JPH0568561A - Genome dna sequence - Google Patents

Abstract

PURPOSE:To provide a genome DNA sequence existing on the downstream side of Fomes japonicus chromosome-coding protein LZ-8 having immunosuppressive activity derived from Fomes japonicus and having a gene of LZ-8-2 as e.g. an immunosuppressive agent high in homology with LZ-8. CONSTITUTION:Fomes japonicus mycelia is ground in liquid nitrogen and suspended in a 10mM tris-HC1 buffer solution (pH 7.5) and added with a surfactant. Protease K is added to the resultant suspension, treated at 37 deg.C for 2hr, added with 5M NaCl and extracted with phenol. The resultant aqueous layer is extracted with chloroform/isoamyl alcohol (24:1) followed by addition of ethanol to precipitate genome DNA, which is, in turn, separated and treated with ribonuclease A to remove RNA, and the genome DNA obtained is put to restriction enzyme treatment and then packaged in phage to make a library, which is then screened with a probe to screen positive clone, from which DNA is collected, thus obtaining the objective genome DNA sequence of the formula.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the genus Ganoderma (Ganode).
rma) Mycelium-derived protein with immunosuppressive ability (LZ-8)
The present invention relates to the LZ-8-2 gene, which is present downstream of the gene encoding Ganoderma lucidum and has high homology with LZ-8.

[0002]

[Prior Art] Ganoderma lucidum
The Polygonales (Polyporales)
Basidiomycete belonging to the group, also called Reishi, has been prized as a herbal medicine since ancient times, and as one of the ingredients of Kampo medicine,
It is also widely used as a health food.

The present inventors previously extracted and purified the protein LZ-8 having immunosuppressive ability from the mycelium of Ganoderma lucidum, identified its amino acid sequence, and encoded the amino acid sequence.
The cDNA was cloned (Japanese Patent Laid-Open No. 64-27490, Japanese Patent Laid-Open No. 2-27490).
124899, The Journal of Biological Chemistry vol.
264, 472-478 (1989), The Journal of Biological Che
mistry vol.264, 16372-16377 (1989) and The Journal
of Biological Chemistry vol.266, 2486-2493 (199
1)). Furthermore, a genomic DNA substantially containing a region controlling the transcription and termination of the structural gene of LZ-8 was cloned and its sequence was determined (Japanese Patent Application No. 1-309257, The Journal).
of Biological Chemistry vol.266, 2486-2493 (199
1)).

LZ-8 consists of 110 amino acids in which the N-terminal amino acid is acetylated, and has a molecular weight of 12,420 including the N-terminal acetyl group. The physiological activities of LZ-8 include allergic type I (anaphylaxis) and
It is known to inhibit type II (insulin-dependent diabetes mellitus), type III (arthus reaction) and type IV (mixed lymphocyte reaction). From this, LZ-8 is considered to be effective in treating allergies, and research is being conducted.

[0005]

As described above, LZ-8 is useful as an immunosuppressant, but it is important for drug development to find a new immunosuppressive protein. Therefore, an object of the present invention is to provide a novel immunosuppressive protein and a gene encoding the same.

[0006]

[Means for Solving the Problems] Therefore, when the present inventors cloned the genomic DNA of Ganoderma lucidum using an oligonucleotide probe derived from the LZ-8 gene, a structural gene LZ-8 different from the LZ-8 gene was obtained. -2 isolated,
Furthermore, they succeeded in expressing this and obtaining a new protein, and completed the present invention.

That is, the present invention provides a genomic DNA sequence having the LZ-8-2 structural gene represented by SEQ ID NO: 1 and an LZ-8-2 protein encoded by the genomic DNA sequence.

The genomic DNA sequence of the present invention is disclosed in, for example, Japanese Patent Laid-Open No.
Genomic DNA was extracted from the mycelium of Ganoderma lucidum described in 4-27490 or JP-A-2-124899, and the genomic DNA was extracted from the mycelium.
It is obtained by preparing a library and cloning it from the library using the probe method.

[0009] Ganoderma lucidum used in the present invention may be any mycelium identified according to the primary color Japanese fungus pictorial book (Nursing company version) and the Japanese mycological journal by Seiya Ito (Yokendo version). However, there is a possibility that there will be variations in the production amount depending on the bacterial strain or there may be regional differences in the collected bacterial strains. Ga deposited as FERM BP-1826)
It is preferred to use noderma lucidum No.16.

Extraction of Ganoderma lucidum genomic DNA from Ganoderma lucidum is carried out according to a conventional method. To prepare a genomic DNA library from the obtained genomic DNA, for example, the genomic DNA may be completely digested with a restriction enzyme EcoRI and packaged into λ phage using a commercially available packaging kit.

To clone the LZ-8-2 gene from the genomic DNA library, it is preferable to use an oligonucleotide probe derived from the LZ-8 gene.

The obtained LZ-8-2 gene is similar to the LZ-8 gene, but there are some clearly different parts,
It is a completely new gene.

In order to express the LZ-8-2 gene and produce the LZ-8-2 protein, an expression vector is constructed by ligating a promoter or the like to the LZ-8-2 gene by a conventional method. Various host cells may be transformed with and the transformed cells may be cultured.

[0014]

INDUSTRIAL APPLICABILITY The genomic DNA sequence of the present invention encodes LZ-8-2 protein similar to LZ-8 protein useful as an immunosuppressant, and is useful for producing LZ-8-2 protein.

[0015]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto. Example 1: Preparation of genomic DNA of LZ-8-2 and determination of its sequence Preparation of genomic DNA of Ganoderma lucidum Mycelium of Ganoderma lucidum (wet weight about 20 g) was crushed in liquid nitrogen, and 10 ml of extraction buffer ( 10 mM Tris-HCl pH 7.
It was suspended in 5, 10 mM NaCl, 25 mM EDTA; hereinafter referred to as TE buffer), and SDS was added so that the final concentration was 1%. Then add 11 mg of Protease K to this suspension,
It was treated at ° C for 2 hours. 1 ml of 5M NaCl was added to this suspension, and then phenol extraction was repeated 5 times. The aqueous layer was extracted twice with chloroform / isoamyl alcohol (24: 1), and 2.5 times the amount of ethanol was added to precipitate the genomic DNA. After washing this precipitate with 70% ethanol, 10 ml TE
Dissolved in buffer. Since this sample contains a large amount of RNA, in order to remove it, 200 μg of ribonuclease A was added and incubated at 37 ° C. for 30 minutes to decompose the RNA.
To remove Ribonuclease A, add 2 with phenol again.
It was extracted twice and the aqueous layer was extracted twice with chloroform / isoamyl alcohol (24: 1). Finally, about 200 μg of genomic DNA was obtained.

Preparation of genomic DNA library About 10 μg of the genomic DNA obtained above was completely digested with the restriction enzyme EcoRI, and the DNA fragments were fractionated by 0.8% agarose gel electrophoresis. A gel fragment of a 5-8 kb fraction was cut out and a DNA fragment was extracted from this gel. About 1/3 of the recovered DNA was ligated to 1 μg of λgt10 arm (Stratagene), and Gigapack Gold (Stratagene) was added.
Packaging kit (manufactured by the company) was used to package λ phage according to the method described in the attached protocol. This library contained approximately 70,000 clones.

Cloning of LZ-8-2 gene 13 plates (φ90m
m) plaques on each side of Biodyne A (Biodyne
A, made by Pall Co.) and nitrocellulose (BA85, S & S Co.) (transferred from one plate to two types of filters). Each filter is 0.
The phage DNA was denatured by treatment with 4N NaOH and 0.8M NaCl for 2 minutes, neutralized with 1M Tris-HCl pH 7 and 1.5M NaCl for 5 minutes, air-dried, and further incubated at 80 ° C for 2 hours to filter the DNA. Fixed on top. The following two kinds of oligonucleotide probes were used for screening of clones having the LZ-8-2 gene.

[0018]

[Chemical 1]

These are synthesized based on the information of the amino acid sequences of two kinds of peptides (probe 1 and probe 2) contained in the tryptic digest of LZ-8. These are [γ- ³² P] ATP. The 5'end was labeled with and a polynucleotide kinase to prepare a probe. The above DNA-immobilized filter is 5 × SSPE (1 × SSPE is 0.18MN
aCl, 10 mM sodium phosphate, 1 mM EDTA), 10 × Denhard
6 in a solution containing t's, 0.1% SDS and 100 μg / ml tRNA
Prehybridize at 5 ° C for 2-5 hours, 5 × SSPE, 5
× Denhardt's, 0.3% SDS, 100 μg / ml tRNA, ³² P-
Hybridization was carried out at 37 ° C. for 3 hours in a solution containing an oligonucleotide probe (2 × 10 ⁶ cpm / ml). At this time, Bi
The filter of odyne A uses probe 1 as the probe,
The nitrocellulose filter hybridized with probe 2 as a probe. After hybridization, the filter is 3M tetramethylammonium chloride, 2 mM
It was washed twice in a solution containing EDTA and 50 mM Tris-HCl pH 8.0 at 4 ° C for 30 minutes each, 30 minutes in the same solution, and finally 20 minutes in the same solution at 45 ° C. The autoradiograms of these filters were examined and compared, and plaques that were positive with the two types of probes were purified. As a result of such screening, 5 strong signals were found. Therefore, the phage corresponding to each signal was purified. Finally, one clone was confirmed by analysis of its nucleotide sequence to contain LZ-8-2 genomic DNA.

Determination of nucleotide sequence of LZ-8-2 gene Genomic DNA containing the LZ-8-2 gene obtained above (6.8 k
b) was digested with various restriction enzymes and the digested fragment was digested with M13mp18 and
Subcloned into M13mp19. Then, using the dye-labeled primer, an ABI DNA sequencer (model 370
Based on A), the nucleotide sequence was determined as shown in FIG. In FIG. 1, a portion of the LZ-8-2 gene that is considered to be an exon is indicated by a rectangle. The part with the pattern in the rectangle is the coding region of the LZ-8-2 protein. Arrows indicate the direction and distance of nucleotide sequencing. The black circle at the starting point of the arrow indicates the position of the primer chemically synthesized for nucleotide sequence determination. Further, Sp, X, Sa, K, S, H are restriction enzymes SphI, XhoI, SalI, KpnI, SacI and HindIII, respectively.
Is represented. As a result, the protein coding region of the LZ-8-2 gene is as shown in SEQ ID NO: 1, and the sequence of the entire gene obtained is as shown in SEQ ID NO: 2.

Features of LZ-8-2 gene In SEQ ID NO: 2, nucleotide numbers 27 to 37 (ACCACCGTGG
T), base number 426 to 436 (ATAATGATTAT) and base number
495 to 508 (TGAGCCCGGATTCA) are palindromic sequences, with base numbers 222 to 225 (ATTG) and base numbers 301 to 304.
(ATTG) and base numbers 446 to 449 (ATTG) are CAA in the opposite direction.
It is a T-sequence, base numbers 450 to 453 are positive CAAT sequences, base numbers 538 to 543 (TATAAA) are TATA boxes, and base numbers 590 to 596 (CCGCCC) are GC boxes. It The transcription start point is base number 629.
Is estimated to be C. The sequence of base numbers 673 to 744 is the sequence on the 5'terminal side of this sequence (base numbers 672 to 674).
Of AGGT) and the 3'terminal sequence (base numbers 727-732)
Since CTGACCG and AG of base numbers 733 to 734 are consensus sequences of splice sites, they are presumed to be introns. The sequences of base numbers 1162-1178 are homologous to the sequence immediately before the polyA addition site of the LZ-8 gene. A at base number 1181 is presumed to be a polyA addition site.

Structure of LZ-8-2 gene-Comparison with LZ-8 gene The number of amino acids encoded by the LZ-8 gene is 111 including the translation initiation methionine, and immediately before the translation initiation methionine codon. The length of an intron is 61 bases. In contrast, the LZ-8-2 gene has 112 encoded amino acids, and the intron-like sequence at the same position as the LZ-8 gene has a length of 62 bases. There are 205 bases between the TATA-box and the translation initiation methionine codon. Their structures are very similar to the LZ-8 gene. First, when the homology was examined, it was about 83% in the nucleotide sequence (277 out of 333 nucleotides of LZ-8) and about 75% in the amino acid sequence (of LZ-8).
Of the 111 amino acids, 83 are homologous in sequence.

A characteristic sequence RRYRR (R is a purine base and Y is a pyrimidine base) found near many transcription initiation points in yeast is also found in the LZ-8-2 gene as in the LZ-8 gene. (Starting at base 627). Furthermore, LZ-8
The sequence homologous to the sequence immediately before the polyA addition site of the gene is L
It starts from the 1160th base of the Z-8-2 gene. or,
In the vicinity of the putative polyA addition site, LZ-8-2 mRNA
It is expected that a structure similar to that of -8 mRNA as shown in Fig. 2 will be produced. As described above, the positions of the sequences constituting the LZ-8-2 gene are very similar to the LZ-8 gene.

However, the LZ-8-2 gene has a sequence that is considered to be involved in transcription, which is not present in the LZ-8 gene, that is, one GC box and three reverse CAAT boxes. If the LZ-8 gene and LZ-8-2 gene are differentially expressed in the mycelium and fruiting body of Ganoderma lucidum,
This GC box and three reverse CAAT boxes may be involved in this phenomenon.

Example 2: Expression of LZ-8-2 gene in vitro Expression of LZ-8-2 gene in vitro The LZ-8-2 gene obtained in the above-mentioned example was prepared by using SacI and HindIII.
A vector containing the SP6 promoter containing the nucleotides 635 to 1161 of SEQ ID NO: 2 after digestion with
It was incorporated into pAM18 (Amersham). Using this plasmid as a template, RNA was synthesized according to the protocol attached to the SP6 RNA synthesis system. Then, using this RNA as a template, a protein was synthesized using a cell-free protein synthesis system of rabbit reticulocyte hemolysate. Upon synthesis, 3H-leucine was added and LZ-8-2 was labeled.

The protein synthesized as described above is denatured by SDS treatment in a reduced state and subjected to polyacrylamide gel electrophoresis, or polyacrylamide gel electrophoresis in an undenatured state without SDS treatment, and further Amplify. (Ame
Fluorescence was performed using rsham).
The results are shown in Fig. 3 (denaturation by SDS) and Fig. 4 (undenatured). The acrylamide concentration was 16% in all cases.

RN synthesized in vitro from LZ-8-2 gene
There are several AUGs in A that differ from the original start codon. Among them, other than LZ-8-2 protein, there is a possibility of translation, and the total number of amino acids constituting the protein is the closest to the total number of amino acids of LZ-8-2 (112) ( 115) was designated as LZ-8-2 'protein (starting from ATG at position 703 of SEQ ID NO: 2), and whether or not it was expressed was examined. Table 1 shows a comparison of the constituent amino acids of LZ-8, LZ-8-2 protein and LZ-8-2 'protein.

[0028]

[Table 1]

When the protein expressed in vitro was subjected to electrophoresis in a denatured state, it showed almost the same electrophoretic mobility as LZ-8 (FIG. 3). This is expected from the number of constituent amino acids shown in Table 1 and the homology of amino acid sequences. Further, as shown in FIG. 3, most of the labeled proteins are occupied by proteins that are considered to be LZ-8-2 or LZ-8-2 'proteins (otherwise, a cell-free protein synthesis system). Hemoglobin, which is thought to be due to the residual rabbit reticulocyte mRNA, was found.

On the other hand, when the protein expressed in the test tube was electrophoresed without denaturation, one main band was LZ-8.
It migrated slightly faster than that (Fig. 4). LZ-8-2 protein and LZ-8
Since the number of basic amino acids (Arg + Lys) and acidic amino acids (Asp + Glu) is different from that of -2 'protein (Table 1), there is a difference in electrophoretic mobility in electrophoresis in the native state. It should be. Despite that, only one band appears, so this band is either LZ-8-2 or LZ-8-2 '. Therefore, AUG which becomes the translation initiation codon of LZ-8-2
To remove LZ-8-2 'from translation by removing about 40 bases upstream from it, and synthesizing the protein in a cell-free synthesis system, and performing electrophoresis in the native state. As a result, it migrated to the same position as the band in FIG. 4 (data not shown). From the above, it is considered that the main product synthesized from RNA containing several translation initiation codons is LZ-8-2.

Reference Example The results of examining the binding of the expressed LZ-8-2 protein to the glycoprotein in comparison with LZ-8 are shown. Binding activity to glycoprotein (1) Measurement by nitrocellulose filter method Spot 10 to 20 μg of glycoprotein (mucin, fetuin or IgA) on a square nitrocellulose filter (BA85 manufactured by S & S Co.) with a side length of about 3 mm, and air-dry. Fixed. This filter was soaked in 10 mg / ml BSA solution for 1 to 2 hours, and then treated with 125I-LZ-8 or 3H-LZ-8-2 protein in 0.25M Tris-HCl (pH 7.5) and 0.5M NaCl buffer. Thermal insulation (about 18
C, about 17 hours). Then filter 10 mM Tris-HC
The cells were washed 5 times with a buffer solution containing 1 (pH 7.5), 0.15M NaCl and 0.05% Tween 20. And 125I bound to the filter
Alternatively, 3H was measured by a well type γ counter and a liquid scintillation counter, respectively.

As a result, among the 3H-LZ-8-2 used, about 7,000 cpm was 20 μg of mucin and about 2,000 cpm was 10 μg.
Bound to Fetuin. Similar experiments performed with 125I-LZ-8 showed similar trends in the difference in the amount of LZ-8-2 bound between mucin and fetuin. In addition, 10
No binding of μg to IgA could be confirmed.

(2) Measurement by gel shift method analyzed by PAGE 10 μg of glycoprotein (mucin, fetuin, or IgA) in 5M Tris-HCl (pH 7.5) buffer, or bovine serum albumin or ovo as a negative control Albumin and 125I-LZ-8 or 3H-LZ-8-2 protein and incubation (about 18
C., about 17 hours), and then PAGE was performed without denaturation. After the electrophoresis is completed, the protein in the gel is fixed, and then Amplif is used for fluorography.
It was treated with y (Amersham) and dried. -8 this gel
It was exposed to an X-ray film at 0 ° C. The results are shown in Fig. 5 (LZ-
8) and FIG. 6 (LZ-8-2).

From FIG. 6, the LZ-8-2 protein did not bind to ovalbumin, only slightly to IgA, slightly to fetuin, and most to mucin. It is recognized that The above results agree very well with the results obtained by the filter method. On the other hand, in the case of LZ-8, almost all of the added LZ-8 undergoes a shift (Fig. 5). Similar results can be obtained by using LZ-8 which was synthesized in vitro by the same method as LZ-8-2 protein. That is, it is considered that the difference between FIG. 5 and FIG. 6 reflects the difference in the binding ability of LZ-8 and LZ-8-2 to the glycoprotein. However, this result indicates that the LZ-8-2 protein has an activity of binding to a glycoprotein.

Based on the above results and amino acid sequence, LZ-8
Considering that the -2 protein has a high homology with the LZ-8 protein, it is suggested that the LZ-8-2 gene is actually expressed in Ganoderma lucidum and may play a role there. It also suggests that LZ-8-2 may have similar biological activity as LZ-8.

[0036]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 336 Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Genomic DNA Sequence ATGCCCTCCA ACACCGCTCT GATCTTCAAG CTCGCCTGGG ACCTGAAGAAGCTCTGGTTC 60 GACTACACCC CCACGCCACCACACACACACACACACACTCAACCTCTACCACC ATCT. TACCGCGTCG TCGCCAATGG ACGGGGCCTC 180 GGCGTGCGCA CCTCGAACGC GGTCGCGTGC GACGGCTCGC AGTTCGTCAA CTTCCTCGAG 240 TATCACTCTG GCCTCGGCAT CGAGGACACG GAGACGATCC AGGTGTACAT CGTCGACCCT 300 GAGACCGGCG ACGACCACCT CATCGCCCAGAG

SEQ ID NO: 2 Sequence length: 1255 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Ganoderma lucida (Ganoderma)
Lucidum) Strain name: No. 16 sequence AACGCCCAGC GCAGCTCGTA GTTCTAACCA CCGTGGTGTA CACGCGTTTG GCTTCATCCT 60 TCGATTCTCA TCAACTCGAT CATCGCCGTC TCCCGAAATT AGCCTCGACA CGTCTTTCGG 120 TGGTGGATTC GTGATACCTC ACGGAGTTCT GGCGGCCTCA TGGGTTTCCT ACTGACGGAC 180 CGGGGTGTTA CACATAAGTG CGTTCATTCC TCATGTCAAA CATTGTACCC CGTTTCCAAA 240 GCTAACGACG GTTTCATAAC GTGTTTCCTG CCTTGGCTCA GAGCTCCGGT TTTTTTCTTA 300 ATTGTGCGTG TTTATATCGC ATTCAGCTCA CCAAAAAGGT CGTGCGGTAT AGGAAGCATG 360 GCACTACGTT TGAATGTGGC TATTCATAAC TACCGTCCCG ACCCCTAGGT GAACGCCATG 420 TGAAGATAAT GATTATGGTG GTCCTATTGC AATTTCAACC CGGCGGTACA ACTCAATGGG 480 CCTCGCACAT CGTATGAGCC CGGATTCACG TGATGGCGGA AGAGCTCTGA CTGCCTGTAT 540 AAAAGGGCAG CAGACGAGGC CAGCGGACTT CAGCACCAAC TACTACACTC CCGCCCAGTA 600 AGTCGCACAC TACCTCACTT GAGAGGGACG AGCTCATTGA CCGCTCTGTA GTCGCAATCT 660 CTTTGAGTTA CAATCAACCA AGGTTTGGCG GCGCCCTCGT TCATGCCCTC CCCCTCCAAC 720 ATATCCCTGA CCGCTGCCCC GCAGAATC ATG CCC TCC AAC ACC GCT CTG ATC 772 Met Pro Ser Asn Thr Ala Leu Ile 5 TTC AAG CTC GCC TGG GAC CTG AAG AAG CTC TCG TT C GAC TAC ACC CCG 820 Phe Lys Leu Ala Trp Asp Leu Lys Lys Leu Ser Phe Asp Tyr Thr Pro 10 15 20 AAC TGG GGC CGC GGC AAC CCC TCC ACC TAT ATC AAC AAC GTG ACC TTC 868 Asn Trp Gly Arg Gly Asn Pro Ser Thr Tyr Ile Asn Asn Val Thr Phe 25 30 35 40 CCG AAG GTC CTG ACC GAC AAG GCG TAC ACG TAC CGC GTC GTC GCC AAT 916 Pro Lys Val Leu Thr Asp Lys Ala Tyr Thr Tyr Arg Val Val Ala Asn 45 50 55 GGA CGG GGC CTC GGC GTG CGC ACC TCG AAC GCG GTC GCG TGC GAC GGC 964 Gly Arg Gly Leu Gly Val Arg Thr Ser Asn Ala Val Ala Cys Asp Gly 60 65 70 TCG CAG TTC GTC AAC TTC CTC GAG TAT CAC TCT GGC CTC GGC ATC GAG 1012 Ser Gln Phe Val Asn Phe Leu Glu Tyr His Ser Gly Leu Gly Ile Glu 75 80 85 GAC ACG GAG ACG ATC CAG GTG TAC ATC GTC GAC CCT GAG ACC GGC GAC 1060 Asp Thr Glu Thr Ile Gln Val Tyr Ile Val Asp Pro Glu Thr Gly Asp 90 95 100 GAC CAC CTC ATC GCC CAG TGG AAC 1084 Asp His Leu Ile Ala Gln Trp Asn 105 110 TAGGAGGGAG ACGGTGACTG ACCCGAGACG GCCTGGTGGG CCACGGAAGA GGGAGCGTCT 1144 *** GCGACGCGTCGCCTCTAAGC TTTCTTGTAC CAGTACTCT C GTTGTGACCG TGTAACAATT 1204 GTAAATCGAC CTTGCTGTAT GCCTACGGCC TTCGCCGTCC ATCCTTGTGT G 1255

[Brief description of drawings]

FIG. 1 is a method for determining the nucleotide sequence of the LZ-8-2 gene.

FIG. 2 is a secondary structure expected to be formed in the 3 ′ untranslated region of LZ-8-2 mRNA.

FIG. 3 is SDS-polyacrylamide gel electrophoresis of LZ-8-2 protein synthesized in vitro.

FIG. 4 is a polyacrylamide gel electrophoresis of native LZ-8-2 protein synthesized in vitro. “Boundary”
Is the boundary between the concentrated gel and the 16% separating gel.

FIG. 5 is a polyacrylamide gel electrophoresis showing the binding between LZ-8 protein and glycoprotein.

FIG. 6 is a polyacrylamide gel electrophoresis showing the binding of LZ-8-2 protein and glycoprotein synthesized in vitro.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 645)

Claims (4)

[Claims] 1. A genomic DNA sequence having the LZ-8-2 structural gene represented by SEQ ID NO: 1. 2. The genomic DNA sequence represented by SEQ ID NO: 2 having the LZ-8-2 structural gene and a region controlling the transcription and termination of the gene. 3. An LZ-8-2 protein encoded by the genomic DNA sequence according to claim 1. 4. The LZ-8-2 protein according to claim 3, wherein the amino acid sequence is shown in SEQ ID NO: 2.