JP2847753B2 - Novel proteins and genes encoding them - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel protein and a gene encoding the same.

More specifically, the present invention relates to a protein having an action of suppressing cell membrane damage caused by complement and a gene (cDNA) encoding the same.

(Problems to be Solved by the Prior Art and the Invention) Complement is a reaction system containing a group of proteases present in blood and body fluid. When the foreign body invades the living body, the complement system is activated, and some of the complement components deposited in response to the foreign body promote poor phagocytosis by poor phagocytes or cause dissolution of the foreign body, and actively remove foreign bodies. work. In addition, various peptide fragments of complement components generated in the process of complement activation are:
It acts on various cells such as activating lymphocytes, migrating neutrophils, and promoting mast cell degranulation, and is involved in various immune and inflammatory reactions.

Although complement responds rapidly to foreign bodies, complement activation does not proceed on its own normal cells. However, for example, at the inflammation site of a disease such as chronic joint, rheumatism, systemic lupus erythematosus, glomerulonephritis, etc., complement recognizes its own tissue as a foreign body, and reacts with the cell membrane of the tissue to induce an inflammatory response. At the same time, it is thought that they promote phagocytosis by poor phagocytes or cause cell damage such as dissolution of the cell membrane to exacerbate the inflammatory response.

In recent years, the existence of a protein that suppresses the activation of autologous complement on the autologous cell membrane has been revealed. For example, Decay accelerating factor (DAF) having a molecular weight of 68 Kd [J. Exp. Me
d., 160 , 1555-1578 (1984)] and Homologou with a molecular weight of 65 Kd.
s restriction factor (HRF) [PNAS, 83 , 6975-6979 (1
986)] has been obtained as a complement inhibitory factor present on human cell membranes. However, the functions of these DAFs and HRFs in the living body have not yet been elucidated.

The present inventors have studied to obtain a membrane protein which has a function of suppressing complement activation on an autologous cell membrane in addition to these factors. First, the present inventors immunized mice with human erythrocytes, took out the spleen of the mouse, and fused the spleen cells with mouse myeloma cells (P3U1) using polyethylene glycol to obtain a large number of antibody-producing hybridomas. ,
From among the antibodies (culture supernatants) produced by these hybridomas, cells that produce antibodies that react with human erythrocytes that have been treated with neuraminidase in advance and also cause hemolysis by human complement are repeatedly screened. An antibody-producing hybridoma was obtained, and a monoclonal antibody 1F5 antibody was obtained [Article of the General Meeting of the Immunological Society of Japan, 17 , 498 (1987), Abstracts of the Complement Symposium, 24 , 180]
(1987)].

Further, some of the present inventors prepared 1F5 antibody affinity sepharose using this 1F5 antibody, and purified 1F5 antigen from human erythrocyte membrane (Japanese Patent Application No. 63-172187).
No.). The resulting antigen was a glycoprotein containing phosphatidylinositol and having a molecular weight of 20 to 25 Kd, and had an activity to suppress complement-induced cell damage. The purified protein had the following partial amino acid sequence.

However, for example, when examining the detailed function of 1F5 antigen in a living body or the effect on inflammation caused by complement activation, a large amount of 1F5 antigen protein is required, but a large amount of antigen is required. It is not always industrially easy to prepare proteins from erythrocyte membranes and the like.

(Means for Solving the Problems) Accordingly, the present inventors have intensively studied to produce the antigen protein in large quantities by recombinant DNA technology, and for the first time, developed a gene encoding a 1F5 antigen protein useful for this purpose. As a result of separation and acquisition, the present invention was completed.

That is, the gist of the present invention resides in a 1F5 antigen protein which suppresses cell membrane damage caused by complement and has an epitope reactive with a 1F5 antibody, and a gene encoding the protein.

Hereinafter, the present invention will be described. As shown in FIG. 1, the 1F5 replacement protein of the present invention is a protein consisting of 128 amino acids, and has a function of suppressing complement-induced cell membrane damage. Of course, in the present invention, antigen proteins of the present invention include those obtained by modifying such as removing, modifying or adding some amino acids within a range that does not impair the function of suppressing cell membrane damage caused by complement. .

Examples of the gene encoding the 1F5 antigen include, for example,
Those having a base sequence as shown in FIG. 2 are exemplified. still,
In the figure, other complementary base sequences are omitted and are represented by "----", and only a single strand is described.

A DNA fragment of a gene encoding a protein having a function of suppressing cell membrane damage caused by complement of the present invention can be obtained, for example, by the following method.

As a cDNA library for screening a gene encoding the 1F5 antigen protein, a human-derived peripheral blood lymphocyte cDNA library, a T cell cDNA library, K562
Cell cDNA library, mononuclear cDNA library, placenta
A cDNA library or the like can be used, and a placental cDNA library is particularly preferred. These libraries are sold by Clonetech. In addition, a cDNA library may be prepared according to a conventional method if it is a cell line expressing the 1F5 antigen. In this case, in particular, MT-2 cDNA library, MOL
T2 cDNA libraries are preferred.

From such a cDNA library, λ phage was prepared by the method of Tomizawa et al. [(Experimental method for bacteriophage], Iwanami Shoten, 9
9-174, (1970)].
The formed plaque is selected by a plaque hybridization method ("Molecular Cloning", Cold Spring Harbor Laboratory, 320-328, (1982)) using an oligonucleotide having a nucleotide sequence deduced from the amino acid sequence of the 1F5 antigen protein as a probe. Thus, the desired cDNA can be easily obtained.

As a probe used in the plaque hybridization method, a partial cDNA fragment of a gene encoding a 1F5 antigen obtained by a polymerase chain reaction (hereinafter abbreviated as PCR) method is used [Scienc
e), 239 , 487–491, (1988)]. For example, corresponding to 27-32 of the amino acid sequence of Figure 1 + strand DNA primer ^{5 'CA}
A / G TG C / T TAC C / T AAC / T TG C / T CC ³ '(17 nucleotides) and a minus strand primer ⁵ ' CA A / G TG C / T TC corresponding to the amino acid sequence 65 to 70 PCR was performed using A / G AAC / T TTCCA ³ '(17 nucleotides) to produce the 131 orthonucleotide fragment shown in FIG. 3 corresponding to the amino acid sequence 27 to 70 in FIG. 1 and used as a probe. Used. Also 1F5
A synthetic oligonucleotide may be prepared based on the DNA base sequence deduced from the amino acid sequence of the antigen and used as a probe.

Further, phages were grown from the plaques positive for screening by the method of Tomizawa et al.
DNA was purified by the method of atis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, 85, (1982)), cut with an appropriate restriction enzyme and, for example, Eco RI, etc., and cloned into a plasmid such as pUC18, pUC19, etc. Sa
The nucleotide sequence of the target cDNA segment was determined by the dideoxy method of Nger et al. [Proceedings of National Academy Sciences USA (Proc. Natl. Acal. Sci. USA), 74 , 5463, (1977)]. Can decide.

The nucleotide sequence of the cDNA fragment thus obtained (secondary
Figure) is a full-length protein encoding a protein consisting of 128 amino acids (the N-terminal 25 amino acids are signal peptides) including the nucleotide sequence corresponding to the partial amino acid sequence (26-70) of the purified 1F5 antigen shown in FIG. It is a fragment consisting of 387 base pairs, including the full length structural gene of 1F5 antigen.

The cDNA fragment obtained as described above is directly or after modifying the 5 'end thereof into a known expression vector and inserted downstream of the promoter by a method known per se, and then the expression vector into which the cDNA is inserted is E. coli. A known method is introduced into a known cell such as a yeast, an animal cell host or the like.

The protein having the action of suppressing cell membrane damage caused by complement obtained by expressing the above cDNA in the above host is isolated and purified from the host by a known method.

(Examples) The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples as long as the gist of the present invention is not exceeded.

Example 1 Preparation of Partial cDNA Fragment of 1F5 Antigen by PCR Method K562 cell cDNA as a substrate is prepared according to a conventional method.
That 1 × 10 ⁹ cells equivalent K562 cells than guanidine thiocyanate - lithium method [DeNA chloride (DN
A), 2, 329, RNA having poly (A) according to (1983)]
Was prepared as follows. An equivalent amount of 1 × 10 ⁹ cells was prepared using 5 M guanidine thiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH
7) and 40% of a solution consisting of 8% (v / v) β-mercaptoethanol. 20 ml of this lysate is gently placed on 10 ml of a 5.7M cesium chloride solution in a centrifuge tube, Hitachi RPS2
RNA was collected as a precipitate after hydrochloric acid at 27,000 rpm for 20 hours using an 8-2 rotor.

Precipitate this RNA with 0.1% sodium lauryl sulfate, 1 mM
It was dissolved in 10 ml of a solution containing DTA and 10 mM Tris-hydrochloric acid (pH 7.5), extracted with phenol-chloroform, and recovered by ethanol precipitation. About 3.95 mg of the obtained RNA was dissolved in 1 ml of a solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. After incubation at 65 ° C for 5 minutes, 0.1 ml of 5M sodium chloride was added. The mixture was treated with an oligo [dT] cellulose column [PL Biochemical (PL Biochem).
mical), chromatography (column volume 0.5 ml)
To The adsorbed mRNA having poly (A) was eluted with a solution composed of 10 mM Tris-acetic acid (pH 7.5) and 1 mM EDTA to obtain about 100 μg of mRNA having poly (A).

First, 10 μg of poly (A) mRNA was added to an RT buffer [20 mM Tris-
Hydrochloric acid (pH 8.8), 0.1 M potassium chloride, 12 mM magnesium chloride, 2 mM manganese chloride] dissolved in 50 μl, and oligo dT primer d (T) 12-18 [PL Biochemical (P-L Biochemical)] Add 8 μg, 95 ° C, 3
Heated for minutes for transformation. This was gradually cooled to room temperature, and the random primer was annealed. 10mM4NTP for this
10 μ and 225 u of reverse transcriptase (manufactured by Takara Shuzo Co., Ltd.) were added, and water was added to make a total of 100 μ, and reacted at 42 ° C. for 1 hour.

Using the above reaction solution 50μ, 10mM NAD2μ, 10mM4dNTP1
0μ, RNaseH5u, Escherichia coli ligase 1u, Escherichia coli DNA polymerase I6.3u, 10 times concentration of T4 DNA ligase buffer [0.1M Tris-HCl (pH7.5) 0.1MDTT, 60mM magnesium chloride]
10 μl was added to make a total of 100 μl, and reacted at 37 ° C. for 1 hour to synthesize double-stranded DNA.

The double-stranded DNA obtained as described above was extracted with the same amount of water-saturated phenol, the phenol in the aqueous layer was removed with ether, and ethanol precipitation was performed.

The resulting precipitate was dissolved in 50 μl of water, and the concentration of T4DN was 10 times higher.
A polymerase buffer [0.33M Tris acetic acid (pH7.9) 0.66M
Potassium acetate, 0.1M magnesium acetate, 5mMDTT) 10μ
, 10 mM 4dNTP10μ, T4 DNA polymerase 6u, 10
The reaction was carried out at 37 ° C. for 1 hour to obtain DNA having double-stranded blunt ends. This was subjected to phenol extraction and protein removal as described above, followed by ethanol precipitation to purify DNA, followed by air drying.

₂ μg of the cDNA thus obtained was dissolved in 1 ml of H ₂ O and used as a substrate cDNA. PCR was performed using a Perkin Elmer Cetus DNA Thermal Cysler, based on instructions for use, and a gene amp kit (Ge
ne Amp ^TM DNA Amplification Reagent Kit, Takara Shuzo)
Was performed using That is, 10 μl of substrate cDNA (equivalent to 20 ng) and a 10-fold concentration of a reaction buffer [500 mM potassium chloride, 100
mM Tris-HCl (pH 8.3), 15 mM magnesium chloride, 0.1
% (W / v) gelatin] 10μ, 1.25mM4dNTP16μ, 20μ
M primers # 1 as 27 to 32 of the amino acid sequence of FIG.
+ Strand DNA primer ⁵ 'CA A / G TG C / T TAC corresponding to
/ TAC / TTCGC / TCC ³ '(17 nucleotides) 5µ, 20µM-strand DNA primer ⁵ ' corresponding to amino acid sequence 65-70 5'CAA / GTGCC / TTCA / GAC / Add 5 μT TTCCA ³ ′ (17 nucleotides) and 0.5 μTaq DNA polymerase to make a 100 μ system. The reaction was pre-treated at 94 ° C for 10 minutes, followed by 35 cycles of incubation at 94 ° C for 1 minute (denaturation step), 42 ° C for 2 minutes (annealing step), 72 ° C for 1.5 minutes (extension step), Further incubation was performed at 72 ° C. for 7 minutes. The obtained reaction solution was further precipitated with ethanol, dissolved in 50 µ of water, and dissolved in a 10-fold concentration of T4 DNA polymerase buffer [0.33 M Tris-acetic acid (pH 7.
9), 0.66M potassium acetate, 0.1M magnesium acetate, 5mMD
TT] 10 μm, 10 μm 4dNTP 10 μm, and T4 DNA polymerase 6 u were added to form a 100 μm system and reacted at 37 ° C. for 1 hour to obtain a double-stranded blunt end. As a result of analyzing the obtained reaction solution by 5% polyacrylamide electrophoresis, a band of about 150 bp was recognized. A band of this approximately 150 bp, was excised from the acrylamide gel in a conventional manner, a commercially available primer ⁵ by the dideoxy method and inserted into the Sma I site of pUC12 cloning vector ^{'CAGGAAACAGCATGAC 3'} and ^{5 'AGTCACG}
The nucleotide sequence was determined using ACGTTGTA ³ '. As a result, the nucleotide sequence of the PCR fragment was as shown in FIG. 3, a 131 bp fragment, the deduced amino acid sequence corresponding to 27 to 70 in FIG. 1, and the amino acid sequence determined from the purified 1F5 antigen protein. Completely matched the sequence. The insert was cut from this recombinant plasmid with Kpn I and Bam HI, excised from acrylamide gel,
Labeling Method [Analytical Biochemical Story (An
al. Biochem.), 132 , 6, (1983)].
The probe was used for library screening. That is, water was added to 100 ng of this DNA fragment to make it 32 μm, and 95 ° C.
After heat denaturation for 2 minutes, the mixture is rapidly cooled in ice. Further, an OLB buffer [0.25 M Tris-HCl (pH 8), 0.025 M magnesium chloride, 0.4% β-mercaptoethanol, 0.1 mM
dATP, 0.1 mM dTTP, 0.1 mM dGTP, 1 MHEPES (pH 6.6), oligo primer pdN ₆ (manufactured by P-LBiochemical, 15 units) 10 μ, 10 mg / ml bovine serum albumin 2 μ, α- ³² P
dCTP (300 ci / mmol) 5μ, DNA polymerase I, large fragment (Klenow Fragment) 2u] 1μ were added, and 37
The reaction was performed at 30 ° C. for 30 minutes, and this reaction solution was used as a probe.

Example 2 Screening of cDNA for 1F5 antigen As a λ phage cDNA library to be screened, a 34-week-old human placenta cDNA-λgt11 library (prepared by Clonetech) was used. This cDNA library was used for E. coli Y109
0 strains were infected to form plaques. Using the probe prepared in Example 1 from about 1.5 million plaques, seven positive clones were finally obtained by the following plaque hybridization method.

 First, Y1090 infected with λgt11 [Proceedings
National Academy of Sciences
Su (Proc. Natl. Acad. Sci. USA),80, 1194, (198
3)] in a Petri dish with upper soft agar kept at 42 ° C
And then left at 42 ° C. for 8 hours. Next, set the dish at 4 ° C for 30
After cooling for a minute, a nitrocellulose filter (manufactured by S & S)
BA-83 pore size 0.2μm) rise on it and hold for 2 minutes
did. Place another filter and hold for 5 minutes
Was. 0.1N hydroxylation of these nitrocellulose filters
Soak twice in 1.5M sodium chloride solution for 20 seconds
2 × SSCP [30 mM sodium citrate, 240 mM chloride
Sodium, 26 mM potassium phosphate, 2 mM EDTA (pH 7.
2)] -0.2 M Tris-HCl (pH 7.4) solution for 20 seconds
After neutralization twice, drying was performed at 80 ° C. for 2 hours. In addition these
Filter 2x SSC (450mM sodium chloride, 45mM
(Sodium phosphate) at 65 ° C for 30 minutes
3 × SSC-10 × D
enhardt's (0.2% Ficoll, 0.2% polyvinylpyrroli
Don, 0.2% BSA) Immerse in a solution at 65 ° C for 1 hour.
Hybridization solution [1 × Solution A (50 mM
Tris-hydrochloric acid (pH 7.8), 10 mM EDTA, 1 M sodium chloride
10% Denhardt's) -0.1% SDS, Herring Tees
stis) DNA at 250 µg / ml] at 65 ° C for 30 minutes. The above processing
Put the filter in a plastic bag and create it in Example 1.
Hybridization containing 0.1 ng / ml PCR probe
Solution and sealed. Keep this filter at 50 ° C, 15
Hybridize under the condition of time and dissolve 2 × SSC-0.1% SDS
Wash twice with the solution at room temperature for 5 minutes, then at 60 ° C for 15 minutes
Wash and wash twice with 0.1 x SSC-0.1% SDS at 60 ° C for 15 minutes
did. Saran wrap these filters (Asahi Kasei
Wrapped in autoradiography
Was.

As a result of such primary screening, 33 phage showing positive signals were obtained. These positive plaques were subjected to signal plaque fission three times. Plaque hybridization was performed in the same manner for all three times, and finally seven positive plaques were obtained.

Next, these phages were cultured in large quantities and the DNA was purified. First, add Y1090 bacteria to NZ medium (10 g of NZ amine, 5 g of sodium chloride, and 5 mM magnesium chloride in water 1 to pH 7.2).
Was cultured overnight in 10 ml. 1ml of this
moi (multiplicity of infection)
After infecting the phage to 0.1, leave at 37 ° C. for 10 minutes, transfer to NZ medium 1, and wait for 7 to 8 hours until the bacteria are lysed.
After shaking culture at 5 ° C., 5 ml of chloroform was added, and shaking was further continued for 30 minutes. Next, remove the bacterial cell residue at 6500 rpm.
Removed by centrifugation for
g and 70 g of polyethylene glycol, and dissolved well. After standing at 4 ° C. overnight, the precipitate obtained by centrifugation at 6500 rpm for 20 minutes was collected, water droplets were well removed, and the precipitate was washed with 20 ml of TM buffer solution (10 mM Tris-HCl). (PH7.5) ・ 5mM magnesium chloride]
And then added DNase I. RNase A to a concentration of 10 μg / ml, and allowed to react at 37 ° C for 1 hour. Then, 20 ml of chloroform was added and stirred, and polyethylene glycol was dissolved in chloroform. Removed. This aqueous layer was further subjected to ultracentrifugation at 28000 rpm for 60 minutes to obtain a phage particle pellet. The pellet was dissolved in 1 ml of TM buffer, and ρ = 1.45 to 1.50 by density gradient centrifugation of cesium chloride (33000 rpm, 20 hours).
A fraction containing phage particles was obtained. After dialysis against the TM buffer overnight, 100 μg of proteinase K was added thereto and reacted at 37 ° C. for 1 hour. Thereafter, the same amount of water-saturated phenol was added and phenol extraction was performed slowly. After centrifugation at 6500 rpm for 10 minutes, the aqueous layer was taken out, placed in a dialysis tube, and dialyzed against water at 4 ° C. overnight. In this way, about 5 mg of DNA was obtained.

These phage DNA was digested with Eco R I, all have inserts of 1.2～1.8Kb, was digested with various restriction enzymes and examining the digestion pattern, Eco RI common about 500bp all -It was found to have a BamHI fragment. Therefore choose four of these phage DNA, and cloned its Eco R I insert and Eco R I- Bam H I again insert into Eco R I site and Eco R I- Bam HI site of pUC119 each cloning vector, dideoxy Commercially available primer ⁵ 'CAGGAAACAGCTATGA
^{C 3 ', 5' AGTCACGACGTTGTA 3} ' and corresponds to 27-32 of the amino acid sequence of Figure 1 + strand DNA primer ^5' CAGTG
CTACAA C / T TG C / T CC ³ ',-strand DNA primer ⁵ ' corresponding to amino acid sequence 42-47 'GTCAGCAGTTGGGTTAGG ³ ',
Corresponding to 60-65 of the amino acid sequence + strand DNA primer ^{5 'GTGTATAACAAGTGTTGG 3',} amino acid sequence 65-70
-Strand DNA primer ⁵ 'CAGTGCTCGAACTTCC corresponding to
By combining the A ³ ', and their nucleotide sequences were determined for each using them. As a result, all four types of cDNAs had the nucleotide sequences shown in FIG. That is, a full-length cDNA of the 1F5 antigen protein gene consisting of a total of 387 base pairs encoding a protein consisting of 128 amino acids including a signal peptide of 25 amino acids at the N-terminal was obtained. PC
The base sequence of the primer portion of the DNA fragment obtained by the R method is different from the base sequence of the cDNA because the primer used when performing PCR was a mixture, so a primer different from the true base sequence was selected. It is considered that the signal has been amplified.

Reference Example [1] Expression of 1F5 antigen protein (1) Preparation of plasmid for expression of 1F5 antigen protein [lambda] phage c encoding 1F5 antigen obtained in Example 2
Maniatis et al.'S method ("Molecular cloning") from DNA clones, Cold Spring Harbor Labor
atory), pages 76-85 (1982)). As described in Example 2, a DNA fragment of about 1.3 kb of the restriction enzyme EcoRI-EcoRI was obtained from the obtained lambda phage DNA, and this DNA fragment was digested with the restriction enzyme EcoRI of plasmid pUC119.
It was inserted into the cleavage site and transformed into H. bacillus HB101 according to a conventional method. Among the subclones of 1F5 antigen thus obtained, E
The subclone pUIF10 containing the coR I-EcoR I fragment is the entire region encoding the 1F5 antigen protein, ie, the protein translation initiation site.
Since it contains the region from ATG to the termination codon TGA, the plasmid DNA was recovered and purified, and the DNA fragment of the region encoding the 1F5 antigen protein was taken out and incorporated into the expression vector. First, Ma from E. coli of subclone pUIF10
niatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 86-96 (19
According to 82)), plasmid DNA was recovered and purified to obtain a large amount. On the other hand, plasmid DNA of the expression vector pKCRH II-EcoR I (Nature 307: 604-608 (1984))
Is preliminarily cleaved with a restriction enzyme EcoRI in accordance with a conventional method, and its end is subjected to dephosphorylation treatment with alkaline phosphatase.
Next, the plasmid DNA of PUIF10 was cut with the restriction enzyme EcoRI in the usual manner, separated by agarose gel electrophoresis, and the DNA band at about 1.3 kb was recovered from the gel, purified, and coded for the 1F5 antigen protein. A DNA fragment of the region to be prepared was prepared. This and the above-described EcoR I cleavage site were ligated to a dephosphorylated expression vector DNA by a T4 DNA ligase reaction according to a conventional method, whereby the expression vector pKCRH II-EcoR I restriction enzyme EcoR I cleavage site of 1F5 antigen cDNA was used. Into which plasmid pKCRH II E-HRF was inserted. Escherichia coli HB101 was transformed using the plasmid thus prepared according to a conventional method to obtain a recombinant Escherichia coli having the plasmid pKCRHII E-HRF. From the obtained recombinant Escherichia coli, the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, 86
Page-96 (1982)), the plasmid DNA is recovered and purified, and the expression plasmid pKCRH II E into which the 1F5 antigen cDNA has been inserted.
-HRF was obtained in large quantities.

Using the obtained plasmid, the method of Ausubel et al. (Current Protocols in Molecular Biology), Green Publishing Associates and Wheelie Interscience (Greene Publishing Associates and W
iley-Interscience) 9.2.1-9.2.2 (19
In 87)), COS cells were transfected to transform COS cells. That is, first DME in a 9 cm diameter petri dish
COS cells in M medium were cultured. Next, the medium was removed from the Petri dish, 4 ml of 10% NuSerum-DMEM medium was added thereto, and a solution prepared by mixing 10 μg of plasmid DNA with 160 μm of a 10 mg / ml DEAE-dextran solution in advance and preparing 240 μm was added dropwise, followed by gentle addition. Mixed. This was allowed to stand at 37 ° C. for 2 hours in the presence of 5% carbon dioxide, after which the DNA-DEAE dextran solution was removed, the cells were washed twice with PBS, and 10% DMSO solution was spread over 3 ml of the cells and left for 2 minutes and 30 seconds. It was left still. PB this again
After washing twice with an S solution, an ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing 10% FCS was placed in an 8 ml petri dish and cultured overnight at 37 ° C. in the presence of 5% carbon dioxide. On the next day, after about 18 hours had elapsed, the culture was replaced with an ERDF medium containing no FCS, and the culture was continued.
After 8 hours, the cells were collected, solubilized with an SDS battery electrophoresis sample solution, and subjected to Western blotting according to a standard method to confirm the expression of 1F5 antigen protein.

(Effect of the Invention) The DNA fragment according to the present invention can be introduced into a cloning site of an expression vector by a conventional method to obtain a 1F5 antigen or a 1F5 antigen-like substance or a fusion protein containing the same by expression using this as a template. it can.

The resulting recombinant 1F5 antigen may be a therapeutic agent for inflammation and the like caused by complement activation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid sequence of the 1F5 antigen protein. FIG. 2 is a view showing the nucleotide sequence of cDNA encoding 1F5 antigen protein. However, in the figure, "---" represents a base sequence complementary to the above base sequence. The last "TAA" indicates a stop codon. FIG. 3 is a diagram showing the nucleotide sequence of a DNA fragment obtained by the PCR method.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Toshio Takizawa 1000 Kamoshidacho Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Kasei Stock (72) Inventor Atsushi Kondo 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Corporation Research Institute (72) Inventor Hidechika Okada 1-5-1, Kokuma, Jonan-ku, Fukuoka, Fukuoka ( 58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 15/12 C07K 14/705 C12P 21/02 C12N 5/00 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A novel protein represented by the following amino acid sequence and having an action of inhibiting cell membrane damage caused by complement. 2. A gene encoding the protein according to claim 1. 3. A gene encoding the protein according to claim 1, which is represented by the following nucleotide sequence.