successfully in CHO cells. 2-3) Confirmation of biological activity: A sample of the human MACIF protein C77 as partially purified from the culture supernatant resulting from mass culture of the transformed CHO cells by the purification procedure described in the above (2)-2-2) was subjected to buffer exchange for SGVB²⁺, followed by MAC formation inhibiting activity assay, which was performed as follows: Human C5-C6 complex (stored frozen and thawed before use; C 5,6 f) was prepared from human C5 and C6 by the method of Dessauer et al. [Dessauer, A. et al., Acta Pathologica Microbiologica Scandinavia , Section C, Supplement 284, 92 , 75-81 (1984)] and admixed with a guinea pig erythrocyte suspension (10⁷ cells/ml), and the mixture was incubated at 33°C for 5 minutes. Then, after addition of C7, incubation was performed further for 15 minutes to give a guinea pig erythrocyte-human complement C5-7 complex (hereinafter referred to as "EC5-7 intermediate"). The EC5-7 intermediate suspension (1.5x10⁸/ml) was admixed with C8, C9 and a sample solution to make the total volume 1 ml (1.5x10⁷/ml). Incubation was carried out at 37°C for 1 hour. In parallel, a control suspension prepared in the same manner but without addition of any sample solution was incubated simultaneously. The reaction mixture was centrifuged at 2,000 x g for 5 minutes, the supernatant was measured for absorbance (at 414 nm) for hemolysis percentage calculation. Thus, the number of sites (Z) per erythrocyte was calculated by the method of Hammer et al. [Hammer, C.H. et al., Journal of Biological Chemistry , 256 , 3995-4005 (1981)] and the ratio in percentage of the Z value to the Z value for the control was taken as an index of activity. As shown in Fig. 9, the human MACIF protein C77 expressed in CHO cells showed MAC formation inhibiting activity in a dose-dependent manner. The inhibitory activity was neutralized by a mouse monoclonal antibody to erythrocyte-derived, naturally occurring human MACIF. From the above results, it is evident that the human MACIF protein C77 expressed in CHO cells and secreted into medium has the same activity as that of human erythrocyte-derived, naturally occurring MACIF. 3) Expression of a human MACIF protein (C70) in CHO cells and confirmation of its biological activity: 3-1) Construction of a recombinant plasmid for gene expression in CHO cells: The procedure was the same as that described in (2)-1-1) except that a DNA fragment of the formula (XIV) phosphorylated at the Eco 0109I site was used in expression plasmid construction. 3-2) Confirmation of gene expression in CHO cells: A human MACIF antigen was detected, by the procedure of (2)-1-2), in the culture supernatant obtained with cells transformed with the above recombinant vector. This result indicates that a human MACIF protein comprising the peptide composed of the 1st to 70th amino acid residues had been expressed successfully in CHO cells. 3-3) Confirmation of biological activity: The MAC formation inhibiting activity of the human MACIF protein (C70) expressed in CHO cells was measured using the culture supernatant obtained by mass culture of the transformed CHO cells and following the procedure of (2)-2-3). As shown in Fig. 10, C70 showed the activity in a dose-dependent manner. From the results mentioned above, it is evident that the human MACIF protein (C70) expressed in CHO cells and secreted into medium has the same activity as that of human erythrocyte-derived, naturally occurring MACIF.

Claims: Claims for the following Contracting States : AT, BE, CH, LI, DE, DK, FR, GB, IT, LU, NL, SE A gene coding for a peptide having the following amino acid sequence: wherein X is H, Met, or the following amino acid sequence: and Y is OH, or A recombinant expression vector containing a gene according to claim 1 wherein said gene is efficiently bound to a regulatory gene DNA capable of allowing expression of said polypeptide. A recombinant expression vector according to claim 2 which can be replicated and maintained in host cells in which the phosphatidylinositol anchor (PI anchor) can be attached to the C terminus of polypeptides. A cell transformed with recombinant expression vector according to claim 2 or 3. A peptide defined by the following amino acid sequence: wherein X is H or Met; and Y is OH, or A peptide defined by the following amino acid sequence: wherein X is H or Met. Claims for the following Contracting States : ES, GR A method of preparing a gene coding for a peptide having the following amino acid sequence: wherein X is H, Met, or the following amino acid sequence: and Y is OH, or which includes isolating a clone containing cDNA coding for said peptide from a cDNA library prepared from mRNA obtained from said peptide producing cells, and isolating the cDNA from the thus isolated clone. A method of preparing a gene coding for a peptide having the amino acid sequence defined in claim 1 which includes chemical synthesis of nucleic acids. A method according to claim 1 and 2. A method of preparing a recombinant expression vector containing a gene coding for a peptide having the amino acid sequence defined in claim 1 which comprises inserting said gene into a suitable expression vector, wherein said gene is efficiently bound to a regulatory gene capable of allowing expression of said polypeptide. A method according to claim 4 wherein the recombinant expression vector can be replicated and maintained in host cells in which the phosphatidylinositol anchor (PI anchor) can be attached to the C terminus of polypeptides. A method according to claim 4 or 5 which further includes transforming a cell with said recombinant expression vector. A method of preparing a peptide defined by the following amino acid sequence: wherein X is H or Met; and Y is OH, or which comprises expressing a gene coding for said peptide in a suitable host. A method of producing a peptide defined by the following amino acid sequence: wherein X is H or Met, which comprises expressing a gene coding for said peptide in a suitable host.

Inventors: FURUICHI KIYOSHI, ITO KATSUHISA, SUGITA YUJI, TAKAYAMA MAKOTO, TAKEMOTO TOSHIYUKI, TOMITA MOTOWO, YAMAJI NOBORU, YANO SHINYA, YUSAKAWA KO, FURUICHI, KIYOSHI, ITO, KATSUHISA, SUGITA, YUJI, TAKAYAMA, MAKOTO, TAKEMOTO, TOSHIYUKI, TOMITA, MOTOWO, YAMAJI, NOBORU, YANO, SHINYA, YUSAKAWA, KO
Applicants: YAMANOUCHI PHARMA CO LTD, YAMANOUCHI PHARMACEUTICAL CO. LTD.
Application year: 1990
Main classification: C12N15
IPC codes: C07K4, C12N15, C12N1, C07K14, C12R1, C12P21, A61K38, C12N5