JP2006087435A - New polypeptide and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring (1→3)-β-D glucan, to provide a (1→3)-β-D glucan-sensitive factor, and to provide a DNA encoding the factor. <P>SOLUTION: A DNA and an amino acid sequence of the a subunit of a (1→3)-β-D glucan-sensitive factor originated from Tachypleus tridentatus amebocyte and having strong affinity with the (1→3)-β-D glucan of fungous cell walls. The peptide is useful as an agent for clinically diagnosing fungi, an anti-microbial agent combined with an anti-fungal agent, or an agent for removing fungi. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a polypeptide represented by the amino acid sequence of a (1 → 3) -β-D-glucan sensitive factor (hereinafter referred to as “Factor G”) derived from horseshoe crab / amebosite and DNA encoding the same, The present invention relates to a polypeptide represented by the amino acid sequence of a subunit and DNA encoding the same.

Conventionally, a method for measuring endotoxin using horseshoe crab, amebocyte lysate is known. This method is based on the coagulation of lysates with trace amounts of endotoxin, for example on the order of 10 ⁻⁹ g, and by subsequent biochemical elucidation this reaction consists of a stepwise activation mechanism of several coagulation factors. (Also referred to as cascade reaction) (Non-patent Document 1). FIG. 1 shows this coagulation cascade reaction in Japanese horseshoe crab (Tachypleus tridentatus). In addition, regarding the three types of serine protease precursors (factor C, factor B, and coagulase precursor) and gelling protein (coagulogen) shown in FIG. 1, their structures have already been clarified by cDNA cloning and the like. (Non-Patent Documents 2 to 4).
On the other hand, this lysate is known to react with (1 → 3) -β-D-glucan present in the cell walls of fungi and yeasts in the order of 10 ⁻⁸ to 10 ⁻⁹ g and cause gelation. (See FIG. 1). Factor G exists as a factor that interacts with this (1 → 3) -β-D-glucan and initiates the gelation reaction. Factor G, like other factors, is a serine protease precursor, and is revealed to be a glycoprotein in which a 72 kDa a subunit and a 37 kDa b subunit are linked non-covalently. (Presented at the 64th Annual Meeting of the Japanese Biochemical Society in 1991).
In addition, the a subunit of factor G has a binding site specific for (1 → 3) -β-D-glucan, and the b subunit has a serine protease region, and (1 → 3) -β- It is believed that when D-glucan binds to the a subunit, factor G is activated to express the function of serine protease. However, the entire structure of factor G has not yet been clarified.
Nakamura Takanori et al., Japanese Bacteriological Journal, 38, 781-803 (1983) T.Muta et al (1991) J. Biol. Chem., 266,6554-6561 T. Muta et al (1991) J. Biol. Chem., 265, 22426-22433 T. Miyata et al (1986) J. Biochem., 100, 213-220

The present invention has been made in consideration of the situation as described above, and isolates a factor G having a binding site derived from horseshoe crab / amebosite, particularly (1 → 3) -β-D-glucan. It is intended to purify and reveal its entire structure.
An object of the present invention is to provide a polypeptide containing a (1 → 3) -β-D-glucan binding site of factor G and DNA encoding the same.
Another object of the present invention is to provide a polypeptide of the G subunit a subunit and a DNA encoding the same.
Furthermore, the subject of this invention is providing the polypeptide of G which contains a subunit, and DNA which codes it.

Therefore, the present invention provides a double-stranded DNA comprising a single-stranded DNA encoding a polypeptide containing at least one motif structure represented by the amino acid sequence Gln-Gln-Trp-Ser or a DNA complementary to the DNA. The present invention relates to DNA and a polypeptide represented by the amino acid sequence.
The present invention also provides a double-stranded DNA comprising a single-stranded DNA encoding a polypeptide represented by the following amino acid sequence or a sequence having homology thereto, or a DNA complementary to the DNA, and the amino acid sequence: It is related with polypeptide shown by.
SerHisGluProLysTrpGlnLeuValTrpSerAspGluPheThrAsnGly
IleSerSerAspTrpGluPheGluMetGlyAsnGlyLeuAsnGlyTrpGly
AsnAsnGluLeuGlnTyrTyrArgArgGluAsnAlaGlnValGluGlyGly
LysLeuValIleThrAlaLysArgGluAspTyrAspGlyPheLysTyrThr
SerAlaArgLeuLysThrGlnPheAspLysSerTrpLysTyrGlyLysIle
GluAlaLysMetAlaIleProSerPheArgGlyValTrpValMetPheTrp
MetSerGlyAspAsnThrAsnTyrValArgTrpProSerSerGlyGluIle
AspPheIleGluHisArgAsnThrAsnAsnGluLysValArgGlyThrIle
HisTrpSerThrProAspGlyAlaHisAlaHisHisAsnArgGluSerAsn
ThrAsnGlyIleAspTyrHisIleTyrSerValGluTrpAsnSerSerIle
ValLysTrpPheValAsnGlyAsnGlnTyrPheGluValLysIleGlnGly
GlyValAsnGlyLysSerAlaPheArgAsnLysValPheValIleLeuAsn
MetAlaIleGlyGlyAsnTrpProGlyPheAspValAlaAspGluAlaPhe
ProAlaLysMetTyrIleAspTyrValArgValTyrGlnAspAlaSerThr
SerSerProValGlyAspThrSerLeuAspGlyTyrTyrPheValGlnAsn
ArgHisSerGluLeuTyrLeuAspValThrAspAlaSerAsnGluAspGly
AlaPheLeuGlnGlnTrpSerTyrSerGlyAsnGluAsnGlnGlnPheAsp
PheGluHisLeuGluAsnAsnValTyrLysIleThrAsnLysLysSerGly
LysSerLeuAspValTyrAsnPheGlyThrGluAsnGlyValArgIleGln
GlnTrpSerTyrGlyGlyAlaArgAsnGlnGlnPheThrValGlnSerVal
GlyAspGlyTyrTyrLysIleIleProArgGlySerGlyLysIleValGlu
ValAlaAspPheSerLysAspAlaGlyGlyLysIleGlnGlnTrpSerAsp
AsnAsnGlnLeuSerGlyGlnTrpLysLeuIleLysSerLysSerTyrSer
LysLeuIleGlnAlaGluSerTyrPheAspSerSerLysValGlnLeuGlu
AspThrSerAspValGlyGlyGlyLysAsnValLysCysAspAsnGluGly
AlaTrpMetAlaTyrLysAspIleAspPheProSerSerGlyAsnTyrArg
IleGluTyrArgValAlaSerGluArgAlaGlyGlyLysLeuSerLeuAsp
LeuAsnAlaGlySerIleValLeuGlyMetLeuAspValProSerThrGly
GlyTrpGlnLysTrpThrThrIleSerHisThrValAsnValAspSerGly
ThrTyrAsnLeuGlyIleTyrValGlnArgAlaSerTrpAsnIleAsnTrp
IleLysIleThrLysIleProGluGlnSerAsnLeuAsnGlnGlyArgArg
AsnSerLysLeuIleGlnAlaGluSerTyrPheSerTyrSerGluValGln
LeuGluAspThrLeuAspValGlyGlyGlyLysAsnValLysCysAspLys
GluGlyAlaTrpMetAlaTyrLysAspIleAspPheProSerSerGlySer
TyrArgValGluTyrArgValAlaSerGluArgAlaGlyGlyLysLeuSer
LeuAspLeuAsnAlaGlySerIleValLeuGlyMetLeuAspIleProSer
ThrGlyGlyLeuGlnLysTrpThrThrIleSerHisIleValAsnValAsp
LeuGlyThrTyrAsnLeuGlyIleTyrValGlnLysAlaSerTrpAsnIle
AsnTrpIleArgIleThrLysVal
To date, four kinds of horseshoe crabs are known. In the four kinds, the structural parts necessary for the functional expression of coagulogen which is a component of amebosite are very similar to each other (73 to 99%). Based on this, it is predicted that the polypeptide of the present invention is similar in amino acid sequence among the four species of horseshoe crabs to the same extent as in the case of coagulogen.
That is, when there is homology between polypeptides, the same or similar function is expressed by the similar amino acid sequence even though the amino acid sequences are not identical. Therefore, those skilled in the art can easily understand that the present invention encompasses not only the amino acid sequence shown above but also amino acid sequences having homology thereto.

The present invention will be described in detail below.
First, factor G is subjected to various column chromatography operations from a horseshoe crab blood cell extract, for example, affinity by dextran sulfate-Sepharose CL-6B and concanavalin A-Sepharose 4B (Con A-Sepharose 4B). It can be purified by chromatography and gel filtration chromatography using Sephacryl S-200HR (T. Morita etal (1981) FEBS Lett., 129 (2), 318-321, Japanese Patent Publication No. 3-399). ). Furthermore, the a subunit and b subunit constituting the factor G are obtained by denaturing factor G with a denaturing agent (surfactant, etc.) and fractionating it by high performance liquid chromatography (HPLC) using a molecular sieve. Can do. The partial amino acid sequence of each subunit can be obtained by determining the amino acid sequence of a peptide fragment obtained by enzymatic digestion after reduction / alkylation of each subunit using a peptide sequencer or the like. In addition, by using an oligonucleotide synthesized based on the partial amino acid sequence of each subunit described above from a cDNA library prepared from poly (A) ⁺ RNA isolated from horseshoe crab blood cells, or an antibody to each subunit, etc. CDNAs encoding each subunit were isolated and their nucleotide sequences were determined by the dideoxy chain termination method (Proc. Natl. Acad. Sci. USA 74,5463-5467 (1977) Sanger, F. et al.) Etc. Can be determined. Further, the amino acid sequence of each subunit can be determined based on the base sequence determined in this way and the partial amino acid sequence.
By the above method, DNA encoding a polypeptide that is a subunit of factor G represented by SEQ ID NO: 1 (AGC... GTG; base numbers 114 to 2075) and the corresponding amino acid sequence (Ser. ... Val; amino acid numbers 1 to 654) were clarified.
Moreover, it was clarified that the a subunit of factor G has a domain structure from its amino acid sequence.
That is, a glucanase domain having a sequence similar to the sequence at the carboxyl terminal side of β-1,3-glucanase was present on the amino terminal side of the a subunit (Pro... Ala; amino acid numbers 4 to 236) ( FIG. 2). On the other hand, a repeating structure composed of 126 amino acids (Ser... Ile; amino acid numbers 391 to 516 and Ser... Val; amino acid numbers 529 to 654) exists on the carboxyl terminal side. The sequence was similar to the amino-terminal sequence of xylanase Z (FIG. 3). Furthermore, between these glucanase domains and xylanase domains, there was a “QQWS (Gln-Gln-Trp-Ser)” motif, and a sequence similar to that of xylanase A was repeated three times (Leu ・ ・··· Leu; amino acid numbers 247 to 293, Glu ··· Val; amino acid numbers 294 to 340 and Gly ··· Ser; amino acid numbers 341 to 387) (Fig. 4).
In FIGS. 2 to 4, each amino acid is represented by one letter.
In the present invention, these peptides can be produced and collected by a so-called genetic engineering technique in which a recombinant DNA vector containing DNAs encoding these peptides is prepared, incorporated into a host, cultured or bred.
Hereinafter, specific examples of the present invention will be described, but the present invention is not limited thereto.

1. Purification of Factor G (1) Preparation of blood cell extract 400 ml of 0.02M Tris-HCl buffer solution, pH8.0 (containing 50 mM sodium chloride) is added to 113 g of blood cells of horseshoe crab (Tachypleus tridentatus), and a high-speed homogenizer (trade name, Hiscotron, (Nippon Seimitsu Kogyo Co., Ltd.) for 3 minutes and then centrifuged at 8000 rpm for 30 minutes to obtain a supernatant. Furthermore, 300 ml of the same buffer was added to the precipitate generated during centrifugation, and the supernatant was obtained again by homogenization and centrifugation as in the above operation. The operation of adding the same buffer to the precipitate and centrifuging and collecting the supernatant was further repeated twice, and all the supernatants obtained by the centrifugation were collected to obtain 1250 ml of blood cell extract.
(2) Purification method of factor G from blood cell extract
Dextran Sulfate-Sepharose CL-6B Column Chromatography 1250 ml of the above extract was equilibrated in advance with a buffer for extraction. Dextran Sulfate-Sepharose CL-6B Column (5X17.8cm) (See Preparation Example 2 in the publication), and after washing thoroughly with the same buffer, the excess protein adsorbed on the column was washed away with 0.02 M Tris-HCl buffer, pH 8.0 (containing 0.15 M sodium chloride). . Subsequently, the protein adsorbed on the column was eluted with 0.02 M Tris-HCl buffer, pH 8.0 (containing 0.25 M sodium chloride) to obtain a factor G fraction.
b. Concanavalin A-Sepharose 4B Column Chromatography The above-mentioned Factor G fraction was equilibrated with 0.02M Tris-HCl buffer, pH 8.0 (containing 0.25M sodium chloride) in advance, a Con A-Sepharose 4B column (Pharmacia Biotech ( The column was thoroughly washed again with 0.02M Tris-HCl buffer, pH 8.0 (containing 0.5M sodium chloride). After washing, the protein adsorbed on the column was eluted with 0.02 M Tris-HCl buffer, pH 8.0 (containing 0.5 M sodium chloride and 0.5 M methyl-α-D-glucoside) to obtain a factor G fraction.
c. Sephacryl S-200 HR column chromatography The above-mentioned factor G fraction was concentrated by ultrafiltration, and then a Sephacryl S-200HR column (Pharmacia Biotech Co., Ltd.) equilibrated in advance with 0.05 M sodium phosphate buffer, pH 6.5. )) (2.7 × 98 cm) and the protein was eluted with the same buffer. The fraction of the protein that eluted last in this chromatography and showed little absorption at a wavelength of 280 nm was collected. The protein thus obtained was used as the final purified preparation of factor G. Finally, about 300 μg of factor G in terms of bovine serum albumin was obtained from 113 g of blood cells.

2. Determination of partial sequence of a subunit of factor G (1) Separation of a subunit and b subunit of factor G a, a factor G necessary for cDNA cloning of a subunit and b subunit of factor G, In order to clarify the partial amino acid sequence of the b subunit, the a subunit and the b subunit of factor G were first separated. The factor G obtained in 1 above is precipitated with methanol and chloroform for both desalting and concentration (Methods in Enzymology vol.182, p78-79, (1990)), and then 2% SDS (dodecyl). Sodium sulfate) solution and heated at 100 ° C. for 5 minutes. The heated sample was subjected to gel filtration with TSKgel G3000SW, which was linked to two using 0.1 M sodium phosphate buffer, pH 7.0 (containing 0.1% SDS) as the mobile phase, thereby allowing a factor G a , B subunits were separated.
(2) Enzymatic digestion of factor G a subunit Trichloroacetic acid (TCA) to a final concentration of 17.4% (W / V) in the fraction of factor G a subunit obtained in 2 (1) above Was added to precipitate the G subunit a subunit. According to the method of Paul Matsudaira (A Practical Guide to Protein and Peptide Purifica-tion for Microsequencing, p42-43, 1989, Academic Press, Inc.), the a subunit of the factor G was converted to 0.4M ammonium bicarbonate (8M And then reductive alkylation with iodoacetamide, and after adding water to dilute the urea concentration to 4M, lysyl endopeptidase, 1:60 enzyme-substrate weight ratio Koji Junyaku Kogyo Co., Ltd. was added and the enzyme was digested at 37 ° C for 20 hours. This digest was added to μBondasphere 5μC8-300Å (2.1X150mm) (Waters) equilibrated with 0.06% (V / V) trifluoroacetic acid in advance, and the column was thoroughly washed. After 5 minutes, 0% (V / V), 24% (V / V) after 65 minutes, 56% (V / V) after 125 minutes, and 80% (V / V) after 135 minutes. Using this method, the adsorbed protein was eluted at a flow rate of 0.2 ml / min. Each peptide eluted at this time was monitored by ultraviolet absorption at 210 nm, and all were collected.
(3) Determination of partial amino acid sequence The amino acid sequence of each obtained peptide was determined using a gas phase sequencer 477A (Applied Biosystems Japan Co., Ltd.). The results are shown in Table 1.

ただし、Xaa は自然界に存在するアミノ酸のいずれかを示す。

Xaa represents any one of the naturally occurring amino acids.

ここに示すオリゴヌクレオチド A′及び B′はそれぞれ、AおよびBに相当する配列あるいは相補的配列の全ての可能性を包括している (但し、 AのPhe のコドン（TT(C/T))中の３番目のヌクレオチド(T, C)は除かれている) 。 3. Synthesis of oligonucleotides Among the amino acid sequences determined in Table 1, two amino acid sequences A: -Glu-Asp-Tyr-Asp-Gly-Phe-, B: -Trp-Val-Met-Phe-Trp- Using Met-, A was sense-translated and B was antisense-translated, and a restriction enzyme recognition sequence and 2 bases for DNA protection were added to the 5 'end, as shown below. And a mixture of 26 oligonucleotides were synthesized using a DNA synthesizer 380A (Applied Biosystems Japan Co., Ltd.).

The oligonucleotides A 'and B' shown here cover all the possibilities of sequences corresponding to A and B or complementary sequences (provided that the codon of A's Phe (TT (C / T)) The third nucleotide (T, C) is removed).

4). Preparation of poly (A) ⁺ RNA containing mRNA encoding factor G Since factor G is purified from horseshoe crab blood cells, poly (A) ⁺ RNA was isolated from horseshoe crab blood cells.
(1) Preparation of total RNA About 11 mg of total RNA was isolated from 11.8 g of horseshoe crab blood cells using the AGPC method (see Experimental Medicine Vol. 9, p1937 to 1940).
^{(2) poly (A) +} RNA Oligotex-dT 30 Super kit from total RNA of approximately 2mg of the total RNA prepared separate poly using (Nippon Roche (Ltd.)) (A) ⁺ RNA was isolated. Further, once isolated poly (A) ⁺ RNA, the same operation was repeated once again in order to further increase the purity. Thus, 34.5 μg of high-purity poly (A) ⁺ RNA was obtained from 2 mg of total RNA by operating the Oligotex-dT 30 Super kit twice.

5. Using Amersham cDNA synthesis kit from 5μg of poly ^{(A) +} RNA of the horseshoe crab Preparation of blood cells cDNA library (1) poly obtained in Synthesis said fourth cDNA ^{(A) +} RNA 34.5μg, the cDNA Synthesized.
(2) Preparation of cDNA Library A horseshoe crab blood cell cDNA library was prepared from the cDNA synthesized in (1) using the Amersham cDNA cloning system λgt10 adapter method.

6). CDNA cloning of the a subunit of factor G Using the poly (A) ⁺ RNA obtained in 4 (2) above as a template and further using the oligonucleotide synthesized in 3 above, the PCR method (Saiki, RK et al, Science 239, 487-491, 1988), and a cDNA fragment encoding a part of the a subunit of factor G was amplified. Using this multi-prime-DNA labeling kit (Nippon Gene Co., Ltd.) labeled with [α- ³² P] dCTP as a probe, the cDNA library prepared in 5. (2) above was screened. As a result, two positive clones containing about 2,400 bp of insert cDNA having the longest length were obtained. The insert cDNAs of these clones were exactly the same except that the lengths of the 5 ′ end side and the 3 ′ end side differed by several bases. In addition, the base sequences of both were determined, and the composite base sequence including the start codon and poly (A) ⁺ tail showed a size of 2,408 bp.

7). Determination of cDNA base sequence encoding a subunit of factor G 6. The insert cDNA obtained in (1) was incorporated into the pUC118 vector and the pBluescript II SK vector. In order to determine the entire nucleotide sequence of cDNA cloned into pUC118 vector and pBluescript II SK vector, subcloning by restriction using restriction enzyme recognition site on cDNA fragment and kilo-sequence deletion kit (Takara Shuzo Co., Ltd.) went. DNA Sequencer 370A (Applied Biosystems Japan Co., Ltd.) (Smith, LM et al (1986) Nature 321, 674-679) using a nucleotide primer fluorescently labeled with the base sequence of cDNA in the clone produced by the above method Was used to determine.
The nucleotide sequence of the a subunit cDNA of factor G determined as described above and the amino acid sequence clarified therefrom are shown in the sequence listing of SEQ ID NO: 1. This amino acid sequence contained all the partial amino acid sequences determined in 2 (3) (Table 1), and the insert cDNA whose base sequence was determined this time surely encoded the a subunit of factor G. .

8). Expression and purification of factor G or its subunits All or part of the gene encoding factor G a subunit from the clones obtained above is sonicated, restriction enzyme treated or other known in the art Cut out by the method and incorporate into an appropriate vector. Vectors constructed for gene recombination from phages or plasmids that can grow autonomously in host microorganisms or cells, including appropriate promoters, SD sequences, translation initiation codons ATG, or more appropriate structural genes Such a thing is suitable. More transformants can be obtained by transferring the constructed vector into an appropriate host organism or cell. Examples of the host include various E. coli strains, various yeast strains, animal cells such as oocytes (CHO) of animals such as mice, rats, and Chinese hamsters, plant cells, insect cells, and animal and plant insect hosts. Stable production of a large amount of G factor or a subunit thereof (hereinafter referred to as G factor, etc.) by culturing the transformant thus obtained in a nutrient medium or by breeding with a nutrient diet Can do. The culture or breeding conditions of the transformant can be appropriately changed within a range in which factor G or the like is produced, but the conditions generally known in this technical field may be used as the host used grows. As for G-factor and the like in culture or breeding, a culture solution containing cells or cells or an extract obtained by crushing an individual by an appropriate mechanical method can be collected and used as it is. However, in general, when a factor G or the like is present in a culture solution or an extract according to a conventional method, the solution containing the factor G or the like is separated from cells, cells or individual fragments by filtration or centrifugation. After use. When factor G or the like is present in the microbial cells, cells or in the organ membrane of the individual, the cultivated cells, cells or debris are collected by means such as filtration or centrifugation of the obtained culture or debris, This product is separated and collected by solubilizing factor G or the like by adding a mechanical method, an ultrasonic method such as sonication or lysozyme, or a chelating agent such as EDTA and / or a surfactant. Isolation of factor G and the like from the solution containing factor G and the like thus obtained is possible by following a generally known protein purification method.
At this time, if factor G or the like is expressed as a chimeric polypeptide, it can be easily isolated using the properties of the other protein. For example, it can be easily isolated by affinity chromatography utilizing the affinity with the other protein.
Actually, the factor G a subunit can be expressed by the following method.
An oligonucleotide having the sequence 5′-AGCCACGAACCAAAGGTGCA-3 ′ is synthesized, and the 5 ′ end is phosphorylated. Next, this oligonucleotide is hybridized with a single-stranded DNA prepared from a plasmid in which a gene encoding the a subunit of factor G is incorporated, and subjected to an extension reaction with a DNA polymerase such as Klenow fragment. The portion is cleaved with a DNA nuclease such as Mung bean nuclease to obtain double-stranded DNA. This double-stranded DNA is cleaved with Sph I restriction enzyme, and the resulting sticky ends are blunted with T4 DNA polymerase, Klenow fragment or Blunting kit. This double-stranded DNA was previously cleaved with Nco I restriction enzyme, and the resulting sticky end was incorporated into pTV118N (including ampicillin resistance gene) blunted with T4 DNA polymerase, Klenow fragment or Blunting kit. This plasmid is designated E. coli. E. coli JM109 or E. coli. Introducing into E. coli MV1184, transformants are obtained. At this time, the necessary transformant can be selected by a general method utilizing the properties of the plasmid. That is, the cells were cultured on LB plates containing ampicillin (pre-coated with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) / isopropylthiogalactoside (IPTG)) Pick up and select the white colonies into which the plasmid is introduced. Furthermore, a plasmid having the correct sequence can be obtained by screening using a synthetic oligonucleotide of 5′-AAACAGACCATGAGCCCACGAACCA-3 ′ as a probe. Furthermore, it can be further confirmed that the plasmid was correctly constructed by DNA sequencing using the RV-N primer (Takara Shuzo Co., Ltd.) as a sequencing primer. A plasmid having the correct sequence has been introduced. E. coli JM109 or MV1184 is cultured in 3% Nutrient broth (Nissui Pharmaceutical Co., Ltd.) containing 100 μg / ml ampicillin and 1 mM IPTG at 30 ° C. for 24 hours. The a subunit of factor G is purified from this culture supernatant or bacterial cell extract by a commonly used method. At this time, purification can be performed more easily by affinity chromatography using a ligand having affinity for the antibody or the a subunit of factor G.

1. Determination of partial amino acid sequence of factor G b subunit (1) Isolation of factor G b subunit By separating factor G into a subunit and b subunit in (1), b subunit was obtained in the same manner.
(2) Enzymatic digestion of b subunit of factor G 2. From the b subunit of factor G obtained in (1) above, Peptide fragments were obtained by the same method as (2).
(3) Determination of partial amino acid sequence The amino acid sequence of each obtained peptide was determined as 2. It determined similarly to (3). The results are shown in Table 2.

ここに示すオリゴヌクレオチドC'およびD'はC およびD に相当する配列あるいは相補的配列の全ての可能性を包括している（但し、C およびD のC 末端アミノ酸のLys ならびにD のThrのそれぞれコドン(AA(A/G)および(AC(A/C/G/T)) の３番目のヌクレオチド（それぞれA,G およびT,C,A,G)は除かれている）。 2. Synthesis of oligonucleotides Among the determined amino acid sequences, two amino acid sequences C: -Asn-Glu-Gln-Cys- (Asn) -Lys, D: -Met-Tyr-Gln-Asn-Pro-Thr- Using the sequence, back-translation of C into sense and D into antisense is performed. In the same manner, a mixture of 25 oligonucleotides C ′ and D ′ as shown below was synthesized. In the C,-(Asn)-indicates that it was estimated as Asn.

The oligonucleotides C ′ and D ′ shown here cover all the possibilities of sequences corresponding to C 1 and D 2 or complementary sequences (provided that C 1 and D 2 C terminal amino acids Lys and D 3 Thr, respectively) The third nucleotide of the codons (AA (A / G) and (AC (A / C / G / T)) (A, G and T, C, A, G respectively removed).

4). CDNA cloning of the G subunit b subunit Using poly (A) ⁺ RNA obtained in (2) as a template,
Further, using the oligonucleotide synthesized in the above 2, the following 6. Experiments were performed in the same manner as described above to obtain positive clones containing 1,979 bp cDNA. Analysis of the base sequence of the insert cDNA of this clone revealed that it contained the base sequence corresponding to the amino acid sequence of the peptide derived from the G subunit b subunit obtained in 1. (2) above, and further added a poly A addition signal. And the size of this clone seems to be a full-length cDNA.

5. Determination of cDNA base sequence encoding factor G b subunit The base sequence of the factor G b subunit cDNA is the same as that of Example 1. It was determined by the same method. As a result of the examination as described above, the nucleotide sequence of the cDNA of the G subunit of the G factor that has been clarified and the amino acid sequence of the G subunit of the G subunit revealed therefrom are shown in the sequence listing of SEQ ID NO: 2.
Since this partial amino acid sequence (Table 2) determined in 1 (3) of Example 2 was included in this amino acid sequence, the insert cDNA whose base sequence was determined this time surely identifies the b subunit of factor G. I was coding.
After transferring the recombinant DNA vector containing the cDNA encoding the polypeptide collected in Example 2 to a replicable host microorganism, an appropriate animal cell, insect or insect cell, the vector marker and (1 → 3) -β- The polypeptide of the present invention can be collected by culturing or breeding microorganisms, animal cells, insects or insect cells carrying the recombinant DNA vector obtained by screening using D-glucan binding ability as an index.

In the present invention, the a subunit of factor G of horseshoe crab amebocyte lysate was isolated and purified, and its amino acid sequence and the cDNA base sequence encoding it were determined.
As a result, this sequence contains the sequence of (1 → 3) -β-D-glucan binding site, and using these cDNAs, a polypeptide corresponding to the binding site is obtained by genetic engineering techniques. Can do. The resulting polypeptide exhibits the following various effects.
(1) In recent years, with increasing number of immunocompromised patients and aging, Candida,
Opportunistic fungal infections by secondary pathogens that are usually weakly pathogenic, such as Aspergillus, have increased markedly. However, in particular, deep mycoses such as visceral organs due to fungi are difficult to diagnose clinically unless they are invasive except for some special disease types, and are therefore often diagnosed for the first time after autopsy ( Microbiology Dictionary, 1989, Gihodo).
The above-mentioned deep mycosis is diagnosed by using the polypeptide containing the (1 → 3) -β-D-glucan binding site or (1 → 3) -β-D-glucan as a radioisotope, enzyme, fluorescent substance. This can be carried out by measuring (1 → 3) -β-D-glucan by a ligand-receptor assay using a substance labeled with a labeling substance such as a luminescent substance. Alternatively, the polypeptide can be bound to a solid phase such as a microplate, a test tube, or a bead, and (1 → 3) -β-D-glucan can be specifically captured and detected. . From such a measurement method, fungal-derived (1 → 3) -β-D-glucan in a body fluid can be specifically measured, and diagnosis of deep mycosis can be performed easily and rapidly.
(2) In addition, a drug in which an antifungal agent is bound to a polypeptide containing a (1 → 3) -β-D-glucan binding site has a strong affinity for a lesion, and (1 → 3) -β-D -Glucan can be a new selective antifungal agent that specifically kills focal fungi present in the cell wall.
(3) Furthermore, with the recent development of gene manipulation techniques, yeast is frequently used as a host for a vector containing a gene encoding a target protein when the target protein is expressed. This is because glycoproteins can be produced and can be cultured in large quantities like bacteria. However, as a problem, the product often contains a residue of yeast having a very small amount of (1 → 3) -β-D-glucan as a constituent component, and its quantification and removal are required. In this case, by performing affinity chromatography using an affinity carrier obtained by binding a polypeptide containing a (1 → 3) -β-D-glucan binding site to a support, removal of yeast residues specifically, Alternatively, quantification can be performed, and confirmation of the absence of yeast cell components in the product after removal can also be achieved by using the (1 → 3) -β-D-glucan binding site as described in (1). By carrying out the quantification method using the contained polypeptide, it is possible to confirm more simply and with higher specificity than when using horseshoe crab, amebocyte, lysate.

FIG. 1 shows the mechanism of endotoxin sensitivity and (1 → 3) -β-D-glucan sensitivity of horseshoe crab blood cells. FIG. 2 shows the amino acid sequence of the glucanase domain (amino acid numbers 4 to 236) of the a subunit of factor G. In the figure, FGA represents the amino acid sequence of amino acid numbers 4 to 236 of the a subunit of factor G, and βG1 A1 represents the amino acid sequence of amino acid numbers 421 to 682 of β-1,3-glucanase. FIG. 3 shows the domain of amino acid numbers 391 to 654 of the a subunit of factor G. In the figure, FGA represents the amino acid sequence of amino acid numbers 391 to 654 of the a subunit of factor G, and Xyn Z represents the amino acid sequence of amino acid numbers 298 to 434 of xylanase Z. FIG. 4 shows the domain of amino acid numbers 247 to 387 of the a subunit of factor G. In the figure, FGA represents the amino acid sequence of amino acid numbers 247 to 387 of the a subunit of factor G, and Xln A represents the amino acid sequence of amino acid numbers 351 to 477 of xylanase A.

[Sequence Listing]

Claims (1)

A method for measuring (1 → 3) -β-D-glucan using a polypeptide (amino acid number 391-654 in Sequence Listing 1) which is a xylanase domain represented by the following amino acid sequence:
SerLysLeuIleGlnAlaGluSerTyrPheAspSerSerLysValGlnLeu
GluAspThrSerAspValGlyGlyGlyLysAsnValLysCysAspAsnGlu
GlyAlaTrpMetAlaTyrLysAspIleAspPheProSerSerGlyAsnTyr
ArgIleGluTyrArgValAlaSerGluArgAlaGlyGlyLysLeuSerLeu
AspLeuAsnAlaGlySerIleValLeuGlyMetLeuAspValProSerThr
GlyGlyTrpGlnLysTrpThrThrIleSerHisThrValAsnValAspSer
GlyThrTyrAsnLeuGlyIleTyrValGlnArgAlaSerTrpAsnIleAsn
TrpIleLysIleThrLysIleProGluGlnSerAsnLeuAsnGlnGlyArg
ArgAsnSerLysLeuIleGlnAlaGluSerTyrPheSerTyrSerGluVal
GlnLeuGluAspThrLeuAspValGlyGlyGlyLysAsnValLysCysAsp
LysGluGlyAlaTrpMetAlaTyrLysAspIleAspPheProSerSerGly
SerTyrArgValGluTyrArgValAlaSerGluArgAlaGlyGlyLysLeu
SerLeuAspLeuAsnAlaGlySerIleValLeuGlyMetLeuAspIlePro
SerThrGlyGlyLeuGlnLysTrpThrThrIleSerHisIleValAsnVal
AspLeuGlyThrTyrAsnLeuGlyIleTyrValGlnLysAlaSerTrpAsn
IleAsnTrpIleArgIleThrLysVal

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