JP2001103977A - STRUCTURAL GENE OF SWINE Fas LIGAND - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protein, especially a swine Fas ligand, having a Fas ligand activity, and a DNA encoding the protein, and a DNA complementary to the DNA. SOLUTION: This protein has (a) the amino acid sequence shown by sequence 2, or (b) an amino acid sequence modified by deleting, substituting, or adding an amino acid or more from, in, or to the amino acid sequence, and has a Fas ligand activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein having Fas ligand activity (particularly, porcine Fas ligand), a DNA encoding the protein, an RNA complementary to the DNA, and the DNA.
The present invention relates to an expression vector, a transformant transformed with the expression vector, and an antibody that reacts with the protein.

[0002]

2. Description of the Related Art Fas ligand (Fas-L) is a type 2 membrane protein belonging to the TNF family and is mainly composed of activated T cells and N cells.
Induces apoptosis by binding Fas antigen to various cells produced from K cells and expressing Fas antigen (T. Suda et al., Cell, 75, 1169-1178, T. Suda
et al., J. immunol., 154, 3806-3813). Fas antigen and Fas-L
Apoptosis through binding of is important in removing virus-infected cells and cancer cells, turning on activated lymphocytes to suppress excessive immune responses, and suppressing autoimmune reactions by removing autoreactive lymphocytes Plays a role.

In recent years, studies using pig organs as donors for human organ transplantation (P. Wolf et al., Vet. Re.
s., 28, 217-222, review) and studies of transplanting pig tissues and cells for the treatment of neurological diseases such as Alzheimer's disease (DB Jacoby et al., Artif. Organ, 21, 1192-1).
198, AE Willing et al., Mol. Med. Today, 4, 471-
477), and Fas-L is considered to be one of the important key factors in these studies as a factor suppressing the immune response.

[0004] A report on transplantation of cells or organs expressing Fas-L (RC Duke et al., Transplant.
roc., 31, 1479-1481) and that it was possible to transplant Fas-L-expressing porcine Sertoli cells into rat brain without using immunosuppressants (S. Saporta et al.). ., Exp.
Neurol., 146, 299-304). To date, humans (T.Taka
hashi et al., Int. Immunol., 6, 1567-1574), monkeys (WW
ang et al., Hum. Immunol., 59, 599-606), cat (T. Mizu
no et al., Vet.Immunol.Immunopathol., 65,161-172),
Fas-L of mouse (DHLynch et al., Immunity, 1, 131-136) and rat (T. Suda et al., Cell, 75, 1169-1178)
The gene has been isolated and identified, but the porcine Fas-L gene has not been isolated and identified to date.

[0005] The reasons for the delay in the isolation and identification of the porcine Fas-L gene include the limited number of cells or tissues in which Fas-L is expressed, and the T cell line (eg, Jurkat) in humans and mice. (ATCC No. TIB-152) and CTLL-2 (ATCC No. TIB-152)
-214) can be used, but such cell lines are not present in pigs. pig's
Although the isolation of the Fas-L gene is thought to greatly contribute to the development of the above-mentioned research, the isolation of the porcine Fas-L gene is extremely difficult for the reasons described above. Under such circumstances, isolation and identification of the porcine Fas-L gene is eagerly desired.

[0006]

Accordingly, the present invention provides a porcine Fas ligand and a DN encoding the porcine Fas ligand.
A is intended to provide.

[0007]

The present inventors have reported that Fas ligand is highly expressed in fetal thymocytes in late pregnancy in mice (M. Fleck et al., J. Immunol).
L., 160, 3766-3775), and as a result of intensive studies to solve the above-mentioned problems, the thymocytes of fetal pigs obtained from pregnant pigs were converted to concanavalin A, a mitogen.
(ConcanavalinA (ConA)), and by performing RT-PCR using mRNA obtained from the thymocytes as a template,
It was found that the porcine Fas ligand gene could be amplified. The present inventors have succeeded in isolating and identifying a pig Fas ligand gene based on such findings, and have completed the present invention. That is, the present invention includes the following inventions.

(1) A protein represented by the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence of SEQ ID NO: 2 consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and which has Fas ligand activity. (2) DNA encoding the protein described in (1) or a DNA complementary thereto. (3) RNA complementary to the DNA according to (2). (4) An expression vector containing the DNA according to (2). (5) A transformant transformed by the expression vector according to (4). (6) An antibody that reacts with the protein according to (1).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
A first aspect of the present invention is a protein shown in the following (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having a Fas ligand activity protein

[0010] Here, the term "one or more amino acids" is not particularly limited as long as a protein comprising a deleted, substituted or added amino acid sequence has Fas ligand activity. Techniques commonly used at the time of filing, such as site-directed mutagenesis (Nucleic
Acids Res. 10, 6487-6500, 1982), meaning one or several, specifically one to five, preferably one to three amino acids.

The term "protein having a Fas ligand activity" means a protein having an activity of inducing apoptosis by binding to a cell surface receptor molecule, a Fas antigen. The protein of the present invention, as described in detail below,
It can be obtained by introducing an expression vector containing the DNA of the present invention into an appropriate host and expressing it. The second of the present invention is a DNA encoding the protein shown in the above (a) or (b) or a DNA complementary thereto. DNA of the present invention
Is not limited as long as it is a DNA encoding the protein shown in (a) or (b) or a DNA complementary thereto.

Specific examples of the DNA of the present invention include the following DNA (c) or (d) or a DNA complementary thereto. (C) DNA comprising the nucleotide sequence of SEQ ID NO: 1 (d) DNA capable of hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and having a Fas ligand activity DNA encoding

Here, the term "DNA capable of hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions" refers to a colony-hybrid DNA having the DNA of the nucleotide sequence of SEQ ID NO: 1 as a probe. DNA obtained by hybridization, plaque hybridization, Southern blot hybridization, etc.
A means, specifically, D from colonies or plaques
After hybridization using a filter having NA immobilized thereon at 42 to 68 ° C (or at 42 ° C in 50% formamide or at 65 to 68 ° C in an aqueous solution) in the presence of 0.5 to 1 M NaCl, 0.1 ~ 2-fold concentration SSC (saline-sodium citrate) solution (composition of 1-fold concentration SSC solution: 150 mM sodium chloride, 15
(mM sodium citrate) at room temperature to 68 ° C. Specific examples of such DNA include a DNA having at least 90% or more homology with the nucleotide sequence of SEQ ID NO: 1, preferably a DNA having 95% or more homology, and more preferably a 98% or more homology. Can be exemplified.

The method for obtaining the DNA of the present invention is not particularly limited. As a source of the present invention, porcine cells or tissues can be used.However, since cells or tissues expressing Fas ligand are limited, the DNA of the present invention can be obtained from any porcine cells or tissues. It is difficult to get. Therefore, as a source for obtaining the DNA of the present invention, it is preferable to use pig cells or tissues in which Fas ligand is highly expressed. The DNA of the present invention is obtained, for example, by stimulating thymocytes of fetal pigs obtained from pregnant pigs with mitogen Concanavalin A (ConA), and performing RT-PCR using mRNA obtained from the thymocytes as a template. Can be obtained by

The specific method is as follows. The porcine fetus is collected by cesarean section of the pregnant pig, and thymocytes are separated from the porcine fetus. Thymocytes derived from porcine fetuses are stimulated with a mitogen, concanavalin A, and cultured for 2 to 48 hours, and then cultured by a known method (for example, P. Chomczynski and N. Sacci,
Analytical Biochem., 162, 156-159, 1987) obtains total RNA from the thymocytes. A known method (for example, J. sambrook et al., Molecular Cloni) using the obtained total RNA as a template and commercially available synthetic oligonucleotide primers
ng, 2nd Ed., Cold Spring Harbor Laboratory Press,
Perform reverse transcription according to pp.8.11-8.17) to synthesize cDNA.

The DNA of the present invention can be amplified by performing PCR using the obtained cDNA as a template and primers capable of hybridizing to both ends of the porcine Fas ligand gene. At this time, the primer used for PCR is SEQ ID NO: 1.
It can be designed based on the described nucleotide sequence. Primers used for PCR are Fas of other species such as human and mouse.
It can be designed based on the nucleotide sequence conserved between the ligand genes. Specific examples of primers that can be used for PCR include, for example, a primer set consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8. The designed primer can be chemically synthesized according to its base sequence. PCR can be performed according to a conventional method.

The obtained PCR product is, for example, commercially available Origi
Using the nal TA CloningR Kit (Invitrogen), the plasmid was subcloned into the plasmid pCRR2.1 (Invitrogen), and this plasmid was cloned by a known method (for example, J. sambro).
ok et al., Molecular Cloning, 2nd Ed., Cold Spring
It is isolated and purified according to Harbor Laboratory Press, pp.1.21-1.52). Then, the base sequence of the PCR product can be determined using a known method such as the Sanger method or the Maxam-Gilbert method.

The DNA of the present invention can be obtained by chemical synthesis according to a conventional method. The DNA of the present invention can be used for mass production of proteins having Fas ligand activity (particularly, porcine Fas ligand). F using the DNA of the present invention
Production of a protein having as ligand activity
The transformation can be performed by transforming a suitable host cell with an expression vector containing DNA and culturing the transformant in a suitable medium to purify a protein having Fas ligand activity from a culture obtained.

The expression vector containing the DNA of the present invention contains an appropriate promoter upstream of the DNA of the present invention,
Contains a suitable terminator downstream of NA. As an expression vector, for example, pcDNA3.1, pVL1392, pQE30 and the like can be used. In order to facilitate detection of a transformant into which the expression vector has been introduced, an appropriate marker gene or reporter gene may be inserted downstream of the promoter. As marker genes, for example, resistance genes of antibiotics such as tetracycline, ampicillin, kanamycin, neomycin, hygromycin, spectinomycin can be used, and as reporter genes, β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), a gene encoding luciferase (LUX) and the like can be used.

The host cell is not particularly limited as long as it is compatible with the expression vector containing the DNA of the present invention and can be transformed. Commonly used natural cells, artificially established recombinant cells, etc. Various cells, such as cells, can be used. Examples of such host cells include animal cells (eg, COS-7 cells, CHO cells, NIH3T3 cells), insect cells (eg, Sf9 cells, Sf21 cells, Tn5 cells), bacteria (eg, Escherichia coli, Bacillus subtilis) ), Yeast and the like can be used.

The introduction of the expression vector into a host cell can be performed by a known method. For example, a protoplast method, a lithium method, an electroporation method, a calcium chloride method, or a modified method thereof can be used. Culture of the transformant and purification of the porcine Fas ligand from the culture can be performed according to a conventional method. The transformant may be cultured in a medium such as an RPMI medium, an LB medium, and an Express FIVE medium at room temperature to 37 ° C. for 24 to 120 hours.

Methods for purifying proteins having Fas ligand activity from culture include, for example, natural Fas ligand and recombinant Fas expressed using various expression systems.
A method for purifying from a cell extract or a culture supernatant containing an s ligand, for example, by combining methods such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and reverse phase chromatography, etc. When expressing the recombinant Fas ligand, various purification tags (eg, His, GST,
T7, thioredoxin, etc.) and expressing by affinity chromatography using respective affinity columns, immunoassay using immunoaffinity columns prepared using monoclonal or polyclonal antibodies specific to Fas ligand. Examples of the method include purification by affinity chromatography and purification by receptor-ligand affinity chromatography using a column to which Fas is coupled. Note that porcine Fas can be obtained by introducing an expression vector containing DNA encoding porcine Fas into a suitable host cell and expressing it.
Since the nucleotide sequence of DNA encoding porcine Fas is already known (Genebank Accession No. AJ001202), primers are designed based on the nucleotide sequence, and PC
By performing R, DNA encoding porcine Fas can be obtained. DNA encoding porcine Fas can also be obtained by chemical synthesis according to its nucleotide sequence.

As a method for measuring the Fas ligand activity of the obtained protein, for example, target cells expressing Fas labeled with ⁵¹ Cr (eg, Jurkat cells (ATCC No. TI
B-152)) and effector cells (eg, Cos-7 cells) incorporating the DNA encoding the protein are co-cultured (eg, at 37 ° C. overnight), and then the release of ⁵¹ Cr into the culture supernatant is determined. Method for calculating cytotoxic activity by measuring with a gamma counter (David H. Lynch et al., Immunity, 1, 131
-136 (1994)), after adding the protein to Fas-expressing target cells (eg, Jurkat cells (ATCC No. TIB-152)) and culturing for a certain period of time (eg, at 37 ° C. overnight),
2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-
Di-sulfophenyl) ^[2 H] tetrazolium monosodium salt (2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disu
lfophenyl) [ ² H] tetrazolium monosodium salt) and 1-methoxy-5-methylphenazinium methyl sulfate (1
-methoxy-5-methylphenazinium methylsulfate) and further culture for 1 hour, collecting the culture supernatant and measuring the absorbance at 450 nm to calculate the cytotoxic activity (Tanaka et a
l., EMBO J., 14, 1129-1135 (1995)), Fas-expressing target cells (eg, Jurkat cells (ATCC No. TIB-15)
2)), the protein is acted on to cause apoptosis in various ways (eg, flow cytometer, DN
A Ladder detection, TUNNEL staining method, etc.). Among these measurement methods, the above method is suitable for measuring the Fas ligand activity of a membrane-type protein, and the above method is
Suitable for measuring ligand activity. The above method is suitable for measuring both Fas ligand activities of membrane proteins and soluble proteins.

When producing the porcine Fas ligand using the DNA of the present invention, commercially available Transient Mammalian Expressio
n Vectors (In Vitrogen), Baculovirus Expressi
onand purification kit (Pharmingen), ESP Yeas
t Protein Expression system (Toyobo), pET Exp
It is convenient to use a ression system (Toyobo). The DNA of the present invention can be used for producing a protein having Fas ligand activity (particularly, porcine Fas ligand).
It can be used as a probe for detecting porcine Fas ligand gene. Furthermore, based on the nucleotide sequence of the DNA of the present invention, a probe for detecting the porcine Fas ligand gene and a primer for PCR for amplifying the porcine Fas ligand gene can be designed.

As a probe for detecting the porcine ligand gene, RNA complementary to the DNA of the present invention can also be used. RNA complementary to the DNA of the present invention can be obtained, for example, by incorporating the DNA of the present invention into a vector (for example, pCRR2.1) having a T7 or T3 phage RNA polymerase promoter,
It can be obtained by allowing a polymerase to act. The resulting RNA contains enzymatic signals, fluorescent signals,
By labeling with radioisotope, etc., pig Fa
It can be used as a probe for detecting s ligand gene.

The protein of the present invention and the DNA of the present invention
Research to analyze apoptosis pathology in swine virus infection, etc., study to use porcine cells and organs expressing Fas ligand as xenograft donor to human, tumor, virus infection or arthritis It can be used for research on the treatment of pathological conditions of local apoptotic abnormalities and autoimmune diseases, and can also be used as an immunogen for producing antibodies against porcine Fas ligand.

The third aspect of the present invention is an antibody that reacts with the protein shown in the above (a) or (b). The antibody of the present invention may be any of a monoclonal antibody and a polyclonal antibody as long as it can react with the protein shown in the above (a) or (b). A polyclonal antibody can be prepared according to a conventional method using the protein shown in the above (a) or (b) as an immunogen. For example, it can be prepared by using an antigen affinity column from serum immunoglobulin of an animal (eg, rabbit, goat, horse, chicken, etc.) immunized with the protein shown in the above (a) or (b).

A monoclonal antibody can be prepared, for example, by the following steps. (1) Preparation of antigen (2) Collection of immune and antibody-producing cells (3) Cell fusion (4) Selection and cloning of hybridoma (5) Collection of monoclonal antibody Each step will be described below.

(1) Preparation of antigen As the antigen, the protein shown in the above (a) or (b) is used. The protein shown in the above (a) or (b)
For example, it can be prepared using a DNA consisting of the nucleotide sequence of SEQ ID NO: 1.

(2) Collection of immunized and antibody-producing cells The protein obtained as described above was used as an immunogen,
It is administered to mammals, birds, etc. together with the adjuvant. Here, examples of the adjuvant include commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, BCG, Hunters, Titer Mac, keyhole limpet hemocyanin-containing oil, and the like, and these may be used alone. And two or more of these may be used in combination.

As mammals, horses, monkeys, dogs, pigs, goats, sheep, rabbits, guinea pigs, hamsters,
Mouse and the like can be used, and birds such as pigeons and chickens can be used, and mouse, hamster and the like are particularly preferable. As a method of administration, any known method may be used, for example, intravenous administration, subcutaneous administration, or intraperitoneal administration. The amount of antigen immunization per mouse at a time is usually 10 to 1000 μg, preferably 20 to 100 μg.
It is. The interval between immunizations is usually 1 to 3 weeks, preferably 2 weeks, and the number of immunizations is usually 1 to 3 times, preferably 2 times. Two to five days, preferably three days after the last immunization, the antibody-producing cells are collected. The antibody-producing cells to be collected include lymph node cells, spleen cells and the like, and preferably spleen cells.

(3) Cell Fusion As the myeloma cells to be fused with the antibody-producing cells, cell lines derived from various animals such as mice, rats, and humans and generally available to those skilled in the art can be used. As a cell line to be used, a cell line having drug resistance, having a property that it cannot survive in a selection medium (for example, HAT medium) in an unfused state and can survive in a selection medium only in a state fused with an antibody-producing cell, preferable. Generally, 8-azaguanine resistant strains can be used. This cell line, hypoxanthine - deficient in guanine phosphoribosyl Bo transferase (HGPRT ^-), can not grow in HAT medium.

[0033] Such myeloma cells include P3X6
3Ag8U1, P3-X63Ag8, P3 / NS1 / 1-Ag4-1, P3X63Ag8.653, S
Mouse myeloma cell lines such as p2 / O-Ag14, Sp2 / O / FO-2, 2
10. Rat myeloma cell line such as RCY.Ag1.2.3, SKO-007
And other human myeloma cell lines. Cell fusion involves, for example, mixing the myeloma cells with the antibody-producing cells.
It can be carried out by bringing the cells into contact with each other for 1 to 7 minutes at room temperature in a ratio of 1: 5 to 1:10 in the presence of a fusion promoter in a medium such as RPMI1640 medium. At this time, as the fusion accelerator, polyethylene glycol, polyvinyl alcohol, or the like having an average molecular weight of 1,000 to 5,000 can be used. Further, a fusion virus such as Sendai virus may be used.

(4) Selection, screening and cloning of hybridomas After cell fusion, hybridomas are selected. The method for selecting a hybridoma may be a conventional method, and is not particularly limited. Hybridomas can be selected, for example, by culturing the hybridomas in a selection medium (eg, HAT medium). The culture at this time may be in accordance with a conventional method, and is not particularly limited. Usually, the culture may be performed at 37 ° C. for 3 to 7 days.

The screening of a hybridoma producing the desired monoclonal antibody can be performed, for example, by the following method. After adsorbing the protein shown in the above (a) or (b) to each well of the microplate, it is blocked with Block Ace (Dainippon Pharmaceutical) or the like. The culture supernatant of the hybridoma is added to each well of the microplate, and left at room temperature to 37 ° C for 1 to 2 hours. After washing with physiological saline, an appropriately diluted alkaline phosphatase-conjugated goat anti-immunoglobulin is added. After washing with physiological saline, the activity of alkaline phosphatase was measured using ALP Rose (Sinotest), and the wells having the alkaline phosphatase color were subjected to the above (a).
Alternatively, a well containing cells producing an antibody specific to the protein shown in (b) is used. Thereby, the desired hybridoma can be screened. The method for cloning the desired hybridoma from this well may be a conventional method, and is not particularly limited. The cloning of the hybridoma can be performed by, for example, a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like.

(5) Collection of Monoclonal Antibody As a method for collecting a monoclonal antibody from the obtained hybridoma, a usual cell culture method, ascites formation method, or the like can be used. In the cell culture method, for example,
The hybridoma is calf serum-containing RPMI1640 medium, MEM medium, E-RDF medium or animal cell medium such as serum-free medium,
The cells are cultured under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration) for 3 to 7 days, and the desired monoclonal antibody can be obtained from the culture supernatant.

In the ascites formation method, for example, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is administered intraperitoneally to an animal of the same species as a mammal derived from myeloma cells, and then the hybridoma is administered. 1 × 10 ⁶ 〜1 × 10
^Seven , preferably 2 × 10 ^6, are administered intraperitoneally. After breeding the administered mammal for 1 to 2 weeks, preferably 10 days, the target monoclonal antibody can be obtained by collecting ascites or serum.

When antibody purification is required in the above-mentioned antibody collection method, sulfate analysis, ion exchange chromatography using an anion exchanger such as DEAE-cellulose, protein A sepharose and the like are used. A known method such as affinity chromatography, molecular sieve chromatography that sifts according to molecular weight or structure, etc. is appropriately selected, and purification can be performed by using these alone or in combination. Confirmation that the collected monoclonal antibody is the desired monoclonal antibody can be performed, for example, by Western blotting using the protein shown in the above (a) or (b). The antibody of the present invention can be used for separation and purification of porcine Fas ligand from a sample containing porcine Fas ligand, detection and quantification of porcine Fas ligand contained in the sample, and the like.

[0039]

[Example 1]

(1) cDNA synthesis In order to obtain mRNA derived from cells expressing porcine Fas ligand, pig fetuses were collected from pregnant pigs on the 110th day of normal pregnancy, and thymocytes collected from the pig fetus were subjected to mitogen. It was stimulated by a certain concanavalin A (ConA). That is, 10% FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 50 U / ml penicillin, 50 μg / ml streptomycin, and 50 μM β
-Resuspended in RPMI1640 medium (manufactured by Sigma) containing mercaptoethanol to a cell concentration of 1 × 10 ⁶ / ml,
g / ml ConA was added, and the cells were cultured under the conditions of 37 ° C and 5% carbon dioxide for 24 hours. After culturing, a known method (P. Chomczynski and
N. Sacci, Analytical Biochem., 162, 156-159, 1987) to extract and purify Total-RNA. 1 of this Total-RNA
Using μg as a template, cDNA was synthesized using a commercially available kit (Takara RNA PCR kit Ver2.1) manufactured by Takara Shuzo.

(2) Amplification of Porcine Fas-LD2R2 Fragment by PCR In a PCR reaction tube manufactured by Perkinermer, 10 μl of 10 × PCR buffer, 10 μl of 25 mM dNTP mix, 5 U / μl of Ex Ta
q 1 μl of DNA polymerase, cDNA 1 synthesized in (1) above
μl, sense primer D2: 5'-tccccagatctactgggt-3 '
(SEQ ID NO: 3) and antisense primer R2: 5'-aga
ttcctcaaaattgaccag-3 ′ (SEQ ID NO: 4) was added, and the mixture was adjusted to 100 μl with sterile distilled water. The primers D2 and R2 were chemically synthesized based on the known base sequences of human and rat Fas ligand genes.

The sense primer D2 (SEQ ID NO: 3)
Nucleotide sequence of human and rat Fas ligand gene 2
This is a nucleotide sequence corresponding to 7-44 (A of the start codon ATG is 1). The nucleotide sequence of the antisense primer R2 (SEQ ID NO: 4) is 796 of the human Fas ligand gene.
This is a base sequence corresponding to −816 (A of the start codon ATG is set to 1). In this mixture, at 94 ° C. for 30 seconds, 50
30 cycles of 30 seconds at 72 ° C and 60 seconds at 72 ° C
A PCR reaction was performed to obtain a PCR product of about 790 bp.

(3) Cloning of PCR product The PCR product obtained in the above (2) was cloned into a TA-cloning kit (I
After the vector was incorporated into the pCRR2.1 vector using nvitorogen, the vector was introduced into Escherichia coli INVαF '(Invitorogen) to obtain a transformant pFas-LD2R2TA.

(4) Determination of Nucleotide Sequence The clone containing the PCR product was cultured in an LB medium containing 100 μg / ml of ampicillin in an amount of 100 ml, and the plasmid DNA was isolated from Wiza.
rd ^R Plus Midipreps DNA Purification System (Promeg
a) and the nucleotide sequence of the integrated PCR product is Th
ermo Sequenasefluorescent labelled primer cycle se
It was determined using a quencing kit (Amersham pharmacia biotech).

(5) Cloning of 3 ′ region Based on the nucleotide sequence determined in (4) above, a sense primer D3: 5′-cct capable of hybridizing inside thereof
ctggaatgggaagacac-3 ′ (SEQ ID NO: 5) was synthesized. In addition, based on the base sequence of the 3 'untranslated region of the human and mouse Fas ligand genes which have already been known, the antisense primer R3'-2: 5'-gacmtkttgraccytgtggtc-3'
(SEQ ID NO: 6) was chemically synthesized. In the primer R3'-2, the base represented by "m" means "a" or "c", and the base represented by "k" means "g", "t" or "u". , “R” means “g” or “a”, and the base represented by “y” means “t”, “u” or “c”.

The sense primer 3 (SEQ ID NO: 5)
Is the nucleotide sequence corresponding to 478-497 of the pig Fas ligand gene (A of the start codon ATG is 1).
The base sequence of the antisense primer R3'-2 (SEQ ID NO: 6) is a base sequence corresponding to 974-994 of the human Fas ligand gene (A is set to 1 in the start codon ATG). Per
Kinermer's PCR reaction tube, 10 × PCR buffer 10 μl, 25 mM dNTP mix 10 μl, 5 U / μl Ex Taq DN
1 μl of A polymerase, 1 μl of cDNA synthesized in the above (1),
Add 1 μl each of primers D3 and R3'-2, and add 10 ml with sterile distilled water.
0 μl was used. In this mixture, 50 ℃ at 94 ℃ for 30 seconds
30 cycles of 30 seconds at 72 ° C and 30 cycles of
After the R reaction, a PCR product of about 520 bp was obtained. After TA cloning of this PCR product according to the above (3) and (4), its nucleotide sequence was determined, and the nucleotide sequence of the 3 ′ terminal region including the termination codon of the porcine Fas ligand gene was determined.

(6) Cloning of 5 ′ region Based on the nucleotide sequence determined in (5) above, human Fa
Primer D1: 5'- containing start codon of s ligand gene
atgcagcagcccttcaattacc-3 ′ (SEQ ID NO: 7) and pig F
Primer R-EcoR containing stop codon of as ligand gene
I: 5′-ctag gaattc tcagagcttatataagcc-3 ′ (SEQ ID NO: 8) was chemically synthesized. In addition, the nucleotide sequence of the sense primer D1 (SEQ ID NO: 7)
(A of the start codon ATG is defined as 1). In the base sequence of the antisense primer R-EcoRI (SEQ ID NO: 8), “ctag” at the 5 ′ end is an additional sequence for efficiently performing a restriction enzyme treatment, and “gaattc” underlined is a restriction enzyme. EcoRI recognition sequence, "tcaga
gcttatataagcc "is the porcine Fas ligand gene 832-849
(A of the start codon ATG is defined as 1).

In a PCR reaction tube manufactured by Perkinermer,
10 x PCR buffer 10 l, 25 mM dNTP mix 10 l, 5
1 μl of U / μl Ex tack DNA polymerase, 1 μl of the cDNA synthesized in the above (1), 1 μl of each of the primers D1 and R-EcoRI were added, and the mixture was made up to 100 μl with sterile distilled water. In this mixture, a cycle of 94 ° C. for 45 seconds, 50 ° C. for 45 seconds, and 72 ° C. for 60 seconds was repeated 30 times to perform a PCR reaction to obtain an 849 bp PCR product. After TA cloning of this PCR product according to (3) and (4) above, its nucleotide sequence was determined, and
The entire nucleotide sequence of the Fas ligand gene was determined.

(7) Comparison of nucleotide sequence c encoding porcine Fas ligand determined by the above method
The entire nucleotide sequence of DNA is shown in SEQ ID NO: 1, and the amino acid sequence of porcine Fas ligand deduced from the nucleotide sequence is shown in SEQ ID NO: 2. The pig represented by the nucleotide sequence of SEQ ID NO: 1
Escherichia coli into which the cDNA encoding the Fas ligand has been introduced has been deposited as FERM P-17501 with the Institute of Biotechnology and Industrial Technology, National Institute of Industrial Science and Technology (Deposit date: August 5, 1999). Known human, monkey, cat, deduced amino acid sequence of porcine Fas ligand,
As compared with the amino acid sequences of mouse and rat Fas ligand, 85.5%, 81.0%, 83.5%, 76.6% and 7
It showed 5.5% homology.

[0049]

According to the present invention, a protein having Fas ligand activity (particularly, porcine Fas ligand) and a DNA encoding the protein are provided. By the genetic engineering technique using the DNA of the present invention, it becomes possible to produce porcine membrane type Fas ligand and soluble Fas ligand in large quantities. The DNA of the present invention can also be used as a probe for detecting a porcine Fas ligand gene. Therefore, the DNA of the present invention
It is useful for research to analyze the pathology of apoptosis in swine virus infection and the like. Further, the DN of the present invention
By using the nucleotide sequence of A, PCR primers for examining the expression of porcine Fas ligand in various organs and cultured cells can be constructed. In addition, when porcine cells or organs expressing Fas ligand are used as a donor for xenotransplantation into humans, rejection due to an immune reaction may be suppressed, and thus application for this purpose is expected. Furthermore, in humans, it has been considered to be applied to cancer treatment by Fas ligand gene transfer, removal of virus-infected cells, and treatment of rheumatoid arthritis. It is useful for the treatment of pathological conditions of local apoptotic abnormalities such as inflammation or arthritis and autoimmune diseases.

[0050]

[Sequence List] SEQUENCE LISTING <110> Director General of National Institute of Animal Health <120> A gene coding Sus scrofa Fas-Ligand <130> P99-0414 <160> 8 <210> 1 <211> 849 <212> DNA <213> Sus scrofa <220> <221> CDS <222> (1) .. (849) <400> 1 atg cag cag ccc ttc aat tac cca tac ccc caa atc ttc tgg gtg gac 48 Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Phe Trp Val Asp 1 5 10 15 agc agt gct acc tct ccc tgg gcc tcc cca ggc tca gtc ttc ccc tgt 96 Ser Ser Ala Thr Ser Pro Trp Ala Ser Pro Gly Ser Val Phe Pro Cys 20 25 30 cca gct tct gtg cca gga agg cca ggg caa agg agg cca cca cca cca 144 Pro Ala Ser Val Pro Gly Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45 ccg ccg cca ccg cca cca cca cca aca ctc ctg cca tca aga ccg ctg 192 Pro Pro Pro Pro Pro Pro Pro Pro Thr Leu Leu Pro Ser Arg Pro Leu 50 55 60 cct cca ctg cca ccg cca tct ctg aag aag aag agg gac cac aat gca 240 Pro Pro Leu Pro Pro Pro Ser Leu Lys Lys Lys Arg Asp His Asn Ala 65 70 75 80 ggc ctg tgt ctc ctt gtg atg ttc ttc atg gtt ctg gtg gcc ctg gtt 288 Gly Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val 85 90 95 gga ttg ggg ctg ggg atg ttt cag ctc ttc cac cta cag aag gag ctg 336 Gly Leu Gly Leu Gly Met Phe Gln Phe His Leu Gln Lys Glu Leu 100 105 110 act gaa ctc aga gag tct gcc agc caa agg cat aca gaa tca tct ttg 384 Thr Glu Leu Arg Glu Ser Ala Ser Gln Arg His Thr Glu Ser Ser Leu 115 120 125 gag aag caa ata ggt cac ccc aat cta ccc tct gag aaa aag gag ctg 432 Glu Lys Gln Ile Gly His Pro Asn Leu Pro Ser Glu Lys Lys Glu Leu 130 135 140 aga aaa gtg gcc cac tta aca ggc aag cct aac tca aga tcc atc cct 480 Arg Lys Val Ala His Leu Thr Gly Lys Pro Asn Ser Arg Ser Ile Pro 145 150 155 160 ctg gaa tgg gaa gac acc tat gga att gcc ttg gtc tct ggg gtg aag 528 Leu Glu Trp Glu Asp Thr Tyr Gly Ile Ala Leu Val Ser Gly Val Lys 165 170 175 tat atg aag ggc agc ctt gtg atc aat gac act ggg ctg tat ttt gtg 576 Tyr Met Lys Gly Ser Leu Val Ile Asn Asp Thr Gly Leu Tyr Phe Val 180 185 190 tat tcc aaa gtg tac ttc cgg ggt ca t ac tgc aac aac cag ccc ctg 624 Tyr Ser Lys Val Tyr Phe Arg Gly Gln Tyr Cys Asn Asn Gln Pro Leu 195 200 205 agt cac aag gta tac aca agg aac tct agg tat ccc cag gac ctg gtg 672 Ser His Lys Val Tyr Thr Arg Asn Ser Arg Tyr Pro Gln Asp Leu Val 210 215 220 ctg atg gag gga aag atg atg aac tat tgc act act ggc caa atg tgg 720 Leu Met Glu Gly Lys Met Met Asn Tyr Cys Thr Thr Gly Gln Met Trp 225 230 235 240 gcc cgc agc agc tac ctg ggg gct gtg ttc aat ctc acc agc gct gac 768 Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp 245 250 255 cat tta tat gtc aac gta tct gag ctc tct tct gtg gag gaa 816 His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu 260 265 270 tct aag aca ttt ttt ggc tta tat aag ctc tga 849 Ser Lys Thr Phe Phe Gly Leu Tyr Lys Leu 275 280 <210> 2 <211> 282 <212> PRT <213> Sus scrofa <400> 2 Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Phe Trp Val Asp 1 5 10 15 Ser Ser Ala Thr Ser Pro Trp Ala Ser Pro Gly Ser Val Phe Pro Cys 20 25 30 Pro Ala Ser Val P ro Gly Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45 Pro Pro Pro Pro Pro Pro Pro Pro Thr Leu Leu Pro Ser Arg Pro Leu 50 55 60 Pro Pro Leu Pro Pro Ser Leu Lys Lys Lys Arg Asp His Asn Ala 65 70 75 80 Gly Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val 85 90 95 Gly Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu 100 105 110 Thr Glu Leu Arg Glu Ser Ala Ser Gln Arg His Thr Glu Ser Ser Leu 115 120 125 Glu Lys Gln Ile Gly His Pro Asn Leu Pro Ser Glu Lys Lys Glu Leu 130 135 140 Arg Lys Val Ala His Leu Thr Gly Lys Pro Asn Ser Arg Ser Ile Pro 145 150 155 160 Leu Glu Trp Glu Asp Thr Tyr Gly Ile Ala Leu Val Ser Gly Val Lys 165 170 175 Tyr Met Lys Gly Ser Leu Val Ile Asn Asp Thr Gly Leu Tyr Phe Val 180 185 190 Tyr Ser Lys Val Tyr Phe Arg Gly Gln Tyr Cys Asn Asn Gln Pro Leu 195 200 205 Ser His Lys Val Tyr Thr Arg Asn Ser Arg Tyr Pro Gln Asp Leu Val 210 215 220 Leu Met Glu Gly Lys Met Met Asn Tyr Cys Thr Thr Gly Gln Met Trp 225 230 235 240 Ala Arg Ser Ser Tyr Leu Gly Al a Val Phe Asn Leu Thr Ser Ala Asp 245 250 255 His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu 260 265 270 Ser Lys Thr Phe Phe Gly Leu Tyr Lys Leu 275 280 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <400> 3 tccccagatc tactgggt 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <400> 4 agattcctca aaattgacca g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <400> 5 cctctggaat gggaagacac 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <220> <221> m <222> 4 <223> a or c <220> <221> k <222> 6 <223> g, t or u <220> <221> r <222> 10 <223> g or a <220> <221> 14 <222> y <223> t, u or c <400> 6 gacmtkttgr accytgtggt c 21 < 210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <400> 7 atgcagcagc ccttcaatta cc 22 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR <400> 8 ctaggaattc tcagagctta tat aagcc 28

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/08 5/10 G01N 33/53 D C12P 21/08 C12P 21/02 C G01N 33 / 53 C12N 15/00 ZNAA // C12P 21/02 5/00 A (71) Applicant 597169292 Shimoji Yoshihiro 1-13-2 Takezono, Tsukuba, Ibaraki 804-303 (71) Applicant 597169328 Keigo Arai Inashiki, Ibaraki 18-1 Sakuragaoka, Kukizaki-cho, Gunma (72) Inventor Yoshihiro Muneda 2-11-11 Azuma, Tsukuba, Ibaraki 807-707 (72) Inventor Yasuyuki Mori 1-2, Tsukuba, Ibaraki Prefecture 72 (72) Yoshihiro Shimoji, Ibaraki 1-13-13, Takezono, Tsukuba 804-303 (72) Inventor Keigo Arai 18-1 Sakuragaoka, Kusazaki-cho, Inashiki-gun, Ibaraki F-term (reference) 4B024 AA10 BA28 CA04 DA06 EA04 GA11 GA18 GA19 H A01 HA14 4B064 AG01 AG27 CA02 CA10 CA19 CA20 CC24 CE04 CE11 CE12 DA11 DA13 DA15 4B065 AA26X AA90Y AB01 AB02 AC14 CA24 CA43 4H045 AA11 CA40 DA22 DA76 DA86 EA22 EA51 EA53 FA72 FA74 GA22 GA23 GA26

Claims (6)

[Claims] 1. A protein represented by the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence of SEQ ID NO: 2 consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and which has Fas ligand activity. Protein 2. A protein encoding the protein according to claim 1.
DNA or DNA complementary thereto. 3. An RNA complementary to the DNA according to claim 2. An expression vector comprising the DNA according to claim 2. A transformant transformed by the expression vector according to claim 4. 6. An antibody which reacts with the protein according to claim 1.