JPH0515375A - Dna sequence encoding human intrinsic factor - Google Patents

Abstract

PURPOSE:To obtain a sequence useful for production of human intrinsic factor by gene technology by using the sequence as a probe which is complementary with a human gastric cell derived mRNA, encodes a human intrinsic factor and is capable of readily and surely cloning cDNA of human intrinsic factor. CONSTITUTION:A RNA is extracted from human gastric tissue by a common method such as thiocyanguanidine method or hot phenol method and treated by an oligo(dT)cellulose column to prepare a human gastric cell derived mRNA as poly(A)<+>RNA, the mRNA is used as a template to synthesize cDNA with a reverse transcriptase, the cDNA is amplified by PCR method, a cDNA library is prepared by a conventional procedure, the library is cloned by using a probe prepared by synthesizing a DNA encoding a well-known human intrinsic factor amino acid part sequence, a clone containing a DNA encoding a human intrinsic factor is selected and the DNA is recovered to give a DNA sequence which is complementary with a human gastric cell derived mRNA and encodes a human intrinsic factor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to DNA, DNA sequences and proteins and methods for producing proteins.
That is, D effective for cloning the human intrinsic factor gene
The present invention relates to an NA probe, a human endogenous factor cDNA sequence effective for the production of human intrinsic factor by a genetic engineering technique, and a method for producing a protein having an elucidated human intrinsic factor amino acid sequence.

[0002]

BACKGROUND OF THE INVENTION The intrinsic factor is a glycoprotein having a molecular weight of 40 to 50 kd (kilodalton), which is a vitamin B12-binding protein produced in human gastric parietal cells. The physiological function of intrinsic factor is to absorb vitamin B12 through a specific receptor present in the ileum. Intrinsic factor is also known as one autoantigen, and a person having an autoantibody (anti-intrinsic factor antibody) against this factor exhibits vitamin B12 deficiency symptoms such as pernicious anemia and peripheral neuropathy. Therefore, human intrinsic factor is important as a diagnostic agent for diagnosing anti-intrinsic factor antibody and T cells that react with intrinsic factor. Also, human intrinsic factor is vitamin B12 alone.
It is used as an oral preparation of vitamin B12 in the form of an absorption enhancer or an intrinsic factor-vitamin B12 complex. It is known to mediate the absorption of conjugates of vitamin B12 and other substances such as vitamin B12 and proteins and peptides, and has a use as a carrier for promoting absorption of the conjugates. It is also important as a reagent for quantifying vitamin B12 that binds to intrinsic factor.

For use as a diagnostic agent, a vitamin B12 absorption enhancer, a carrier for promoting absorption of a conjugate of vitamin B12 and another substance, or a reagent for quantifying vitamin B12, a large amount of human intrinsic factor is used. It is necessary to prepare Although human intrinsic factor can be separated and purified from gastric juice, it is very difficult to prepare a sufficient amount for the above-mentioned use in terms of availability of raw materials, process and labor.

[0004] At present, a protein which is present only in a trace amount can be produced inexpensively and in large quantities by a genetic engineering technique. However, in order to produce human intrinsic factor by genetic engineering, the necessary human intrinsic factor gene is first isolated. Need to be separated (cloned).

However, until now, a partial amino acid sequence of human intrinsic factor has been known (Hansen Em.
Scandinavian Journal of Clinical and Laboratory Investigation (Scand, J, Clin, Lab.Invest.) Volume 49, Supplement (Suppl.) 194: 19) (SEQ ID NO: 4 in the sequence listing) (SEQ ID NO: 3 is the amino acid sequence of rat intrinsic factor), the complete amino acid sequence is unknown, and the nucleotide sequence of the human intrinsic factor gene is unknown at all.

[0006] Several methods may be used for cloning the gene encoding human intrinsic factor. For example, by using a mixture of DNA probes deduced from the partial amino acid sequence of human intrinsic factor (mixed probe) currently known, a human intrinsic factor cDNA is derived from a cDNA library derived from cells expressing human intrinsic factor. Could be screened. However, the c selected with this mixed DNA probe
DNA cannot be truly confirmed as a human intrinsic factor gene unless its nucleotide sequence is determined. Further, the mixed DNA probe is not suitable for detecting the human intrinsic factor gene. Also, the method of Lathe et al. (Lase et al. (1985) Journal of Molecular Biology (J. Molec. Biol.), 18
The DNA probe predicted and prepared according to Volume 3, page 1) is not always effective for gene cloning. Antibodies to human intrinsic factor are known,
Although it is possible that cloning of a target gene can be achieved by screening a gene expression library using the antibody labeled with a radioactive substance, there is no report that the antibody is truly usable for cloning. Therefore,
Until now, it can be said that there is no known means for reliably cloning the human intrinsic factor gene. A DNA probe effective for reliable cloning of the human intrinsic factor gene is
DN which has the nucleotide sequence of human intrinsic factor gene as it is
A probe. Such a DNA probe can selectively hybridize with the human intrinsic factor gene or mRNA and is highly effective in detecting them.

The problem to be solved by the present invention is to provide a sequence of a DNA probe for easily and reliably cloning such a human intrinsic factor gene,
Further, it is to provide a human intrinsic factor gene cloned using the probe and its nucleotide sequence. Once the human intrinsic factor gene is isolated, it is possible to produce human intrinsic factor and its mutants by genetic engineering for the above-mentioned purposes and uses.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a DN consisting of the base sequence shown in SEQ ID NO: 1 or a part of the base sequence.
A, a DNA having the nucleotide sequence shown by SEQ ID NO: 1 or a part of the nucleotide sequence, a protein having the amino acid sequence shown by SEQ ID NO: 2, and a method for producing the same.

[0009]

[Examples] As a result of intensive studies to solve the above-mentioned problems, the present inventors have utilized a known human intrinsic factor amino acid partial sequence and performed PCR (polymerase chain reaction) method (Molecula 2nd edition).
r Cloning 2nd edition), J. Sambrook (J.
Sambrook, et al., Cold Spring Harbor Laboratory (1989)
); K. Knoth et al. (1988) Nucleic Acids Research 16
Vol. 109, p. 10932) to find a method for amplifying a cDNA which is a part of a gene encoding human intrinsic factor, and determining the nucleotide sequence of this cDNA is effective for reliably cloning the human intrinsic factor gene. The present invention was completed by obtaining a DNA probe and a clone having human intrinsic factor cDNA.

The DNA sequence of the present invention and the method for obtaining the DNA will be described.

First, human intrinsic factor mRNA is prepared.
Human intrinsic factor mRNA is extracted from cells or organs expressing human intrinsic factor, such as the stomach, by a conventional method such as the thiocyanguanidine method or the hot phenol method, and further poly (A) is extracted by an oligo (dT) cellulose column. ⁺ Can be prepared as RNA (Lab Manual Genetic Engineering, edited by Masami Muramatsu, Maruzen 1988
Year).

Next, from the obtained mRNA, a conventional method or a commercially available kit such as Pharmacia cD is used.
CDNA for human intrinsic factor is prepared using the NA synthesis kit.

A primer predicted from the amino-terminal or carboxyl-terminal sequence of the known human intrinsic factor amino acid partial sequence is synthesized. The cDNA thus obtained is amplified by the PCR method using the synthesized mixed primers. Amplification by the PCR method may be carried out by the number of cycles so that a sufficient amount of cDNA can be obtained for subsequent cloning. The amplified cDNA is detected and extracted by ordinary gel electrophoresis such as agarose gel electrophoresis or polyacrylamide gel electrophoresis.

The extracted cDNA can be cloned by using an appropriate vector such as pBR322, pUC18, or the like in an appropriate host such as Escherichia coli, and the nucleotide sequence can be determined by an ordinary method such as Sanger method.

The human intrinsic factor D thus determined
The DNA having the NA sequence is the human intrinsic factor gene and m
It can be used as a useful probe for the detection of RNA. That is, the full-length cDNA of human intrinsic factor is
It can be easily detected from a cDNA library using this probe. Moreover, it can be amplified by the PCR method using the primer having the determined sequence and cloned. The cloned full-length cDNA of human intrinsic factor is characterized by analysis with a restriction enzyme or determination of the nucleotide sequence.

[0016] The full-length cD of the human intrinsic factor thus obtained
The protein of the present invention can be obtained by culturing NA using an appropriate vector and an appropriate host thereto, for example, bacteria such as Escherichia coli and Bacillus, yeast, mold, or animal and plant cultured cells. .

Next, the DNA sequence, the DNA, the protein and the method for producing the protein of the present invention will be explained based on examples, but the present invention is not limited to such examples. Example 1 a. Synthesis of PCR primer for human intrinsic factor cDNA For human intrinsic factor, a partial sequence is known as shown in SEQ ID NO: 4. As a primer for PCR of human intrinsic factor cDNA, a sequence containing the recognition sequence of the restriction enzyme BamHI and the subsequent DNA sequence predicted from the 95th to 103rd amino acid sequence AspVal Pro Gln Met Glu Asn TrpAla of SEQ ID NO: 4 were used. Shown in SEQ ID NO: 5
Sequence containing recognition sequence of 5'-side mixed primer and restriction enzyme PstI and amino acid sequence from 164th to 171st Cys Met Tyr Asn Lys Ile ProVal predicted D
The 3'-side mixed primer represented by SEQ ID NO: 6 having an NA sequence was synthesized using a DNA synthesizer (Applied Biosystems, model 381A). b. Amplification and cloning of human intrinsic factor gene sequence by PCR The cDNA sequence of human intrinsic factor was amplified in vitro by the PCR method. That is, 10 mM Tris-HCl (pH 8.3), 100 mM
Potassium chloride, 1.5 mM magnesium chloride, 0.01% gelatin, 10 μ each of the two kinds of mixed primers synthesized in the above a.
M, DNA extracted from human stomach cDNA library (λgt11) purchased from Clontech, 10 ng, 0.2 mM
100 μl of a reaction mixture containing four deoxynucleotide triphosphates and 2.5 units of Taq DNA polymerase was added at 94 ° C. for 1 hour.
Min, 37 ° C for 2 minutes, 72 ° C for 2 minutes under 35 cycles of PC
R was performed. After the reaction, the size of the PCR product was measured by polyacrylamide gel electrophoresis.
It was confirmed that the base pair DNA was amplified. This about
DNA of 340 base pairs was extracted from the gel and the restriction enzyme Bam was used.
After treatment with HI and PstI, it was incorporated into the BamHI-PstI site of the M13mp19 phage vector (Takara Shuzo),
Subcloning was performed using E. coli as a host. c. Sequencing of Gene Amplified by PCR The sequence of DNA of about 340 base pairs prepared from the positive clone obtained in the above b was determined by a conventional method using dideoxynucleotide triphosphate. As a result, the nucleotide sequence between the two types of primers used for PCR and the deduced amino acid sequence were 48 to 307 to 48 in SEQ ID NO: 1, respectively.
It was determined to be the 9th nucleotide sequence and the 103rd to 163rd amino acid sequences.

This sequence corresponds to the known partial amino acid sequence of human intrinsic factor shown in SEQ ID NO: 4 from Ala 103 to Th 163.
Corresponding to r, the amino acid sequence of the reported human intrinsic factor, Glu at 118 is Ser, and Val at 157 is Me.
It was found that the unidentified sequence from 130 to 138 was Glu Ala Thr Leu Pro Ile Ala Val Arg and the 148th amino acid was Ser. d. Isolation of Human Intrinsic Factor cDNA Clone and Determination of Nucleotide Sequence In order to clone the full-length human Intrinsic Factor cDNA, the human Intrinsic Factor cDNA sequenced in Example 1-c
Was used to screen a human stomach cDNA library (Clontech). That is, the probes shown in SEQ ID NOS: 7 and 8 having the cDNA sequence of human intrinsic factor were synthesized. Screening of about 600,000 recombinant phages with these probes resulted in 1.5 kb (kilobase) and 1.0 kb of insert cDNA 2
Strain positive clones (HI-9, HI-10) were obtained. Of these positive clones, insert cDNA of HI-9
The sequence was determined by a conventional method using dideoxynucleotide triphosphate. As a result, it was determined that the cDNA base sequence of human intrinsic factor and the deduced amino acid sequence are as shown in SEQ ID NO: 1.

When this sequence is compared with the known partial amino acid sequence of human intrinsic factor shown in SEQ ID NO: 4, it is found to be 27 of the known sequence.
The second Thr is Asn, Ala Gln Ile Leu from 82 to 85
Is the sequence of Thr Ser Ser Cys, and Le from 88 to 89 is
u Lys sequence is replaced by Pro Gly sequence and Tyr from 93 to 97
Leu Asp Val Pro sequence is Ser Ile Leu Gln Arg sequence, 118 Glu is Ser, 128 Phe is Asn, 15
Val 7 is for Met, Val 194 is for Met, and 203 is
Gln is Gly and the sequence of Gly Leu from 356 to 357 is As.
n Asn array, 361 Gly is Asn, 368 to 36
It was found that the Gly Val sequence at position 9 was replaced with the Trp Gln sequence.

[0020]

EFFECT OF THE INVENTION Using the DNA of the present invention as a probe, cDNA for human intrinsic factor can be easily and reliably cloned.

With the human cDNA sequence of the present invention, the amino acid sequence of human intrinsic factor can be determined, and the DNA sequence encoding the intrinsic factor can be synthesized from this amino acid sequence.

Further, the human intrinsic factor cDNA of the present invention can be used for the genetic engineering production of human intrinsic factor. As a host, bacteria such as E. coli and Bacillus, yeast,
Molds or cultured cells of animals and plants can be used. Furthermore, it is possible to analyze and clone the human intrinsic factor gene in the genome. The genomic DNA may contain introns and is useful for genetically engineered production of human intrinsic factor using animal cultured cells as hosts.

In the production of human intrinsic factor by genetic engineering, not only a molecule having an amino acid sequence of naturally occurring human intrinsic factor but also a mutant having artificially one or more amino acid substitutions, insertions or deletions , Can produce fusion proteins with other proteins. In addition, a derivative obtained by chemically or enzymatically modifying them can be prepared. Among them, those having different affinity for vitamin B12, those having different affinity for receptors and immunogenicity as compared with natural human factor are included.

The genetically engineered human intrinsic factor and its mutants or their derivatives are vitamin B12.
If it has an affinity with, it can be a reagent for the determination of vitamin B12. As a quantitative method, a competitive binding reaction between vitamin B12 labeled with an enzyme, a fluorescent reagent or an isotope and vitamin B12 contained in a subject can be used.

[0025]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 1197 Sequence type: Nucleic acid chain number: Single strand (or double strand) Sequence type: cDNA to mRNA Hypothetical sequence: No Origin organism name: Homo sapiens (Homo sapiens) Tissue type: direct origin of stomach Library name: human stomach cDNA library (λgt
11) (Clontech) Sequence AGT ACC CAG ACC CAG AGT TCA TGC TCC GTT CCC TCA GCA CAG GAG CCC 48 Ser Thr Gln Thr Gln Ser Ser Cys Ser Val Pro Ser Ala Gln Glu Pro 1 5 10 15 TTG GTC AAT GGA ATA CAA GTA CTC ATG GAG AAC TCG GTG ACT TCA TCA 96 Leu Val Asn Gly Ile Gln Val Leu Met Glu Asn Ser Val Thr Ser Ser 20 25 30 GCC TAC CCA AAC CCC AGC ATC CTG ATT GCC ATG AAT CTG GCC GGA GCC 114 Ala Tyr Pro Asn Pro Ser Ile Leu Ile Ala Met Asn Leu Ala Gly Ala 35 40 45 TAC AAC TTG AAG GCC CAG AAG CTC CTG ACT TAC CAG CTC ATG TCC AGC 192 Tyr Asn Leu Lys Ala Gln Lys Leu Leu Thr Tyr Gln Leu Met Ser Ser 50 55 60 GAC AAC AAC GAT CTA ACC ATT GGG CAG CTC GGC CTC ACC ATC ATG GCC 240 Asp Asn Asn Asp Leu Thr Ile Gly Gln Leu Gly Leu Thr Ile Met Ala 65 70 75 80 CTC ACC TCC TCC TGC CGA GAC CCT GGG GAT AAA GTA TCC ATT CTA CAA 288 Leu Thr Ser Ser Cys Arg Asp Pro Gly Asp Lys Val Ser Ile Leu Gln 85 90 95 AGA CAA ATG GAG AAC TGG GCA CCT TCC AGC CCC AAC GCT GAA GCA TCA 336 Arg Gln Met Glu Asn Trp Ala Pro Ser Ser Pr o Asn Ala Glu Ala Ser 100 105 110 GCC TTC TAT GGG CCC AGT CTA GCG ATC TTG GCA CTG TGC CAG AAG AAC 384 Ala Phe Tyr Gly Pro Ser Leu Ala Ile Leu Ala Leu Cys Gln Lys Asn 115 120 125 TCT GAG GCG ACC TTG CCG ATA GCC GTC CGC TTT GCC AAG ACC CTG CTG 432 Ser Glu Ala Thr Leu Pro Ile Ala Val Arg Phe Ala Lys Thr Leu Leu 130 135 140 GCC AAC TCC TCT CCC TTC AAT GTA GAC ACA GGA GCA ATG GCA ACC TTG 480 Ala Asn Ser Ser Pro Phe Asn Val Asp Thr Gly Ala Met Ala Thr Leu 145 150 155 160 GCT CTG ACC TGT ATG TAC AAC AAG ATC CCT GTA GGT TCA GAG GAA GGT 528 Ala Leu Thr Cys Met Tyr Asn Lys Ile Pro Val Gly Ser Glu Glu Gly 165 170 175 TAC AGA TCC CTG TTT GGT CAG GTA CTA AAG GAT ATT GTG GAG AAA ATC 576 Tyr Arg Ser Leu Phe Gly Gln Val Leu Lys Asp Ile Val Glu Lys Ile 180 185 190 AGC ATG AAG ATC AAA GAT AAT GGC ATC ATT GGA GAC ATC TAC AGT ACT 624 Ser Met Lys Ile Lys Asp Asn Gly Ile Ile Gly Asp Ile Tyr Ser Thr 195 200 205 GGC CTC GCC ATG CAG GCT CTC TCT GTA ACA CCT GAG CCA TCT AAA AAG 672 Gly Leu Ala Met Gln Ala Leu Se r Val Thr Pro Glu Pro Ser Lys Lys 210 215 220 GAA TGG AAC TGC AAG AAG ACT ACG GAT ATG ATA CTC AAT GAG ATT AAG 720 Glu Trp Asn Cys Lys Lys Thr Thr Asp Met Ile Leu Asn Glu Ile Lys 225 230 235 240 CAG GGG AAA TTC CAC AAC CCC ATG TCC ATT GCT CAA ATC CTC CCT TCC 768 Gln Gly Lys Phe His Asn Pro Met Ser Ile Ala Gln Ile Leu Pro Ser 245 250 255 CTG AAA GGC AAG ACA TAC CTA GAT GTG CCC CAG GTC ACT TGT AGT CCT 816 Leu Lys Gly Lys Thr Tyr Leu Asp Val Pro Gln Val Thr Cys Ser Pro 260 265 270 GAT CAT GAG GTA CAA CCA ACT CTA CCC AGC AAC CCT GGC CCT GGC CCC 864 Asp His Glu Val Gln Pro Thr Leu Pro Ser Asn Pro Gly Pro Gly Pro 275 280 285 ACC TCT GCA TCT AAC ATC ACT GTC ATA TAC ACC ATA AAT AAC CAG CTG 912 Thr Ser Ala Ser Asn Ile Thr Val Ile Tyr Thr Ile Asn Asn Gln Leu 290 295 300 AGG GGG GTT GAG CTG CTC TTC AAC GAG ACC ATC AAT GTT AGT GTG AAA 960 Arg Gly Val Glu Leu Leu Phe Asn Glu Thr Ile Asn Val Ser Val Lys 305 310 315 320 AGT GGG TCA GTG TTA CTT GTT GTC CTA GAG GAA GCA CAG CGC AAA AAT 1008 Ser Gly Ser V al Leu Leu Val Val Leu Glu Glu Ala Gln Arg Lys Asn 325 330 335 CCT ATG TTC AAA TTT GAA ACC ACA ATG ACA TCT TGG GGC CTT GTC GTC 1056 Pro Met Phe Lys Phe Glu Thr Thr Met Thr Ser Trp Gly Leu Val Val 340 345 350 TCT TCT ATC AAC AAT ATC GCG GAA AAT GTT AAT CAC AAG ACA TAC TGG 1104 Ser Ser Ile Asn Asn Ile Ala Glu Asn Val Asn His Lys Thr Tyr Trp 355 360 365 CAG TTT CTT AGT GGT GTA ACA CCT TTG AAT GAA GGG GTT GCT GAC TAC 1152 Gln Phe Leu Ser Gly Val Thr Pro Leu Asn Glu Gly Val Ala Asp Tyr 370 375 380 ATA CCC TTC AAC CAC GAG CAC ATC ACA GCC AAT TTC ACA CAG TAC 1197 Ile Pro Phe Asn His Glu His Ile Thr Ala Asn Phe Thr Gln Tyr 385 390 395

SEQ ID NO: 2 Array length: 399 Sequence type: Amino acid Topology: linear Sequence type: Protein Hypothetical array: Yes Array Ser Thr Gln Thr Gln Ser Ser Cys Ser Val Pro Ser Ala Gln Glu Pro                   5 10 15 Leu Val Asn Gly Ile Gln Val Leu Met Glu Asn Ser Val Thr Ser Ser              20 25 30 Ala Tyr Pro Asn Pro Ser Ile Leu Ile Ala Met Asn Leu Ala Gly Ala          35 40 45 Tyr Asn Leu Lys Ala Gln Lys Leu Leu Thr Tyr Gln Leu Met Ser Ser      50 55 60 Asp Asn Asn Asp Leu Thr Ile Gly Gln Leu Gly Leu Thr Ile Met Ala  65 70 75 80 Leu Thr Ser Ser Cys Arg Asp Pro Gly Asp Lys Val Ser Ile Leu Gln                  85 90 95 Arg Gln Met Glu Asn Trp Ala Pro Ser Ser Pro Asn Ala Glu Ala Ser             100 105 110 Ala Phe Tyr Gly Pro Ser Leu Ala Ile Leu Ala Leu Cys Gln Lys Asn         115 120 125 Ser Glu Ala Thr Leu Pro Ile Ala Val Arg Phe Ala Lys Thr Leu Leu     130 135 140 Ala Asn Ser Ser Pro Phe Asn Val Asp Thr Gly Ala Met Ala Thr Leu 145 150 155 160 Ala Leu Thr Cys Met Tyr Asn Lys Ile Pro Val Gly Ser Glu Glu Gly                 165 170 175 Tyr Arg Ser Leu Phe Gly Gln Val Leu Lys Asp Ile Val Glu Lys Ile             180 185 190 Ser Met Lys Ile Lys Asp Asn Gly Ile Ile Gly Asp Ile Tyr Ser Thr         195 200 205 Gly Leu Ala Met Gln Ala Leu Ser Val Thr Pro Glu Pro Ser Lys Lys     210 215 220 Glu Trp Asn Cys Lys Lys Thr Thr Asp Met Ile Leu Asn Glu Ile Lys 225 230 235 240 Gln Gly Lys Phe His Asn Pro Met Ser Ile Ala Gln Ile Leu Pro Ser                 245 250 255 Leu Lys Gly Lys Thr Tyr Leu Asp Val Pro Gln Val Thr Cys Ser Pro             260 265 270 Asp His Glu Val Gln Pro Thr Leu Pro Ser Asn Pro Gly Pro Gly Pro         275 280 285 Thr Ser Ala Ser Asn Ile Thr Val Ile Tyr Thr Ile Asn Asn Gln Leu     290 295 300 Arg Gly Val Glu Leu Leu Phe Asn Glu Thr Ile Asn Val Ser Val Lys 305 310 315 320 Ser Gly Ser Val Leu Leu Val Val Leu Glu Glu Ala Gln Arg Lys Asn                 325 330 335 Pro Met Phe Lys Phe Glu Thr Thr Met Thr Ser Trp Gly Leu Val Val             340 345 350 Ser Ser Ile Asn Asn Ile Ala Glu Asn Val Asn His Lys Thr Tyr Trp         355 360 365 Gln Phe Leu Ser Gly Val Thr Pro Leu Asn Glu Gly Val Ala Asp Tyr     370 375 380 Ile Pro Phe Asn His Glu His Ile Thr Ala Asn Phe Thr Gln Tyr 385 390 395

SEQ ID NO: 3 Array length: 399 Sequence type: Amino acid Topology: linear Sequence type: Peptide Hypothetical array: No origin Organism: Rattus norvegicus (Rattus norvegicus) Array   Met Trp Lys Gly Met Ala Trp Leu Ser Phe Tyr Leu Leu Asn Val Leu           -20 -15 -10   Trp Ala Val Ala Gly Thr Ser Thr Arg Ala Gln Arg Ser Cys Ser Val       -5 1 5 10   Pro Pro Asp Gln Gln Pro Trp Val Asn Gly Leu Gln Leu Leu Met Glu                    15 20 25   Asn Ser Val Thr Glu Ser Asp Leu Pro Asn Pro Ser Ile Leu Ile Ala                30 35 40   Met Asn Leu Ala Ser Thr Tyr Asn Leu Glu Ala Gln Lys Leu Leu Thr            45 50 55   Tyr Gln Leu Met Ala Ser Asp Ser Ala Asp Leu Thr Asn Gly Gln Leu        60 65 70   Ala Leu Thr Ile Met Ala Leu Thr Ser Ser Cys Arg Asp Pro Gly Ser    75 80 85 90   Lys Val Ser Ile Leu Gln Lys Asn Met Glu Ser Trp Thr Pro Ser Asn                    95 100 105   Leu Gly Ala Glu Ser Ser Ser Phe Tyr Gly Pro Ala Leu Ala Ile Leu               110 115 120   Ala Leu Cys Gln Lys Asn Ser Glu Ala Thr Leu Pro Ile Ala Val Arg           125 130 135   Phe Ala Lys Thr Leu Met Met Glu Ser Ser Pro Phe Ser Val Asp Thr       140 145 150   Gly Ala Val Ala Thr Leu Ala Leu Thr Cys Met Tyr Asn Arg Ile Pro   155 160 165 170   Val Gly Ser Gln Glu Asn Tyr Arg Asp Leu Phe Gly Gln Ala Leu Lys                   175 180 185   Val Ile Val Asp Asn Ile Ser Leu Arg Ile Lys Ala Asp Gly Ile Ile               190 195 200   Gly Asp Ile Tyr Ser Thr Gly Leu Ala Met Gln Ala Leu Ser Val Thr           205 210 215   Pro Glu Gln Pro Thr Lys Glu Trp Asp Cys Glu Lys Thr Met Tyr Thr       220 225 230   Ile Leu Lys Glu Ile Lys Gln Gly Lys Phe Gln Asn Pro Met Ser Ile   235 240 245 250   Ala Gln Ile Leu Pro Ser Leu Lys Gly Lys Thr Tyr Leu Asp Val Pro                   255 260 265   Gln Val Thr Cys Gly Pro Asp His Glu Val Pro Pro Thr Leu Thr Asp               270 275 280   Tyr Pro Thr Pro Val Pro Thr Ser Ile Ser Asn Ile Thr Val Ile Tyr           285 290 295   Thr Ile Asn Asn Gln Leu Arg Gly Val Asp Leu Ile Phe Asn Val Thr       300 305 310   Ile Glu Val Ser Val Lys Ser Gly Ser Val Leu Leu Ala Val Leu Glu   315 320 325 330   Glu Ala Gln Arg Arg Asn His Met Phe Lys Phe Glu Thr Thr Met Thr                   335 340 345   Ser Trp Gly Leu Ile Val Ser Ser Ile Asn Asn Ile Ala Glu Asn Val               350 355 360   Lys His Lys Thr Tyr Trp Glu Phe Leu Ser Gly Lys Thr Pro Leu Gly           365 370 375   Glu Gly Val Ala Tyr Tyr Ile Pro Phe Asn Tyr Glu His Ile Thr Ala       380 385 390   Asn Phe Thr Gln Tyr   395

SEQ ID NO: 4 Sequence length: 372 Sequence type: Amino acid hypothetical sequence: Yes Origin organism name: Homo sapiens
s) Sequence Ser Thr Gln Thr Gln Ser Ser Cys Ser Val Pro Ser Ala Gln Glu Pro 1 5 10 15 Leu Val Asn Gly Ile Gln Xaa Leu Met Glu Thr Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Ala 65 70 75 80 Leu Ala Gln Ile Leu Arg Asp Leu Lys Xaa Lys Val Tyr Leu Asp Val 85 90 95 Pro Gln Met Glu Asn Trp Ala Pro Ser Ser Pro Asn Ala Glu Ala Ser 100 105 110 Ala Phe Tyr Gly Pro Glu Leu Ala Ile Leu Ala Leu Cys Gln Lys Phe 115 120 125 Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Ala Lys Thr Leu Leu 130 135 140 Ala Asn Ser Xaa Pro Phe Asn Val Asp Thr Gly Ala Val Ala Thr Leu 145 150 155 160 Ala Leu Thr Cys Met Tyr Asn Lys Ile Pro Val Gly Ser Glu Glu Gly 165 170 175 Tyr Arg Ser Leu Phe Gly Gln Val Leu Lys Xaa Ile Val Glu Lys Ile 180 185 190 Ser Val Lys Ile Lys Asp Xaa Gly Xaa Xaa Gln Asp Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Thr Ser Trp Gly Leu Val Val 340 345 350 Ser Ser Ile Gly Leu Ile Ala Xaa Gly Val Xaa Xaa Xaa Thr Tyr Gly 355 360 365 Val Phe Leu Ser 370

SEQ ID NO: 5 Array length: 35 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical array: Yes Array   GTTGGATCCG AYGTNCCNCA RATGGARAAY TGGGC 35

SEQ ID NO: 6 Array length: 32 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical array: Yes Array   ACACTGCAGA CNGGDATYTT RTTRTACATR CA 32

SEQ ID NO: 7 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical array: No Array   GCAATGGCAA CCTTGGCTCT GACC 24

SEQ ID NO: 8 Sequence length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical array: No Array   GCATCAGCCT TCTATGGGCC CAGT 24

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location G01N 33/53 M 8310-2J // C12P 21/02 C 8214-4B (72) Inventor Toru Sumiya 90-43 Shinnobe, Beppu Town, Kakogawa City, Hyogo Prefecture

Claims (6)

[Claims] 1. A DNA consisting of the base sequence shown in SEQ ID NO: 1 or a part of the base sequence. 2. A cDNA sequence for human intrinsic factor. 3. A DNA having the base sequence represented by SEQ ID NO: 1 or a part of the base sequence. 4. A protein having the amino acid sequence shown in SEQ ID NO: 2. 5. A method for producing a protein having the amino acid sequence represented by SEQ ID NO: 2. 6. A DNA encoding a protein having the amino acid sequence shown by SEQ ID NO: 2.