JPH09294589A - Dna coding for membrane protein of bovine macrophage and vector and transformant containing the dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new DNA, coding for a membrane protein associated with ion permeability of a bovine macrophage containing a specific amino acid sequence and useful for producing a cow having resistance against intracellularly invasive bacteria such as Salmonella bacteria. SOLUTION: A new DNA coding for a protein which is a membrane protein of a bovine macrophage and contains an amino acid sequence expressed by the amino acid numbers 1 to 548 in the amino acid sequence of the formula. For instance, a cow having resistance against intracellularly invasive bacteria such as Salmonella bacteria can be produced by using the DNA. The DNA is obtained by isolating mRNAs from a bovine spleen, synthesizing cDNAs by using the obtained mRNAs as templates, subsequently preparing a cDNA library from the resultant cDNAs according to an ordinary method, screening the library using a probe consisting of a part of a gene coding for a membrane protein associated with ion permeability of a bovine macrophage and collecting the cDNA from a positive clone.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding a bovine macrophage membrane protein, a vector containing the DNA and a transformant, and more specifically, it is effective against bovine intracellular invasive bacteria (for example, Salmonella). DNA encoding a membrane protein involved in ion permeability of various macrophages, a vector containing the DNA, and Escherichia coli transformed with the vector. INDUSTRIAL APPLICABILITY According to the present invention, the structure of a membrane protein involved in ion permeability of macrophages, which is effective against intracellular invasive bacteria, has been elucidated, and by utilizing the DNA encoding this membrane protein, intracellular invasive bacteria can be treated. It is possible to produce cows that are resistant.

[0002]

2. Description of the Related Art For livestock farmers and the like, it is an important subject to take protective measures against intracellular invasive bacteria such as Salmonella in cattle. As a conventional defense measure against intracellular invasive bacteria, a method using a drug such as an antibiotic or a vaccine is mainstream, but it is difficult to completely eliminate these bacteria from the bovine body by this method. Further, in breeding cattle, a variety having sufficient resistance to intracellular invasive bacteria has not yet been produced.

[0003]

The disease resistance of cattle against Salmonella and the like depends on the activity of macrophages capable of killing the bacteria. The activity of this macrophage is DN
Cattle with A predominance and disease resistance are expected to have DNA that activates macrophages. As such a DNA involved in macrophage activation, a gene encoding a membrane protein involved in macrophage ion permeability has been mentioned, and studies on it have been conducted. For example, in mice, a membrane protein involved in macrophage ion permeability (Natural Resistence-Associate) that is effective against intracellular invasive bacteria.
d Macrophage Protein, hereinafter abbreviated as Nramp. )
The gene that encodes E. coli has been elucidated (Vidal et al., C
ell, 73, 469-485 (1993)). The gene was also cloned in sheep, and a part of Nramp was clarified (Pitel et al., 24th International Society of Animal Genetics, 199).
4 years).

However, the existence and function of the gene have not been clarified in cattle. Therefore, if a gene effective against intracellular invasive bacteria such as Nramp gene can be found in cattle, is the macrophage membrane protein encoded by the gene resistant to intracellular invasive bacteria? It is possible to discriminate whether or not it is sensitive, and using this as an index for selection, it is possible to select cows that have the gene that codes for a resistant membrane protein and to produce cows that are resistant to intracellular invasive bacteria. Is. Therefore, it is an object of the present invention to provide a gene encoding a membrane protein involved in ion permeability of macrophages involved in resistance of bovine intracellular invasive bacteria.

[0005]

[Means for Solving the Problems] In order to achieve the above object, the present inventors have prepared a primer with reference to a portion of the sheep Nramp gene whose nucleotide sequence has been elucidated, and used the primer. Polymerase chain reaction (Sa
iki et al., Science, 230, 1350 (1985))
A part of the ramp-like gene was created. This cow Nramp
A membrane protein involved in ion permeability of macrophages involved in resistance to intracellular invasive bacteria in bovine was coded by hybridization from a cDNA library prepared from bovine spleen using a part of the gene The present invention has been completed by isolating the full length of the gene, analyzing the base sequence of the gene, and clarifying the corresponding amino acid sequence.

That is, the invention according to claim 1 is a membrane protein of bovine macrophage, which comprises a protein containing the amino acid sequence from amino acid number 1 to 548 described in SEQ ID NO: 2 of the sequence listing in its amino acid sequence. It is the encoding DNA. The invention according to claim 2 is a recombinant vector containing the DNA according to claim 1, and the invention according to claim 3 is Escherichia coli transformed with the vector according to claim 2.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. First, isolation and identification of a gene encoding a membrane protein involved in bovine macrophage ion permeability will be described. (1) Preparation of bovine spleen cDNA library Bovine spleen cDNA library was prepared from bovine spleen by mRN.
A is extracted and this is used as a template to prepare according to a conventional method.
The mRNA used in the present invention is the same as the mRNA containing Poly (A) (Poly (A) + RNA).

[0008] This method will be described in more detail. After obtaining mRNA from the bovine spleen, the mRNA
CDNA is synthesized using A as a template and oligo (dT) and random hexamer as primers. Then this cd
NA is incorporated into a vector such as λZAPII which is a phage DNA, and this is subjected to in vitro packaging (Kretz, PL, et al. Nucleic Acids Res. 17: 5409).
(1989)) to prepare a cDNA library. λZA
PII is a phage-to-plasmid (Bluescript)
It has a function of converting to tSK (-), and a plasmid can be easily prepared by utilizing this function.

(2) Preparation of bovine Nramp gene probe A primer is prepared with reference to a portion of the sheep Nramp gene whose nucleotide sequence has been elucidated, and the polymerase chain reaction method (Saiki et al. , Scienc
e, 230, 1350 (1985)) to produce part of the bovine Nramp-like gene. A part of this bovine Nramp-like gene is used as a probe for the following screening.

(3) Screening The above cDNA library was spread on a suitable medium plate together with Escherichia coli to form plaques, and then bovine Nra.
Plaque hybridization (Benton, WD and Davis, RW, Scie
nce, 196, 180 (1977)) to isolate positive phages. In the case of the present invention, as a result of such screening, 7 phages were obtained. These phage are transformed into plasmids. That is, E.I. col
When iXL1-Blue was infected with λZAPII and a helper phage, BluescriptS was detected from λZAPII.
Since K (-) is excised, it is used to transform E. coli, such as E. E. coli XL1-Blue etc. are transformed. In this way, a plasmid which is considered to have homology with the bovine Nramp gene is screened, and the desired bovine Nra is selected from the plasmid having a fragment of about 1.8 kb.
A DNA fragment similar to the mp gene is obtained.

Next, the nucleotide sequence of the gene encoding the membrane protein involved in the ion permeability of bovine macrophages isolated as described above is determined. (4) DNA nucleotide sequence determination The nucleotide sequence of the screened cDNA can be analyzed by a conventional method. For example, several cDNA fragments were prepared from the obtained cDNA, and the widely used dideoxy method (Sanger et al., Proc.
Natl. Acad. Sci. USA., 74, 5463-5467 (1977)) and Maxam-Gilbert method (Maxam et al., Proc. Natl. Aca
d. Sci. USA., 74, 560-564 (1977)) and the like to analyze the respective nucleotide sequences and comprehensively analyze them to determine the entire sequence.

Bovine cDNA obtained by this structural analysis
A has a total length of 1787 base pairs, contains the coding region of the mouse Nramp gene, and encodes a protein having 547 amino acids. The obtained bovine cDNA was used as mouse N
When compared with the ramp gene, it was revealed that there is similarity, and the homology at the amino acid level is 98.7.
% (See FIG. 1). Mouse Nramp has tryptophan at the 95th amino acid, but bovine Nramp
As described in SEQ ID NO: 2 in the sequence listing, the amino acid at the 95th amino acid is the translation terminator. Therefore, the translation region is divided, and the actual translation start is amino acid number 1.
From the 47th methionine, the cow Nramp is 10
It is expected to have individual transmembrane regions. Mouse Nramp has been reported to have 12 transmembrane regions (Govoni et al., Genomics, 27, 9-19 (199).
Five)).

[0013]

Next, the present invention will be described in detail with reference to examples. Example 1 Preparation of mRNA After extraction, 250 mg of bovine spleen stored on ice was subjected to ISOGE
Homogenizer with N (Nippon Gene Co., Ltd.) (Polytron, KINEMATIC, Switzerland)
AAG) and homogenized. Then, the mixture was left at room temperature for 5 minutes, added with chloroform and shaken vigorously, and then left at room temperature for 2 minutes. Then 3500 at 4 ° C for 10 minutes
Centrifuge at rpm. The upper layer was transferred to another container, isopropanol was added, the mixture was allowed to stand at room temperature for 5 minutes, and then centrifuged at 4500 for 10 minutes at 3500 rpm.
% Ethanol was added and shaken vigorously, followed by centrifugation at 3500 rpm at 4 ° C. for 6 minutes. The precipitate obtained is 3
After being dried for a minute, it was dissolved in 2 ml of sterilized water and stored at -20 ° C. Thus, from 250 mg tissue, about 1
mg total RNA was obtained.

500 μg of total RNA obtained by the above method
To a final concentration of 0.5M NaCl and 5M N
aCl was added. Further, 50 mg of oligo (dT) cellulose (Collaborative Res.) Was added, shaken for 20 minutes, and centrifuged at 3000 rpm for 6 minutes at room temperature to remove the supernatant. Add 10 ml of binding buffer (2
0 mM Tris-HCl (pH 7.6) /0.5M
NaCl / 1 mM EDTA (pH 8.0) /0.1%
Sodium lauryl sarcosinate) and stir up and down,
After centrifugation at 3000 rpm for 4 minutes at room temperature, the supernatant was removed (this was done twice).

Next, 0.8 ml of low salt wash buffe
r (20 mM Tris-HCl (pH 7.6) / 0.
1 M NaCl / 1 mM EDTA (pH 8.0)) was added, 0.4 ml was placed in a spin column, and the mixture was centrifuged at 4 ° C. for 10 seconds at 14000 rpm to remove the lower aqueous layer (this was performed twice). Furthermore low salt wash buffer
Was added and the mixture was centrifuged at 14,000 rpm for 10 seconds at 4 ° C. to remove the lower layer (this was repeated 3 times).

Then, the spin column was transferred to a new tube, 200 μl of sterilized water was added, and the spin column was kept at 4 ° C. for 10 seconds for 1 second.
Centrifuge at 4000 rpm (this was done twice).
This collected the Poly (A) + RNA fraction. This fraction was ethanol precipitated, dissolved in 50 μl of sterilized water, and stored at −20 ° C. In this way, 500
About 7.2 μg of Poly (A) + R from μg of total RNA
NA was obtained.

Example 2 Preparation of bovine spleen cDNA library Poly (A) + RNA (mRN obtained in Example 1
A) was used as a template, oligo (dT) _12-18 (Pharmacia) and random hexamer were used as primers, and cDNA was used.
A was synthesized. Next, add this to the vector λZAPII (St
(produced by ratagene) and packaged in vitro to prepare a cDNA library. λZAP
II is a 41 kb DNA having one cleavage site by the restriction enzyme EcoRI. The above cDNA was then added to T4 DNA ligase (Stratagene) at 4 ° C.
The DNA strands were ligated for 42 hours in vitro and in vitro packaging was performed using Gigapack II Gold (manufactured by Stratagene). This makes cDNA 100n
From the g, 5 × 10 ⁹ phages were obtained.

Example 3 Isolation of Gene Encoding Membrane Protein (Nramp) Related to Ion Permeability of Bovine Macrophages (1) Preparation of Probe for Bovine Nramp-Like Gene The nucleotide sequence of the sheep Nramp gene was elucidated. The primer is prepared with reference to the part that is present, and the polymerase chain reaction method (Saiki et al., Scienc
e, 230, 1350 (1985)), a part (1 kb) of a bovine Nramp-like gene is produced and used for the following screening.

(2) Screening The cDNA library prepared in Example 2 was used for E. coli XL.
After plating with NZY agar medium having the following composition along with 1-Blue to form plaques, take a replica on a nylon membrane filter, moisten the filter with 0.5M NaOH / 1.5M NaCl and alkali After treatment, 0.5M Tris-HCl (pH
7.5) /1.5 M NaCl, then 2 × S
After treating with SC and air-drying, irradiate with ultraviolet rays to DNA
Was fixed to a nylon membrane filter.

NZY medium (pH 7.5) NZ amine 10 g Sodium chloride 5 g Yeast extract 5 g Magnesium sulfate 2 g Distilled water 1000 ml NZY agar medium: 1.5% bacto agar in NZY medium.
Have.

5 × this nylon membrane filter
Prehybridization was performed at 40 ° C. for 2 hours in a solution containing SSPE / 20% formamide / 0.1 × Denhardt solution / 0.1 mg / ml salmon sperm DNA / 1% SDS. 1.0k of bovine Nramp-like gene already obtained
b was labeled with [α- ³² P] dCTP using a nick translation method kit (Pharmacia), and this was added to the hybridization solution, and hybridization was carried out at 40 ° C. for 2 days. After that, the nylon membrane filter was washed twice in a solution containing 1 × SSPE / 0.5% SDS for 20 minutes at room temperature and twice for 20 minutes at 45 ° C., and then the filter was air-dried, followed by autoradiography ( Laskey, RA, FEBS Letters, 82, 314 (1977))
Was done.

As a result of screening in this way,
Seven positive phages were isolated for the above probe.
These phages were converted into plasmids and used to transform E. coli XL1-Blue. This bacterium has been deposited at the Institute of Biotechnology, Institute of Biotechnology, and the deposit number is FERM BP-5434. Then, this E. coli was cultured on LB agar medium (pH 7.5) having the following composition containing ampicillin at 37 ° C. for 1 day to obtain ampicillin-resistant colonies.

LB medium (pH 7.5) Peptone 10 g Sodium chloride 10 g Yeast extract 5 g Distilled water 1000 ml LB agar medium: LB medium has 1.5% Bacto agar.

(3) Analysis of nucleotide sequence Alkali-SDS method (Birnboim, HC and Doly, J., Nuc
Plasmids were prepared from the above obtained colonies by leic Acids Research, 7, 1513 (1979)). 1 μg of plasmid was added to 50 mM Tris-HCl (pH 7.5) / 10
_{0mM NaCl / 10mM MgCl 2 / 1mM} D
It was digested with a restriction enzyme EcoRI (2 units) in DT at 37 ° C. for 60 minutes for cleavage, and the obtained DNA fragment was subjected to electrophoresis for fractionation. Of these, those having a 1.8 kb fraction were analyzed for their nucleotide sequences by the dideoxy method, and it was confirmed that they were similar to the mouse Nramp gene. In addition, as a result of structural analysis of other DNA fragments,
It was revealed that those having the 1.8 kb fraction had the entire length of the nucleotide sequence corresponding to the bovine Nramp gene.

The base sequence of the 1.8 kb fraction is shown in SEQ ID NO: 1 in the sequence listing. The total length is 1787 base pairs,
The amino acid sequence corresponding to the nucleotide sequence of nucleic acid Nos. 27 to 1670 described in SEQ ID NO: 1 in Sequence Listing is described in SEQ ID NO: 2 in Sequence Listing. In addition, in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, the 95th symbol ** indicates that the corresponding nucleotide sequence is a translation termination and the corresponding amino acid does not exist. The bovine cDNA obtained by this structural analysis has a total length of 1787 base pairs, and includes a coding region of the mouse Nramp gene and encodes a protein having 547 amino acids. When the amino acid sequence of the obtained bovine Nramp was compared with the amino acid sequence of mouse Nramp, the homology at the amino acid level was 98.7% (see FIG. 1). In particular, mouse Nramp has tryptophan at the 95th amino acid, but cow Nramp
Amino acid No. 95 as described in SEQ ID No. 2 in the sequence listing
The th amino acid is the termination of translation. Therefore, the translation region is divided, and the actual translation start is at amino acid number 147.
From the second methionine, bovine Nramp is expected to have 10 transmembrane regions.

[0026]

INDUSTRIAL APPLICABILITY The present invention provides a gene encoding a membrane protein involved in ion permeability of macrophages involved in resistance to bovine intracellular invasive bacteria,
By utilizing this, it becomes possible to produce a cow having resistance to intracellular invasive bacteria.

[0027]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1787 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Direct origin: Bovine spleen cDNA library Sequence characteristics Features Symbol: domain Location: 27..1673 Method of determining features: S sequence CAGAGTATCC TGCCGCCTGC GTCCTC ATG ATT AGT GAC AAG AGC CCC CCG AGG 53 CTG AGC AGG CCC AGT TAT GGC TCC ATT TCC AGC CTG CCT GGC CCA GCA 101 CCT CAG CCA GCG CCT TGC CTG GAG ACC TAC CTG AGT GAG AAG ATC CCC 149 ATT CCC AGC GCA GAC CAG GGT ACA TTC AGC CTG AGG AAG CTG TGG GCG 197 TTC ACG GGG CCT GGT TTC CTC ATG AGC ATC GCT TTC GTT GAC CTC 245 AAC ATT GAG TCC GAC CTT CAA GCT GGC GCT GTG GCT GGG TTC AAA CTC 293 CTC TGG GTG CTG CTC TGA GCG ACT GTG CTA GGT TTG CTG TGC CAG CGG 341 CTG GCT GCC CGG CTG GGC GTG GTA ACA GGC AAG GAC TTG GGT GTC 389 TGC CAT CTC TAC TAC CCC AAG GTG CCC CGC ATC CTC CTC TGG CTG ACC 437 ATT GAG CTG GCC ATT GTG GGC TCA GAT ATG CAG GAA GTC ATC GGG ACG 4 85 GCT ATC TCC TTC AAT CTG CTC TCC GCT GGA CGC ATC CCG CTG TGG GAC 533 GGT GTA CTG ATC ACC ATT GTG GAC ACC TTC TTC TTC CTC TTC TTG GAT 581 AAC TAT GGT TTG CGC AAG CTG GAA GCT TTC TTC GATT CTC CTC CTC ACC 629 ATA ATG GCT TTG ACC TTC GGC TAT GAG TAT GTG GTA GCA CAC CCT TCC 677 CAG GGA GCG CTC CTT AAG GGC CTG GTG CTG CCC ACC TGT CCG GGC TGT 725 GGG CAG CCC GAG CTG CTG CAG GCA GTG GGC ATC GTC GTC GTC ATC ATC 773 ATG CTC CAT AAC ATC TAC CTG CAC TCA GCC TTG GTC AAG TCT AGA GAA 821 GTA GAC AGA ACC CGC CGG GTG GAT GTT CGA GAA GCC AAC ATG TAC TTC 869 CTG ATT GAG GCC ACC ATC GCC CTA TCG ATG TCC TCC TTC ATC GTC AAC CTC 917 TTC GTC ATG GCT GTT TTT GGT CAG GCC TTC TAC CAG CAA GCC AAT GAG 965 GAA GCG TTC AAC ATC TGT GCC AAC AGC AGC CTC CAG AAC TAT GCT AAG 1013 ATC TTC CCC AGG GAC AAT AAC ACT GAT TCA GTG ATT TAT CAA GGA 1061 GGT GTG ATC CTA GGC TGT CTC TTT GGC CCT GCG GCC CTC TAC ATC TGG 1109 GCA GTA GGT CTC CTG GCA GCG GGG CAG AGT TCT ACT ATG ACC GGC ACC 1157 TAT GCA GGA CAG TTC GTG ATG CAG GGT TTC TT AAG CTG CGG TGG TCC 1205 CGC TTC GCT CGG GTC CTT CTC ACG CGC TCT TGC GCC ATC CTG CCC ACT 1253 GTG TTG GTG GCT GTC TTC CGA GAC CTG AAG GAC CTG TCC GGC CTC AAC 1301 GAA CTA CTC AAT GTT CTG CAG AGT CTG CTG CCC TTC GCT GTA CTG 1349 CCC ATT TTG ACT TTC ACC AGC ATG CCA GCT GTC ATG CAG GAG TTC GCC 1397 AAC GGC CGG ATG AGC AAA GCC ATC ACT TCG TGC ATC ATG GCG CTA GTC 1445 TGC GCC ATC AAC CTG TAC TTT TTT ATC AGC TAC CTG CCC AGC CTC CCG 1493 CAC CCT GCC TAC TTC GGC CTT GTG GCT CTG TTC GCA ATA GGT TAC TTG 1541 GGC CTG ACT GCT TAT CTG GCC TGG ACC TGT TGC ATC GCC CAC GGA GCC 1589 ACC TTC CTG TACC CAC AGC CAC AAG CAC TTC TTA TAT GGG CTC CCT 1637 AAC GAG GAG CAG GGA GGC GTG CAG GGT TCC TGG TGA CCGCGGCATC 1683 CAGCAAGCAA AGAGGCAACA GGGCAGACAC AGCAGAGCAA TTGGAGGTCC CTCACTGTGG 1743 TTCTGGATTA CCGGTCTCCAGT

SEQ ID NO: 2 Sequence length: 548 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Ile Ser Asp Lys Ser Pro Pro Arg Leu Ser Arg Pro Ser Tyr Gly 1 5 10 15 Ser Ile Ser Ser Leu Pro Gly Pro Ala Pro Gln Pro Ala Pro Cys Leu 20 25 30 Glu Thr Tyr Leu Ser Glu Lys Ile Pro Ile Pro Ser Ala Asp Gln Gly 35 40 45 Thr Phe Ser Leu Arg Lys Leu Trp Ala Phe Thr Gly Pro Gly Phe Leu 50 55 60 Met Ser Ile Ala Phe Leu Asp Pro Gly Asn Ile Glu Ser Asp Leu Gln 65 70 75 80 Ala Gly Ala Val Ala Gly Phe Lys Leu Leu Trp Val Leu Leu *** Ala 85 90 95 Thr Val Leu Gly Leu Leu Cys Gln Arg Leu Ala Ala Arg Leu Gly Val 100 105 110 Val Thr Gly Lys Asp Leu Gly Glu Val Cys His Leu Tyr Tyr Pro Lys 115 120 125 Val Pro Arg Ile Leu Leu Trp Leu Thr Ile Glu Leu Ala Ile Val Gly 130 135 140 Ser Asp Met Gln Glu Val Ile Gly Thr Ala Ile Ser Phe Asn Leu Leu 145 150 155 160 Ser Ala Gly Arg Ile Pro Leu Trp Asp Gly Val Leu Ile Thr Ile Val 165 170 175 Asp Thr Phe Phe Phe L eu Phe Leu Asp Asn Tyr Gly Leu Arg Lys Leu 180 185 190 Glu Ala Phe Phe Gly Leu Leu Ile Thr Ile Met Ala Leu Thr Phe Gly 195 200 205 Tyr Glu Tyr Val Val Ala His Pro Ser Gln Gly Ala Leu Leu Lys Gly 210 215 220 Leu Val Leu Pro Thr Cys Pro Gly Cys Gly Gln Pro Glu Leu Leu Gln 225 230 235 240 Ala Val Gly Ile Val Gly Ala Ile Ile Met Leu His Asn Ile Tyr Leu 245 250 255 His Ser Ala Leu Val Lys Ser Arg Glu Val Asp Arg Thr Arg Arg Val 260 265 270 Asp Val Arg Glu Ala Asn Met Tyr Phe Leu Ile Glu Ala Thr Ile Ala 275 280 285 Leu Ser Val Ser Phe Ile Val Asn Leu Phe Val Met Ala Val Phe Gly 290 295 300 Gln Ala Phe Tyr Gln Gln Ala Asn Glu Glu Ala Phe Asn Ile Cys Ala 305 310 315 320 Asn Ser Ser Leu Gln Asn Tyr Ala Lys Ile Phe Pro Arg Asp Asn Asn 325 330 335 Thr Val Ser Val Asp Ile Tyr Gln Gly Gly Val Ile Leu Gly Cys Leu 340 345 350 Phe Gly Pro Ala Ala Leu Tyr Ile Trp Ala Val Gly Leu Leu Ala Ala 355 360 365 Gly Gln Ser Ser Thr Met Thr Gly Thr Tyr Ala Gly Gln Phe Val Met 370 375 380 Glu Gly Phe Leu Lys LeuArg Trp Ser Arg Phe Ala Arg Val Leu Leu 385 390 395 400 Thr Arg Ser Cys Ala Ile Leu Pro Thr Val Leu Val Ala Val Phe Arg 405 410 415 Asp Leu Lys Asp Leu Ser Gly Leu Asn Glu Leu Leu Asn Val Leu Gln 420 425 430 Ser Leu Leu Leu Pro Phe Ala Val Leu Pro Ile Leu Thr Phe Thr Ser 435 440 445 Met Pro Ala Val Met Gln Glu Phe Ala Asn Gly Arg Met Ser Lys Ala 450 455 460 Ile Thr Ser Cys Ile Met Ala Leu Val Cys Ala Ile Asn Leu Tyr Phe 465 470 475 480 Val Ile Ser Tyr Leu Pro Ser Leu Pro His Pro Ala Tyr Phe Gly Leu 485 490 495 Val Ala Leu Phe Ala Ile Gly Tyr Leu Gly Leu Thr Ala Tyr Leu Ala 500 505 510 Trp Thr Cys Cys Ile Ala His Gly Ala Thr Phe Leu Thr His Ser Ser 515 520 525 His Lys His Phe Leu Tyr Gly Leu Pro Asn Glu Glu Gln Gly Gly Val 530 535 540 Gln Gly Ser Trp 545

[Brief description of drawings]

1 shows a comparison of the amino acid sequence of the bovine mouse Nramp-like gene and the mouse Nramp gene. The upper row in the figure shows the amino acid sequence of the mouse Nramp gene, and the lower row shows the amino acid sequence of the bovine mouse Nramp-like gene. In addition, ── in the figure indicates that the amino acid does not exist, and if the amino acids are the same,
*, If similar, indicated by a. A comparison table of amino acid names in one-letter code is shown below. One-letter code Amino acid G Glycine A Alanine V Valine L Leucine I Isoleucine S Serine T Threonine D Aspartic acid E Glutamate N Asparagine Q Glutamine K Lysine R Arginine C Cysteine M Methionine F Phenylalanine Y Tyrosine P-H tryptophosphine W tryptone

Claims (3)

[Claims] 1. A bovine macrophage membrane protein,
A DNA encoding a protein containing the amino acid sequence of amino acid numbers 1 to 548 set forth in SEQ ID NO: 2 in the sequence listing in its amino acid sequence. 2. A recombinant vector containing the DNA according to claim 1. 3. Escherichia coli transformed with the vector according to claim 2.