JPH1175840A - Production of recombinant mite allergen derfi - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain recombinant mite allergen DerfI useful as an agent for hyposensitization, having the same allergen DerfI activity as that of natural DerfI, by expressing a gene encoding DerfI with yeast Saccharomyces cerevisiae. SOLUTION: A gene encoding DerfI is expressed with yeast Saccharomyces cerevisiae or a filamentous fungus Aspergillus oryzae such as niaD300 (FERM P-16,368) to produce recombinant mile allergen DerfI. In removing a saccharide chain from the recombinant mile allergen DerfI, preferably EndoHf which is a fused protein of Endoglycosidase H (EndoH) and maltose is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a novel allergen substance useful for hyposensitization therapy, a diagnostic agent, and the like, and more particularly, to the production of a recombinant mite allergen DerfI derived from Dermatophagoides farinae. About the method.

[0002]

2. Description of the Related Art In recent years, allergic patients have been increasing. Typical examples are hay fever, atopic dermatitis,
Atopic asthma can be mentioned. Atby asthma is often caused by house dust. The causative substance that causes allergy is called an allergen. In house dust, a tick-derived protein is known to be the most important allergen.

[0003] Derfl is a glycoprotein contained in the feces of Dermatophagoides farinae contained in house dust, and it is known that a majority of patients with atopic asthma and atopic dermatitis react (Heymann PW, et. al.:J. Allergy Clin. Immunol., 8
3, 1055-1067, 1989), one of the major mite allergens. Regarding Derfl, a purification method from mite culture has already been established (Yasueda H. et al .: Int. Arch.
Allergy Appl. Immunol., 81, 214-223, 1986), and the cDNA encoding it has also been cloned (Dilwor
thR. J. et al .: Clinical and Experimental Allergy,
21, 25-32, 1991). Although there is an expectation to examine the involvement of DerfI in allergy and to use DerfI for desensitization therapy, it was difficult to obtain a large amount of natural DerfI due to the slow growth of mites.

[0004] Therefore, an attempt was made to produce recombinant protein (ShojiH. Et al .: Biosci. Biotech. B
iochem., 60, 621-625 , 1996). A site-specific mutation was introduced into the DerfI cDNA, and DerfIE (-1) K DNA in which the codon corresponding to the C-terminus of the prosequence was replaced with lysine codon from codon glutamate was introduced into a baculovirus expression system. Recombinant Derfl having the same IgE binding activity as native Derfl is produced by producing a recombinant Derfl whose side is replaced with a lysine group from the original glutamic acid group and accurately removing the prosequence using lysyl endopeptidase. (Shoji H. et al .: Biosci. Biotech. Biochem., 61, 1)
668-1673, 1997). However, the sugar chains of recombinant Derfl produced using insect cells were not identical to the sugar chains of native Derfl (Shoji H. et al .: Biosci. Biot.
ech. Biochem., 61, 1668-1673, 1997).

[0005]

As described above, the sugar chain of the recombinant DerfI produced using insect cells is the natural type D
Since it is not the same as the erfI sugar chain, new antigenicity is a concern. In addition, since special equipment is required for culturing insect cells in large quantities, production in a less costly system has been expected. Furthermore, in desensitization therapy or the like, since it is directly taken into the human body, it is preferable to use a safer system including trace substances. Therefore, it has been expected to produce a recombinant Derfl having the same sugar chain or only a part of the natural Derfl sugar chain, and to use a lower-cost and safer expression system.

[0006]

Means for Solving the Problems The present inventor has intensively studied to solve the above problems, is considered to be safe because it is widely used food and brewing, etc., yeasts Saccharomyces cerevisiae (Saccharomyces cerevisia
e), the filamentous fungus Aspergillus oryzae (Aspergillus o
The present invention has been completed by focusing on using ryzae ) as a host. That is, according to the present invention, when producing the recombinant mite allergen Derfl,
a gene encoding a fI, by genetic recombination technology yeast Saccharomyces cerevisiae (Saccharomyces cere
visiae ) or the filamentous fungus Aspergillus oryzae ( As
pergillus oryzae ), the method comprising producing a recombinant mite allergen Derfl.

[0007] It should be noted that, as the filamentous fungus Aspergillus oryzae (Aspergillus oryzae) is, niaD300 (FE
RM P-16368) can be used. When the filamentous fungus Aspergillus oryzae is used as a host, it is preferable to use a bran such as a wheat bran as a culture medium, because it can be mass-produced compactly. Furthermore, in the present invention, the sugar chain is removed from the recombinant mite allergen DerfI produced by the above-described method to thereby remove the sugar chain-removed recombinant mite allergen D
erfI can be produced. Here, in removing sugar chains from the recombinant mite allergen Derfl, it is preferable to use Endo Hf which is a fusion protein of the enzyme Endoglycosidase H (Endo H) and maltose.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the recombinant Derf of the present invention will be described.
I allergen will be described. The recombinant Der of the present invention
The fI allergen is secreted into the medium as a bromide by induction of expression. A solution containing recombinant Derfl containing the prosequence of the present invention was added to Endo Hf
To remove sugar chains. Natural Derf
The sugar chain of I is an N-type sugar chain (Shoji H. et al .: Biosci.
Biotech. Biochem., 61, 1668-1673, 1997), a sugar chain is added to an asparagine group using glucosamine-glucosamine-mannose as a mother nucleus. Endo Hf cleaves between the first and the second glucosamine. That is, although the sugar chain is not completely removed, all the proteins to which the N-type sugar chain is added have this glucosamine, so that only this has no specific antigenicity. The prosequence is then removed by acid treatment or lysyl endopeptidase. Thereafter, high-purity mature recombinant Derfl allergen can be easily obtained by various conventionally known separation and purification.

As described above, in the present invention, since a large amount of a recombinant Derfl allergen having allergen activity equivalent to that of a natural Derfl allergen can be obtained, atopic disease caused by sensitivity to the allergen can be obtained. The present invention can provide a hyposensitizing therapeutic agent effective for treating sexually asthma and the like. In particular, the Endo Hf of the present invention
The recombinant Derfl allergen whose sugar chain has been removed by
It has only glucosamine added to asparagine, which is a part of sugar chain, and contains only the same part as the sugar chain of natural type DerfI allergen, so there is no possibility of becoming a new antigen due to the difference of sugar chain, and desensitization therapy Excellent as an agent.

Next, an example of an operation procedure relating to the above-described method for producing a pro-form of the present invention will be described. The yeast Saccharomyces cerevisiae (hereinafter, referred to as S. cerevisiae ) and the filamentous fungus Aspergillus oryzae (hereinafter, A. oryzae )
Called. In order to express a heterologous protein in ()), it is necessary to introduce a plasmid having a gene complementary to the marker and inserted into the downstream of the transcription promoter, in which the gene is efficiently transcribed, into a host having an appropriate marker. . This marker, transcription promoter, the host is yeast S.
In the case of cerevisiae , anything that works in the genus S. cerevisiae can be used, and when the host is the filamentous fungus A. oryzae , A. oryzae
Anything that works in the genus can be used. The method for inserting a heterologous gene can be arbitrarily performed using a conventionally known genetic engineering technique.

[0011] The prepared plasmid has a host of yeast S. c.
In the case of erevisiae , the host is a filamentous fungus, A. oryzae , to any S. cerevisiae sp.
In any case, any marker whose marker is complemented by the plasmid
What is necessary is just to introduce | transduce into A. oryzae genus, and can introduce | transduce using a conventionally well-known genetic engineering technique. By inducing the transcription activity of the promoter upstream of the heterologous gene contained in the transformant thus expressed, a pro-form of the recombinant mite allergen Derfl can be obtained.

[0012]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Construction of Expression Vectors As shown in FIG. 1, pUC118-DerfIE (-) subcloned into the BamHI site of the vector pUC118.
1) K was digested with the restriction enzyme BamHI, and the fragment containing DerfIE (-1) K was inserted into the BamHI site of pYES2 (Funakoshi). De in the correct direction downstream of the GAL1 promoter
rfIE (-1) K DNA inserted into pYES2
-Derf IE (-1) K. Also, as shown in FIG. 1, pUC118-DerfIE (-1) K was replaced with BamH
The fragment containing DerfIE (-1) K is blunted with a blunting kit (Takara Shuzo), and pU
It was inserted at the Sma I site of NG1. The DNA in which DerfIE (-1) K DNA was inserted in the correct direction downstream of the glaA promoter was designated as pUNG1-DerfIE (-1) K.

(Example 2) Acquisition of a transformant of yeast S. cerevisiae Lithium acetate method (Ito H. et al .: J. Bacteriol., 153, 16)
3-168, 1983), pYES2-DerfIE (-1) K
Was replaced with S. cerevisiae CG379 (α, ade5, his7-2, leu2-
3,112, trp1-289, ura3-52, East Genetic
Stock Center). CG379 is a medium containing adenine, histidine, leucine, and tryptophan (yeast nitrogen base 0.67% (0.67% Ye
Ast nitrogen base without amino acids (manufactured by Difco)), glucose 2%, agar 2%) cannot grow, but the strain into which the plasmid is introduced can grow. Expression of the heterologous gene in the transformant was determined using a galactose medium (yeast
Nitrogen base 0.67% (0.67% East nitrogen
base without amino acids (Difco)), galactose 2%, casamino acids 2% (2% casamino acids) (Difco
)).

[0015] (Example 3) and the transformants producing yeast S. cerevisiae recombinant DerfI allergens which uses the transformant yeast S. cerevisiae was cultured for 48 hours at 30 ° C. by galactose media. Using an anti-Derfl antibody, it was examined whether recombinant Derfl was secreted and produced in the culture supernatant. As a result, no signal was observed as it was, but under denaturing conditions, Endoglycosidase H
Endog, a fusion protein of (Endo H) and maltose
When the sugar chains were removed with lycosidase Hf (EndoHf) (manufactured by Biolabs), a signal was observed at 35 kDa (FIG. 2, panel A).

Next, purification was studied. Molecular weight cutoff 3
The culture supernatant was concentrated using a 10,000 ultrafiltration membrane. Add En to the culture supernatant
do Hf is added to 10 units / ml, and
The mixture was kept warm for a period of time to remove sugar chains. The prosequence was removed using lysyl endopeptidase. High molecular weight proteins were removed by passing through an ultrafiltration membrane having a molecular weight cut off of 30,000. Finally, purification was performed using an anion exchange column (FIG. 2, panel B). As shown in Table 1, the yield was 0.5 mg / L.

(Example 4) Acquisition of a transformant of the filamentous fungus A. oryzae The expression vector pUNG1-DerfIE (-1) K was transformed into Unkles et al. (Unkles SE et al .: Mol. Gen. Genet., 218, 99- 104,
According to the method of 1989), NO ₃ metabolic gene-deficient strain (nia
D300 (FERM P-16368)). The host niaD300 is a selective medium (0.2% NaNO ₃ , 0.
1% K ₂ HPO ₄ , 0.05% MgSO ₄・ 7H ₂ O, 0.05% KCl, 0.001% Fe
SO ₄ , 2% glucose, 1.2 M Sorbitol, 2% agar, pH5.5)
However, since pUNG1 has a NO ₃ metabolism gene (niaD) inserted as a selection marker, a strain in which the introduced plasmid has recombined on the chromosome can grow on a selection medium. Leave it at 30 ° C for 5 days, take the grown bacteria, and subculture three times in the selective medium,
Transformant niaD300-Derfl (FERM P
-16359). Expression of the heterologous gene in the transformant was determined using DPY medium (2% Dextrin, 1% polypepton, 0.5%
yeast extract, 0.5% KH ₂ PO ₄ , 0.05% MgSO ₄ ).

(Example 5) Recombinant Derf in liquid medium
Production of I Transformant niaD300-Der of filamentous fungus A. oryzae
fI (FERM P-16359) with 10 ml of D
PY medium is inoculated, and shake culture (60 rpm) is performed at 30 ° C. for 2 nights.
m). Western analysis of the culture supernatant was performed. As shown in FIG. 3, no signal was observed (FIG. 3, panel A, lane 1). When treated with Endo Hf, a single signal was observed at the position of the precursor (35 kDa) (FIG. 3, lane 2 of panel A). This suggested that a hetero-sugar chain was added.

Next, purification was attempted. Endo Hf was added at 10 units / ml to the culture supernatant to which the expression was induced, and the mixture was kept at 37 ° C. for 3 hours to remove sugar chains.
By passing the culture supernatant directly through a QMA column,
α-amylase and recombinant Derfl were separated. Lysyl endopeptidase was added to a concentration of 10 μg / ml, and dialysis was performed at 4 ° C. overnight against 20 mM Tris HCl (pH 9.0). 20 mM Tris HC
DEAE-Sephac equilibrated to 1 (pH 8.0)
El column (Pharmacia) was charged directly, and 3 times the volume of 20 mM Tris HCl (pH 8.0) was used.
And washed. Recombinant Derfl by NaCl gradient
Was eluted, the fraction containing the recombinant Derfl was detected by Western analysis, and fractions having high purity were collected and used as a purified sample. The purity was 90% on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
% (FIG. 3, panel B, lane 1). The yield was 8 mg / L (Table 1).

(Example 6) Recombinant Derf in solid culture
Production of I To 2 g of wheat bran, 1.6 ml of 0.1 M ammonium sulfate was added,
Heat treatment was performed in an autoclave. A transformant of filamentous fungus A. oryzae niaD300-Derfl (FERMP-16
359) and cultured at 30 ° C. for 2 nights. When the transformant culture was extracted with water and subjected to Western analysis, no signal was observed (FIG. 4, panel A, lane 1). When treated with Endo Hf, anti-Der
Two signals reacting with the fI antibody (35 kDa, 28
kDa) (FIG. 4, panel A, lane 2). Addition of lysyl endoprotease resulted in one signal (28 kDa) (FIG. 4, panel A, lane 3).

Next, purification was attempted. The water extract was filtered with a glass microfiber filter (Whatman) and filtered again with a 0.45 μm membrane filter (Advantec). One-third volume of 1.5 M citrate buffer (pH 5.5) was added to the filtrate. Endo H
f at 10 units / ml, and add 3 units at 37 ° C.
After leaving for a time, the sugar chains were removed. Ammonium sulfate was added to achieve 60% saturation. It was left overnight at 4 ° C. 12000rpm
By centrifuging for 10 min at, and collecting the precipitate fraction,
Contaminants were reduced. The resultant was solubilized with distilled water, and subjected to gel filtration using a G-50 gel filtration column (Pharmacia) to remove low molecular weight substances. Solution exchange at the same time (20m
M phosphate buffer (pH 6.0)), so that it was passed through a DEAE-Sephacel column equilibrated with the same buffer to reduce other proteins.

Lysyl endopeptidase at 10 μg /
ml and add 20 mM Tris HCl (p
H9.0) was dialyzed to remove the pro-sequence of the recombinant Derfl mite allergen. 20 mM Tris
DEAE-equilibrated with HCl (pH 8.0)
Charge the dialysate directly to the Sephacel column,
After washing with the same buffer solution, the recombinant Derfl was eluted with a NaCl concentration gradient, the fraction containing the recombinant Derfl was detected by Western analysis, and fractions with high purity were collected.
It was used as a purified sample (FIG. 4, panel B). SDS-PA
As determined by GE, the purity was greater than 90%. The yield was 48 mg / kg when wheat bran was used.
(Table 1).

[0023]

[Table 1]

(Example 7) IgE binding activity of each recombinant DerfI allergen Each recombinant mite allergen Derfl obtained using yeast S. cerevisiae and filamentous fungus A. oryzae , Der derived from tick
The fI was adsorbed to the filter paper activated with cyanogen bromide at room temperature overnight under light-shielding, and then adsorbed with 0.1 M NaHCO ₃ for 1 hour.
After washing twice, 1M β-ethanolamine (pH 9.
0) was added and left at room temperature for 3 hours. Then 0.1
Once with MNaHCO ₃ , 0.1 M acetate buffer (pH 4.
0) three times with washing buffer (16 mM Na ₂ HPO ₄ , 4
mM NaH ₂ PO ₄ , 0.15 M NaCl, 0.1% T
After washing twice with Ween-20 (pH 7.4), human anti-mite IgE serum was further added and shaken at 37 ° C for 3 hours. Then, it is washed three times with a washing buffer, and the enzyme-linked anti-Ig
E antibody was added and reacted at room temperature overnight. Further, the plate was washed three times with a washing buffer, a substrate solution was added, and the mixture was shaken at 37 ° C for 2 hours. Thereafter, the reaction stop solution was added, and the absorbance at 420 nm was measured to compare the IgE binding activities of each recombinant Derfl allergen and Derfl derived from tick. As shown in FIG. 5, they were equivalent. In addition,
In FIG. 5, A420nm is the absorbance at 420nm, mi
te is derived mite DerfI, fungi recombinant mite allergen DerfI obtained using filamentous fungi A.oryzae, S.ce
revisiae indicates the recombinant mite allergen Derfl obtained using the yeast S. cerevisiae.

[0025]

As described above, according to the present invention, a remarkable effect that a large amount of a recombinant Derfl allergen having allergen activity equivalent to that of a natural Derfl allergen can be obtained can be obtained. Accordingly, it is possible to provide a therapeutic agent for desensitization that is effective in treating atopic asthma and the like that are developed due to sensitivity to this allergen. In addition, the recombinant Derfl allergen of the present invention from which the sugar chain has been removed by Endo Hf has only glucosamine added to asparagine which is a part of the sugar chain, and contains only the same portion as the sugar chain of the natural Derfl allergen. Therefore, there is no possibility of becoming a new antigen due to a difference in sugar chain, and it is extremely excellent as a desensitizing therapeutic agent.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a method for constructing an expression vector.

FIG. 2 is a panel diagram showing that a recombinant Derfl allergen is produced by a transformant of yeast S. cerevisiae .

FIG. 3 is a panel diagram showing that recombinant DerfI allergen is produced in a liquid medium by a transformant of the filamentous fungus A. oryzae .

FIG. 4 is a panel diagram showing that recombinant Derfl allergen is produced in solid culture by a transformant of the filamentous fungus A. oryzae .

FIG. 5 is a graph showing the IgE binding activity of each recombinant Derfl allergen.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 865) (C12P 21/02 C12R 1:69)

Claims (6)

[Claims] Upon producing 1. A recombinant mite allergen DerfI, the gene encoding the DerfI, genetic recombination technology by the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) a recombinant mite allergen DerfI, characterized in that is expressed in Production method. Upon wherein producing recombinant mite allergen DerfI, the gene encoding the DerfI, genetic recombination technology by filamentous fungi Aspergillus oryzae (Aspergillus oryzae) recombinant mite allergen characterized in that it is expressed in DerfI Production method. 3. The filamentous fungus Aspergillus oryzae (Aspe
rgillus oryzae ) is niaD300 (FERM P-16)
No. 368). 4. The production method according to claim 2, wherein the bran is used as a medium. 5. A recombinant mite allergen Derfl produced by the method according to any one of claims 1 to 4.
A method for producing a recombinant mite allergen, DerfI, comprising removing sugar chains from E. coli. 6. In removing sugar chains from recombinant mite allergen Derfl, Endoglycosidase H (Endoglycosidase H) is used.
H) EndoHf, a fusion protein of maltose
The method for producing a recombinant mite allergen Derfl according to claim 5, characterized in that: