JP3475221B2 - Mutant proteins with reduced immunogenicity - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to the substitution of at least one amino acid residue in a protein molecule having immunogenicity,
The present invention relates to a mutant protein, which is deleted or added, that causes a hyporesponsiveness or unresponsiveness of an immune response, particularly a mutant β-lactoglobulin and its gene, and a method for producing a mutant β-lactoglobulin.

[0002]

2. Description of the Related Art Living bodies have acquired various defense mechanisms in the course of evolution in order to protect themselves from foreign substances. For example,
When foreign substances such as toxin proteins and pathogenic microorganisms invade the body, the living body tries to remove them by distinguishing them from foreign substances by a highly developed function called immunity. The essence of the immune function is a function of discriminating between self and non-self and eliminating non-self components, which contributes to maintain homeostasis of the individual.

[0003] However, the immune function originally supposed to be provided for the purpose of protecting the body may, on the contrary, act to be harmful. Allergies and autoimmune diseases are examples.
In recent years, the number of patients with food allergies has rapidly increased due to changes in dietary habits such as a decrease in breastfeeding infants, an increase in animal protein intake, and the use of food additives [Shuichi Uenogawa; Food Allergy Kodansha Blue Bucks (199
2)]. Nevertheless, the underlying cure is not yet established, and the current situation is to refrain from ingesting the causative food.

The most frequent foods causing food allergies are milk, chicken eggs and soybeans, which are called three major allergens. All of these are foods with high nutritional value, but they cause food allergies, and therefore infants and children among allergic patients must refrain from ingesting these foods, which is a serious problem.

Therefore, great expectations are placed on the development of foods that reduce allergies. Further, as a therapeutic method for a patient with a genetic disease in which a certain protein is deficient, a method in which the defective protein is produced using a gene manipulation technique and administered can be considered. In this case, since the protein is a non-self component for patients, it is highly likely that anaphylactic shock, which is one of allergic reactions, will occur when administered multiple times. In order to avoid this, it is necessary to reduce or eliminate the immunogenicity of the protein itself.

[0006] In order to solve these problems, development of a method for reducing the immunogenicity of a protein is desired.

[0007]

The present invention can reduce the immunogenicity (antigenicity) of a protein that causes allergies, even though it is a useful protein contained in food, and can be used as an allergy-reducing food. It is an object of the present invention to provide a mutant protein and a protein drug that does not cause anaphylactic shock.

[0008]

[Means for Solving the Problems] A series of allergic reactions such as antibody production, mast cell degranulation, and inflammatory reaction
It is known that a T cell receptor recognizes a complex of an antigen fragment fragmented in an antigen presenting cell and a major histocompatibility complex (hereinafter referred to as "MHC"), and T cell is activated to start. From this, by controlling the interaction between the antigen, MHC molecule and T cell receptor,
It is believed that it can regulate the immune response.

[0009] Therefore, as a result of intensive studies based on the above problems, the present inventors have found that MH in protein antigen molecules is
It was found that the immunogenicity of a protein of a protein is reduced by substituting, deleting or adding at least one amino acid residue at a site recognized by a C class II molecule and a T cell receptor. It came to completion.

[0010] That is, the present invention provides a mutant protein in which at least one amino acid residue in a protein molecule having immunogenicity is substituted, deleted or added to bring about a decreased response or unresponsiveness of an immune response. is there. Here, examples of the mutant protein include mutant β-lactoglobulin, mutant α-lactalbumin, and mutant casein. Among them, the mutant β-lactoglobulin includes the amino acid sequence of β-lactoglobulin represented by SEQ ID NO: 1 in which the 126th proline is replaced with alanine, and the 126th proline is phenylalanine. Containing a sequence replaced by
The 28th valine contains a sequence in which aspartic acid is substituted.

Furthermore, the present invention is a mutant β-lactoglobulin gene containing a DNA encoding any of the amino acid sequences represented by SEQ ID NOs: 2-4. Furthermore, the present invention is a recombinant DNA in which the mutant β-lactoglobulin gene is integrated. Furthermore, the present invention is a transformant transformed with the recombinant DNA.

Furthermore, the present invention is a method for producing mutant β-lactoglobulin, which comprises culturing the transformant and isolating the mutant β-lactoglobulin from the resulting culture. Hereinafter, the present invention will be described in detail. The present invention elicits the strongest T cell response among certain protein antigens in view of the fact that only a limited number of regions called T cell antigen determinants induce T cell responses among protein antigens. Territory, the most dominant T
Regarding the cellular antigenic determinants, after identifying the amino acid residues involved in the binding to MHC class II molecules, a mutant protein having the amino acid residues substituted, deleted or added was prepared, and its immunogenicity was determined. It is done by looking for a drop.

Examples of the protein used in the present invention include β-lactoglobulin, α-lactalbumin, casein and the like, and β-lactoglobulin is preferable because it is the major and most potent allergen in milk. Hereinafter, β-lactoglobulin (hereinafter “β-L
G ”) will be described as an example.

The T cell antigenic determinant of β-LG, that is, the region inducing the T cell response has already been clarified. Among them, the region in which the T cell response is most strongly induced in C57BL / 6 mice (the most dominant region) T cell antigenic determinants)
Is a peptide corresponding to the 119th to 133rd amino acids in the amino acid sequence of natural β-LG (SEQ ID NO: 1), and among them, the 122nd to 129th amino acid residues are important for its function. Is known [Mamoru Totsuka, Akio Ameya, Kyoko Goto, Shuichi Uenogawa; Proceedings of the 1992 Annual Meeting of the Japanese Society of Agricultural Chemistry 66, 276 (1992)].

Therefore, in the present invention, first, the amino acid residue region of the 119th to 133rd amino acid residues of natural β-LG is used, and the amino acid residue in any part of the region is substituted, deleted or added to obtain β
-To examine whether the immunogenicity of LG is reduced or eliminated, the residues involved in the binding of β-LG-derived peptides to MHC class II molecules were identified, and further, those residues were replaced. It was decided to obtain an analog of the peptide and to investigate in which analog peptide the immunogenicity was lost or diminished.

Next, whether or not the β-LG has immunogenicity is determined by using the recombinant β-LG which has been subjected to substitution or the like by converting the base sequence encoding the identified amino acid residue. I decided to do a test. (1) Identification of amino acid residues involved in binding to MHC class II molecule. First, the peptide used in the present invention is synthesized. That is, in the amino acid sequence of natural β-LG (SEQ ID NO: 1),
A peptide corresponding to 15 amino acid residues from 119th to 133rd, and one amino acid residue at the 122nd to 129th positions among the 119th to 133rd amino acid residues An analog peptide in which is substituted is synthesized. The synthesizing method is a conventional method, and for example, an automatic peptide synthesizer or the like can be used.

Purification of the synthesized peptide is carried out by a conventional purification means such as high performance liquid chromatography and reverse phase chromatography. Then, amino acid analysis and confirmation of the amino acid sequence of the obtained peptide are performed. The peptide synthesized and purified as described above is used in the following MHC class II molecule binding test.

In C57BL / 6 mice, MHC class I
The molecule corresponding to the I molecule is the ^IAb molecule. Therefore, MH
In order to identify the residues involved in the binding to the C class II molecule, the degree to which the above analog peptide inhibits the binding of the biotin-labeled peptide to the ^IAb molecule can be used as an index. MHC class II molecular binding test
As shown in Examples described later, the method is performed according to Jensen's method [Jensen, PE, J. Exp. Med. 174, 1111-1120 (1991)].

The T of β-LG in C57BL / 6 mice
The structure of the cell antigenic determinant is similar to that of other mice (BALB / C, C3H / He
Etc.) is the simplest compared with MHC class
From having only I-A ^b molecule as II molecules, for immunizing C57BL / 6 mice in that it is possible to perform an immune reaction in a simple system, to use for the supply of MHC Class II molecules preferred.

(2) Immunogenicity of analog peptide in which the residue involved in binding to MHC class II molecule is substituted The above is the most dominant T cell antigenic determinant of β-LG in C57BL / 6 mice. Amino acid residues in the amino acid residue region that are involved in binding to class II molecules can be identified. Then, in the present invention, whether or not the peptide has immunogenicity can be determined by examining the immune response to the peptide having low binding property to MHC class II molecule.

Mice are immunized with a peptide that is considered to have low immunogenicity, a lymph node cell proliferation test is performed, and a T cell response to the peptide is examined. For immunization of mice, a peptide which is considered to have low immunogenicity is subcutaneously administered to the back of the mouse along with an adjuvant. Seven days after immunization, lymph node cells are prepared, and the proliferative response to the peptide used for immunization is examined. The lymph node cell proliferation test is performed according to the method of Kurisaki et al. (Kurisaki, J., et al., Eur.J.Immuno), as shown in Examples described later.
L., 16, 236-240 (1986)).

Also, mice are immunized with the above peptides, and antibody production response to the peptides is examined.
For immunization of mice, the peptide is administered to the abdominal cavity of mice with an adjuvant. After the first immunization, the mice are immunized with the same amount of the peptide every two weeks, and blood is collected from the tail of the mouse before the first immunization and one week after each immunization. The amount of specific antibody in the obtained antiserum can be measured by a usual method, for example, enzyme-linked immunosorbent assay (ELISA).

(3) Mutant β-by a genetic engineering method
LG expression and immunogenicity studies. In the present invention, mutant β-L prepared by a genetic engineering method
Production of G is performed as follows. That is, natural β-L
The site of the target amino acid residue of G cDNA (the amino acid residue involved as an antigenic determinant, which became clear from the description of (1) and (2) above) was subjected to site-directed mutagenesis. It is converted and inserted into an expression vector to construct an expression plasmid. The conversion means not only substitution of amino acids but also deletion or addition.

CD obtained by conversion by the mutagenesis method or the like
The NA fragment can be purified by, for example, agarose gel electrophoresis, polyacrylamide electrophoresis, or the like. Next, all or part of the DNA encoding β-LG is digested with an appropriate restriction enzyme (for example, EcoRI, SalI).
Etc.) and ligate it downstream of an appropriate promoter, enhancer, or the like, or insert it between the promoter and the terminator, and then introduce this into a transformable host for transformation.

As the vector DNA used here, a plasmid vector, a virus vector and the like are used. As a plasmid vector, pYG100, pKK223-3
Etc. are preferable, and as the viral vector, pSV2gpt and the like are preferable. As a host, yeast, E. coli, animal cells,
Insect cells and the like can be mentioned, but yeast is preferred.

The nucleotide sequence of DNA encoding β-LG is known (LJ Alexander, et al., Nucl. Acids Re
s.17,6739 (1989)), and can also be obtained by synthesis. In order to obtain the recombinant mutant β-LG of the present invention,
Culturing the transformant in a commonly used medium,
The culture supernatant is collected and purified by a commonly used method.

As the medium, for example, YMM medium (minimum medium for yeast), LB medium (for E. coli culture), RPMI1640
A medium (for animal cell culture) and the like can be mentioned. Culture is usually 30
It can be carried out at 48 ° C. or 37 ° C. for 48 to 120 hours, and if necessary, aeration and stirring can be carried out. After culturing, the transformant is removed by centrifugation or the like to obtain a culture supernatant. Alternatively, the collected transformants are suspended in, for example, a buffer solution, frozen, thawed, boiled, etc., and then centrifuged to obtain a supernatant.

Separation and purification of the gene recombinant type mutant β-LG from the obtained culture supernatant or the above-mentioned supernatant may be carried out according to a generally known protein purification method. For example, salting out, centrifugation, various chromatographies, electrophoresis and the like are appropriately combined for purification. Examples of various chromatographies include gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like. The purity and the approximate molecular weight of the purified product are confirmed by SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis, Western blotting and the like.

The detection of anti-mutant β-LG antibody using the genetically modified mutant β-LG of the present invention is carried out as follows. That is, each antigen of natural β-LG and gene-recombinant mutant β-LG is intraperitoneally administered to a mouse together with an adjuvant, and 2 weeks after that, booster immunization is performed. One week after that, blood was collected and the specific antibody titer in the serum was measured by ELISA.
It is measured by Method A.

[0030]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples. [Example 1] Identification of residues involved in binding of β-LG-derived peptide to MHC class II molecule (1) Synthesis and purification of β-LG partial peptide and its analog peptide Natural β-LG No. 119- Synthesis and purification of a peptide corresponding to the 133rd amino acid residue region (p119-133), and an analog peptide corresponding to this region, each of which has one amino acid residue substitution at the 122nd to 129th positions. went.

In the following, a simple numerical description "mn" means the position from the mth to the nth position in the amino acid sequence of β-LG. Also,
When described as "pm-n", it means a peptide having the m-th to n-th amino acid residues of β-LG. For example, when "p119-133" is described, it means that it is a peptide having 15 amino acid residues from the 119th to the 133rd amino acids of β-LG.

A schematic diagram showing the amino acid sequence of each peptide is shown in FIG. In Fig. 1, symbols with numbers sandwiched between alphabets are the amino acids described on the left side of the numbers (single-letter notation) while shifting the amino acids up to 122-130 in the amino acid residue region of 119-133 of β-LG one by one. The symbol of the amino acid according to the law) is replaced with the amino acid described on the right side of the number. For example, "L122A" means a peptide in which leucine at the 122nd position (4th position out of 15 amino acid residues) is replaced with alanine.

The definitions given here will be used similarly hereinafter. (2) Synthesis of β-LG partial peptide A peptide corresponding to the 119-133 amino acid residue region of β-LG was synthesized as a peptide synthesizer 430A (Applied Biosyste
ms Inc., Foster City, CA) and synthesized by the F-moc method. After the synthesis, the protecting groups of the resin and amino acid side chain were cleaved by the following method.

I) The peptide with resin was put in an eggplant-shaped flask and cooled with ice. ii) Cleavage reagent that has been ice-cooled in advance [0.75 g phenol (Wako Pure Chemical Industries, Ltd.), 0.25 ml 1,2-ethanedithiol (Nacalai Tesque), 0.5 ml thioanisole (Tokyo Kasei), 0.5 ml deionized water, 10 ml trifluoroacetic acid (tr
ifuluoroaceticacid; TFA Applied Biosystems Inc.)
] Was added.

Iii) Return the flask to room temperature and then 1.5
Stir for hours. iv) Pass the contents of the flask through a glass filter No. 13 and cool 30 ml of methyl-t-butyl ether (M
TBE; Tokyo Kasei Kogyo). v) Wash the resin remaining on the filter with 1 ml of TFA, iv)
Peptides were transferred into MTBE as in.

Vi) MTBE in which the peptide was suspended was transferred to a centrifuge tube and centrifuged at 1300 xg for 10 minutes. vii) The supernatant was aspirated and removed, and 30 ml of MTBE was added to the precipitate to suspend it, followed by centrifugation in the same manner as vi). viii) vii) was repeated two more times. The iX) peptide was dissolved in water and lyophilized.

(3) Purification of Synthetic Peptide 119-133 by HPLC β-LG partial peptide 119-13 synthesized as described above
3 was purified by reverse phase HPLC using an ODS column. HPLC
Uses LC-4A (Shimadzu) and the column is Lichroso
rb RP-18 (E. Merck, Darmstadt, FR Germany) was used. The elution conditions were a flow rate of 3 ml / min and 0.1% TFA (App
lied Biosystems Inc.) aqueous solution was subjected to a linear concentration gradient (10% to 50% in 50 minutes) of acetonitrile (Wako Pure Chemical Industries). The elution peak was collected, freeze-dried and used in the subsequent experiments.

(4) Amino acid analysis of synthetic peptide 119-133 The elution peak of HPLC was collected and the freeze-dried sample was hydrolyzed in hydrochloric acid vapor at 130 ° C. for 3 hours. Then, it was dissolved in 0.02N hydrochloric acid and analyzed by an 835 high-speed amino acid analyzer (Hitachi Ltd.) to confirm that it had an amino acid composition of a peptide corresponding to the 119-133 region of β-LG.

(5) Confirmation of Amino Acid Sequence of Synthetic Peptide 119-133 The amino acid sequence of the synthetic peptide was confirmed using a protein sequencer PSQ-1 (Shimadzu Corporation). (6) Biotinylation of peptide 119-133 Peptide 119-133 and NHS-LC synthesized and purified by the above method
-Biotin (Pierce) with 50 mM sodium hydrogencarbonate solution (p
H8.5) and dissolved in a molar ratio of 1: 2.5, 4
Shake for 2 hours at ℃. It was then purified using reverse phase HPLC in the same manner as described in (3). Furthermore, quantification was performed by the same amino acid analysis as in (4).

(7) α _S1 -Casein Partial Peptide A peptide (P10) corresponding to the 136-155 amino acid residue region of α _S1 -casein was synthesized, purified and used in the same manner as in (3). Also, the peptide (P3) corresponding to the 31-50 region,
It was synthesized and purified in the same manner. (8) I-A ^b molecule I-A ^b molecule, from C57BL / 6 mouse spleen cells were solubilized in NP-40, anti-I-A ^b monoclonal antibody M5 / 114.15.2
(Bhattacharya, A., et al., J. Immunol. 127, 2488 (1981))
What was purified using an affinity column on which was immobilized was used.

(9) I- ^Ab molecular binding test The I- ^Ab molecular binding test was carried out by Jensen's method (Jensen, PE, JE).
xp.Med., 174, 1111-1120 (1991)). The outline is shown in FIG. 2 μM biotinylated peptide and 0-400 μM unbiotinylated peptide were added to 0.2% NP-40 (Nacalai Tesque), 1 mM phenylmethylsulfonyl fluoride (Wako Pure Chemical Industries, Ltd.), 2 mM ethylenediaminetetraacetic acid tetrasodium (EDTA; by Wako pure chemical), 5 mM N-ethylmaleimide (Nakarai chemicals) 30 [mu] L of 50mM citric acid / phosphate buffer containing in (pH 5.0), together with the I-a ^b molecule of 40 nM 37
It was left standing at ℃ for 48 hours.

50 μg / ml of affinity purified goat anti-rat immunoglobulin antibody on immunoplate (Nunc)
(Cappel) 0.11M phosphate buffered saline (PBS; disodium hydrogen phosphate 12 water 26.2 g, potassium dihydrogen phosphate 5.0 g, sodium chloride 2.34 g / deionized water 1 L, p
H 7.2) was added in an amount of 100 μl per well, and the mixture was allowed to stand at room temperature for 2 hours.

Each well was washed 3 times with PBS-tween (tween 20; PBS containing 0.5 ml / L of Wako Pure Chemical Industries, Ltd.), and 120 μg / ml
The rat anti-I-A ^b monoclonal antibody M5 / 114.15.2
100 μl was added, and the mixture was left standing at room temperature for 2 hours. After washing as above, 5% skim milk, 0.1% bovine serum albumin (BSA; armour pharmaceutical compan
y), 50 mM Tris-HCl buffer containing 0.1% Tween 20 (pH
7.5) 125 μl was added, and the mixture was left standing at room temperature for 2 hours.

After washing in the same manner as described above, 200 mM containing 5% skim milk, 0.1% BSA, 0.1% Tween20 0.5% NP-40
What was diluted to 200 μl with Tris-HCl buffer (pH 7.5) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing in the same manner as above, alkaline phosphatase-labeled streptavidin (Zym diluted with PBS-tween 150 times) was used.
ed) (100 μl) was added, and the mixture was allowed to stand at 25 ° C. for 1 hour.

After washing in the same manner as in the above, 100 μl of 1 mg / ml disodium p-nitrophenylphosphate (Tokyo Kasei) / diethanolamine-hydrochloric acid buffer solution (pH 9.8) was added as a substrate, and the mixture was reacted at room temperature and the absorbance at 405 nm was measured. The value was measured.
The results are shown below. In C57BL / 6 mice, MHC
Corresponding to class II molecules are ^IAb molecules. I-
The results of the ^Ab molecular binding test are shown in FIG.

In FIG. 3, p119-133 (“●” in both FIGS. 3a and 3b) inhibited the binding of biotinylated p119-133 to the IA ^b molecule in a concentration-dependent manner. This indicates that there is competition for binding to the ^IAb molecule. On the other hand, the peptide P126A in which the 126th proline was replaced with alanine (“Δ” in FIG. 3a) and the peptide P126 in which the 126th proline was replaced with phenylalanine
126F (“Δ” in FIG. 3b) had a lesser degree of inhibition compared to p119-133. Peptide V128D (“◯” in FIG. 3b) in which the 128th valine (hereinafter referred to as “128Val”; the same applies to other amino acids) was replaced with aspartic acid had a weaker degree of inhibition.

[0047] These three analog peptide binding to I-A ^b molecule of biotinylated p119-133 To the same extent inhibit the p119-133 required a concentration of more than 10 times. Therefore, P126A, P126F, V128D is coupled with the I-A ^b molecule was found to be lower by 10 times or more as compared with P119-133.

Example 2 Immunogenicity of Analog Peptides Substituting Residues Relating to Binding to MHC Class II Molecules In Example 1, immunization with peptides P126A and V128D having low binding to MHC class II molecules. The origin was analyzed.
First, the ability of these analog peptides to induce T cell responses
It was compared with the ability of P119-133 to induce T cell response. Next, the ability of these analog peptides to induce antibody production was analyzed, and P1
Compared with the case of 19-133.

(1) Immunization of Mice C57BL / 6 mice (female, 8 weeks old; Charles River Japan) were subcutaneously p119-133, P126A and V128D subcutaneously on the sole of each hind foot.
50 μL of each of the emulsions containing 1 was administered so that the amount of peptide was 10 μg or 100 μg per animal. Each peptide was dissolved in sterile PBS at 0.2 mg / ml or 2 mg / ml, and Freund's complete adjuvant (CFA; Difco) containing an equal amount of H37Ra was killed to give a water / oil emulsion. .

(2) Lymph node cell proliferation test One week after immunization of the mouse by the method of (1), lymph nodes in the groin and below the knee were excised to prepare a lymphocyte single cell suspension. The medium was the same as that described in Example 1
PMI1640 with 1% of normal mouse serum was used. The cell suspension was diluted to a concentration of 4 × 10 ⁶ cells / ml and cultured in the well of 96 well in the presence of antigen. After 3 days, add 1 μCi / well of ³ H-thymidine, and add 18-22
After a period of time, the cells were filtered on the filter with a cell harvester (Abe Scientific Co., Ltd.). ³ H-thymidine incorporated into cells was measured by a TRICAR B1500 liquid scintillation counter (Packard). For scintillation cocktail, 4 g of toluene (Wako Pure Chemical Industries) and 1,4-bis [2- (5-
Phenyloxazolyl)]-benzene (Wako Pure Chemical Industries) 0.1 g
What melt | dissolved was used.

(3) Immunization and Blood Collection of Mice C57BL / 6 mice (female, 8 weeks old; Charles River Japan) were intraperitoneally administered with 10 μg of each of p119-133, P126A and V128D. In addition, mice were immunized every two weeks with the same amount of peptide. Each peptide is sterile PB
It was dissolved in S to a concentration of 200 μg / ml, and the same volume of adjuvant was added to give a water / oil emulsion. CFA (Difco) was used for the first immunization, and Freund's incomplete adjuvant (IFA; Difc) was used for the second and subsequent immunizations.
o) was used. Blood was collected from the tails of mice before the first immunization and one week after each immunization. The time schedule for immunization and blood collection is shown in FIG.

(4) Measurement of the amount of specific antibody in serum The amount of specific antibody in the antiserum obtained in (3) was measured by enzyme immunoassay (E
LISA). The method will be described below. To each well of a 96-well polystyrene microtiter plate (Nunc), 100 μl of an antigen peptide dissolved in PBS to a concentration of 10 μg / ml was added, and the mixture was allowed to stand at 4 ° C. overnight.

After washing each well with PBS, 1%
(200 μl) of BSA (armour pharmaceutical company) prepared by dissolving in 0.1M citrate buffer (pH 6.4) was added, and the mixture was allowed to stand at room temperature for 2 hours. After washing each well with PBS-tween, 1% BSA
And 100 μL of antiserum diluted with PBS-tween (PBSTB) containing 5 mM EDTA were added, and the mixture was allowed to stand at room temperature for 2 hours.

After washing each well with PBS-tween,
Alkaline phosphatase-labeled anti-mouse immunoglobulin G, A, M antibodies (Cappel) 100 diluted with PBSTB
μl was added, and the mixture was shielded from light and allowed to stand at room temperature for 2 hours. After washing each well with PBS-tween, 100 μl of 1 mg / ml disodium p-nitrophenyl phosphate (Tokyo Kasei) / diethanolamine monohydrochloride buffer (pH9.8) was added and reacted at room temperature, and the absorbance at 405 nm Was measured.

The results are shown below. First, P126A or V1
The results of lymph node cell proliferation test of mice immunized with 26D are shown in FIG. In addition, FIG. 5 is a diagram when 10 μg of each peptide was administered per animal. In FIG. 5, a is immunized with p119-133,
b is immunized with P126A and c is immunized with V128D.

Immunization with p119-133 strongly evoked a proliferative response to p119-133 (FIG. 5a). In contrast, when immunized with P126A, almost no proliferative response to P126A was observed (Fig. 5b). Also, V1
Immunization with 28D evoked a T cell response against V128D (Fig. 5c). However, when immunized with V128D, the proliferative response to each immunogen was significantly lower at lower concentrations of added immunogen in vitro than when immunized with p119-133. In order to confirm this phenomenon, the same experiment was conducted with a finer antigen concentration, and again, the above tendency was observed (FIG. 6).

Similar results were also obtained when 100 μg of the peptide was administered per animal (FIG. 7). The SI value in FIG. 7 is an average value of the results obtained by using 4 mice. Next, FIGS. 8 to 10 show the results of tests in which the antibody production inducing ability in mice when p119-133, P126A and V128D were used as antigens was compared.

When p119-133 was immunized, the amount of specific antibody in the serum was significantly increased after the second immunization. On the other hand, when immunized with P126A, almost no increase in the amount of specific antibody in serum was observed in the two immunizations.
The same was true when immunized with V128D. 126A and V128D were immunized again and the amount of specific antibody in the serum was measured, but production of specific antibody was hardly observed even after the third immunization.

From the above, when the peptides P126A and V128D, which had low binding properties to MHC class II molecules, were used as antigens, the T cell response inducing ability and the antibody production inducing ability were p119-1.
Since it was lower than that of 33, it was found that the immunogenicity of both peptides was lowered.

[Example 3] The immunogenicity 126Pro of mutant β-LG obtained by a genetic engineering technique was clarified to be involved in binding to MHC class II molecules in Example 1, and in Example 2 It was revealed that substituting it with alanine reduces the immunogenicity of the peptide. In the present invention, 126Pro
In which β was replaced with alanine (hereinafter referred to as “P126Amu
t))), which is within the same T cell antigenic determinant, but which is a mutant β-LG in which 129Asp, which is a residue not involved in binding to MHC class II molecule, is replaced with alanine.
(Hereinafter referred to as “D129Amut”) is a yeast secretory expression system for β-LG that has already been established [Totsuka, M., Katakura, Y., S
himizu, M., Kumagai, I., Miura, K.and Kaminogawa, S., Agr
ic.Biol.Chem. 54, 3111-3116 (1990)],
Antibody production response to these mutant β-LG
-Compared to that for LG.

(1) The synthetic DNA prepared for the introduction of the synthetic mutation of the oligonucleotide was SEQ ID NO: 5 for preparing P126Amut and SEQ ID NO: 6 for preparing D129Amut.
Oligonucleotides having the respective sequences described in 1. were synthesized using an automatic DNA synthesizer (Cyclone, Millipore). The synthesized oligonucleotide was cut out from the synthesis column with 1 ml of 27% ammonia solution and purified using an oligo pack EX cartridge (Millipore).

(2) The site-directed mutagenized bovine β-LG cDNA was cleaved with restriction enzymes SacI and SmaI, and a T4D was prepared using a DNA blunt end kit (Takara Shuzo).
The cDNA fragment was blunt-ended using the 5 '→ 3' polymerase activity of NA polymerase. SalI linkers were attached to both ends of the fragment, and the fragment was inserted into the SalI site of the plasmid vector pUC118 to obtain pBAML. E. coli JM
Single-stranded DNA was prepared by introducing it into 109 strains and further infecting with helper phage M13KO7. Using this as a template, a sculpter in vitro mutagenesis system (Amersham) was used to introduce mutations at the corresponding positions of the target amino acid residues to prepare cDNAs for each mutant β-LG. That is, the 5'end of the synthesized oligonucleotide was phosphorylated with polynucleotide kinase. This phosphorylated synthetic oligonucleotide was annealed to single-stranded DNA serving as a template. Klenow fragment in a solution containing dCTPαS and DN by T4 DNA ligase
The A chain was extended and ligated. Restriction enzyme NclI
Was used to make a break in the chain on the template side. The nicked strand was partially digested with exonuclease III. DNA
The template-side chain digested with polymerase I and T4 DNA ligase was resynthesized based on the mutant-type chain. Escherichia coli TG1 strain was transformed with the obtained plasmid. After preparing a plasmid from the obtained transformant, Dye Deoxy Cycl
By determining the nucleotide sequence using the e-Sequencing Kit (Applied Biosystems) and fluorescent DNA sequencer (373A, Applied Biosystems), select the one that has been converted to the target sequence, and the entire nucleotide sequence is correct. Was confirmed.

(3) Construction of Expression Plasmid Yeast expression vector pYG100 [Kumagai, I. and Miura, K.,
J. Biochem., 105,946 (1989)], Faculty of Engineering, University of Tokyo, Kumagaya
Obtained from Dr. Izumi. The cloning site between the promoter and terminator of glyceraldehyde 3-phosphate dehydrogenase (GAPD) possessed by pYG100 was cleaved with restriction enzyme SalI, and then DNA was recovered by phenol extraction and ethanol precipitation. On the other hand, both 5'ends were dephosphorylated using Escherichia coli alkaline phosphatase.

The plasmid obtained in (2) was treated with the restriction enzyme SalI to obtain a mutant β-LG cDNA fragment. This cDNA fragment was ligated with pYG100 treated as described above using T4 DNA ligase to transform Escherichia coli DH5α strain. From the obtained transformant, plasmid D
NA is extracted and the restriction enzymes HindIII or HindIII and P
By digesting with stI and examining the size of each fragment by agarose gel electrophoresis, one in which only one copy of the cDNA fragment was inserted into the expression vector pYG100 in the correct direction was selected. The resulting P126Amut expression plasmid was designated as pYP126A, D12
The 9Amut expression plasmid was designated as pYD129A (Fig. 11).

(4) Yeast and its medium Yeast Saccharomyces cerevisiae AH22 strain (a, leu2-3,
leu2-112, his4-519, can1) were used. The AH22 stock was acquired from Dr. Izumi Kumagai, Faculty of Engineering, The University of Tokyo. As a selective medium for yeast culture, YMM medium (0.7% Yeast Nitrogen
Glucose concentration in base w / o Amino Acids (Difco), 2% glucose) added with 20 mg / ml L-histidine (hereinafter referred to as “YMM ( ⁺ His) medium”) and YMM medium with a glucose concentration of 10%. And 20 mg / ml L-histidine,
A medium containing 5 g / l ammonium sulfate, 1 g / l dipotassium phosphate, 0.6 g / l potassium chloride, 0.5 g / l magnesium sulfate, 0.02 g / l ferrous sulfate (hereinafter referred to as “10GM-YMM
( ⁺ His) medium ") was used.

(5) Transformation of yeast and transformation of the cultured yeast are carried out by the alkali metal method [Ito, H., Fukuda, Y.,
Murata, K. And Kimura, A., J.Bacteriol., 153,163 (198
3)] After transformation, the colonies appearing after culturing at 30 ° C for 5 to 7 days were inoculated into 2 ml of YMM ( ⁺ His) medium and incubated at 30 ° C for 48 hours.
The culture was shaken for 72 hours. This is the amount of YMM ( ⁺ Hi
s) medium and inoculate 96 g of 15 L of 10GM-YMM ( ⁺ His) medium.
Cultivation was performed at 30 ° C for ~ 120 hours.

(6) The purified culture fluid of the mutant β-LG was centrifuged at 10,000 × g for 10 minutes, and the supernatant was 0.8 μm.
Then, the cells were completely removed by filtration with a 0.45 μm membrane filter. The culture supernatant was concentrated to 500 ml by subjecting it to ultrafiltration (molecular weight cut: 10,000). To this, 50 mM imidazole-hydrochloric acid buffer solution (pH 6.
7) was added in an equal amount and concentrated to 500 ml. This was repeated 4 times. A 50 mM imidazole-hydrochloric acid buffer solution (pH 6.
It was applied to an anion exchange column using DEAE-Sephacel (Pharmacia) that had been equilibrated in advance in 7). Elution was performed with a linear concentration gradient of sodium chloride from 0 to 0.5M. The fraction containing the mutant β-LG was dialyzed against deionized water and freeze-dried. This sample was dissolved in 0.15 M ammonium bicarbonate aqueous solution, fractionated by gel filtration FPLC column (Superose 12, Pharmacia), and the fraction corresponding to β-LG was freeze-dried to obtain the purified mutant β-LG.
Got Detection of mutant β-LG at each operation step was performed by the sandwich ELISA method described in (7).

(7) Sandwich ELISA method 2 μg / ml of anti-β-LG monoclonal antibody (hereinafter referred to as “mAb”) 62A6 [Kamin] was added to each well of a 96-well polystyrene microtiter plate (Nunc).
ogawa, S., Shimizu, M., Ametani, A., Hattori, M.,
Ando, O., Hachimura, S., Nakamura, Y., Totsuka, M.
and Yamauchi, K. (1989) Biochim. Biophys., Acta,
PBS containing 998, 50 (1989)] was added in an amount of 100 μl, and the mixture was allowed to stand at 4 ° C. overnight.

After washing each well three times with PBS-tween, 100 μl of a sample solution diluted with PBS was added, and the mixture was allowed to stand at room temperature for 2 times.
Let stand for hours. After washing in the same manner as above, 100 μl of PBS-tween containing anti-β-LGmAb61C1 labeled with alkaline phosphatase
Was added, and the mixture was shielded from light and allowed to stand at room temperature for 2 hours. The alkaline phosphatase labeling of mAb61C1 was carried out by the one-step glutaraldehyde method [Takao Tasaka, Yoshihiro Hamashima, Immunological Experimental Procedure X,
Japan Immunological Society, p. 3165 1981]. The dilution ratio at the time of use was determined by conducting a preliminary experiment each time it was prepared. After washing in the same manner as in (1), 100 mg of 1 mg / ml disodium nitrophenyl phosphate / diethanolamine-hydrochloric acid buffer (pH 9.8) was added and reacted at room temperature, and the absorbance value at 405 nm was measured.

(8) SDS polyacrylamide gel electrophoresis Purified mutant β-LG was electrophoresed on the basis of Laemmli's method [Laemmli, UK, Nature 227, 680 (1970)] containing 1% SDS. It was carried out under reducing conditions using a polyacrylamide gel. After migration, stain this with a staining solution (0.25%
It was stained with Coomassie Brilliant Blue, 10% acetic acid, 10% methanol). Decolorization was performed by immersing in (7% acetic acid, 10% methanol) and shaking.

(9) Western blot analysis By the method of (8), electrophoresis was performed, and the protein in the gel was adsorbed to the PVDF membrane (imopirone, millipore) using a plotting device (milliblot-SDE, Millipore). It was That is, the PVDF membrane was immersed in methanol for 20 seconds, and then immersed in anodic buffer solution # 2 (25 mM Tris, 20% methanol, pH 10.4) for 5 minutes, and one piece of filter paper was used for anodic buffer solution # 1 (0.3 M Tris,
20% methanol, pH 10.4), # 2 and cathode buffer (25
It was soaked in mM Tris, 40 mM 6-aminohexanoic acid, 20% methanol, pH 9.4). The filter paper soaked in the anode buffer solution # 1, the filter paper soaked in the anode buffer solution # 2, the PVDF membrane, the gel, and the filter paper soaked in the cathode buffer solution were overlaid in this order to assemble the device. The current was adjusted so that an initial current of 25 mA / cm ² was applied to the gel area, and transfer was performed for 30 minutes.

After the transfer of the protein to the PVDF membrane was completed, anti-β-LGmAb21B3 was used as the primary antibody and antibody staining was performed using the ECL Western plotting detection kit (Amersham). The results of the production of mutant β-LG are shown below (Fig. 12). From the result of the SDS polyacrylamide gel electrophoresis pattern, the purified mutant β-LG was found to be the natural β-LG.
It showed a single band with the same mobility as LG and was found to have the correct molecular weight. In addition, as a result of Western blot analysis, it was revealed that this band found by electrophoresis had a binding site for anti-β-LG mAb, and it was confirmed that it was a mutant of β-LG (the result of Western blot analysis And β-L in each lane
The shadow seen in the lower left of the band indicating G is a non-specific shadow generated during exposure. )

(10) Purification of natural β-LG From milk obtained from Holstein strains of genetically modified β-LG of AA, the method of Aschaffenburg et al. [Aschaffenburg. R. and
Drewry, J., Biochem. J. 65, 273 (1957)], to prepare crude β-LGA, which was used in DEAE-Sephacel.
Purified by subjecting it to ion exchange chromatography.

(11) Immunization and Blood Collection of Mice C57BL / 6 mice (female, 8 weeks old) were purchased from Charles River Japan. The purified mutant β-LG was intraperitoneally administered to C57BL / 6 mice together with CFA at 40 μg / animal. Two weeks later, the same amount of the mutant was intraperitoneally administered together with IFA. One week after the second immunization, blood was collected and its serum was obtained. For P126A mutant, 1
A booster immunization was performed again one week later, and one week later, blood was collected.

(12) Measurement of amount of specific antibody in serum The amount of specific antibody in serum was measured by the ELISA method. The measurement was performed according to the method shown in (4) of Example 2.
However, in the operation step of, the concentration of natural type and mutant β-LG adsorbed on the microtiter plate was 100μ.
g / ml. The results of the antibody production test against the mutant β-LG are shown below (Fig. 13).

When immunized with native β-LG, a specific antibody that binds to native β-LG is produced,
A small amount of antibody to p119-133 was also produced. D129
In the case of immunization with Amut, production of specific antibodies against natural β-LG and the immunogen D129Amut was almost the same as in the case of natural β-LG immunization. However, when immunized with P126Amut, almost no production of natural β-LG and a specific antibody that binds to P126Amut as an immunogen was observed. From this result, it is possible to reduce the immunogenicity of the protein by converting at least one amino acid residue of the protein antigen, particularly by replacing one residue of the residue involved in binding to MHC class II in this example. It was shown that it was possible.

[0077]

INDUSTRIAL APPLICABILITY According to the present invention, a mutant protein having reduced immunogenicity can be provided. INDUSTRIAL APPLICABILITY The present invention is industrially extremely useful because it can be used for hypoallergenic foods, antiallergic foods, and the like, and can also be used for safe proteinaceous pharmaceuticals with a low risk of causing anaphylactic shock.

[0078]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 162 Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Protein sequence:

SEQ ID NO: 2 Sequence length: 162 Sequence type: Number of amino acid chains: Single-stranded topology: Linear sequence type: Protein sequence:

SEQ ID NO: 3 Sequence length: 162 Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Protein sequence:

SEQ ID NO: 4 Sequence length: 162 Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Protein sequence:

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GTCCACCTCA GCGGTCCTGA C 21

SEQ ID NO: 6 Sequence length: 26 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: CAGGGCCTCG TCAGCCACCT CCGGGG 26

C

[Brief description of drawings]

FIG. 1 is a diagram showing an amino acid sequence of an analog peptide.

FIG. 2 is a diagram showing a schematic view of an IA ^b molecular binding test method.

FIG. 3 is a view showing a result of an I- ^Ab molecule binding test.

FIG. 4 is a diagram showing a schedule of immunization and blood collection of mice.

FIG. 5 is a diagram showing the results of a mouse lymph node cell proliferation test.

FIG. 6 is a diagram showing the results of a mouse lymph node cell proliferation test.

FIG. 7 is a diagram showing the results of a lymph node cell proliferation test in mice.

FIG. 8 is a view showing the results of a comparative test of antibody production inducing ability in mice.

FIG. 9 is a view showing the results of a comparative test of antibody production inducing ability in mice.

FIG. 10 is a diagram showing the results of a comparative test of antibody production inducing ability in mice.

FIG. 11 is a diagram showing the construction of an expression plasmid for mutant β-LG.

FIG. 12 is a diagram showing the results of electrophoresis and Western blot analysis of purified mutant β-LG.

FIG. 13 is a diagram showing the results of a comparative test of the antibody production inducing ability of purified mutant β-LG in mice.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/19 C12P 21/02 C12R 1: 865) C12N 15/00 A (C12P 21/02 C12R 1: 865) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09-15/90 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (5)

(57) [Claims] 1. Amino acid sequence of the β-lactoglobulin represented by SEQ ID NO: 1, wherein the 126th proline is replaced with alanine, the 126th proline is replaced with phenylalanine, or 128th valine comprises the amino acid sequence replaced by aspartic acid, mutant β- lactoglobulin resulting in low responsiveness or unresponsiveness of the immune response. 2. A mutant β-lactoglobulin gene comprising a DNA encoding any of the amino acid sequences represented by SEQ ID NOs: 2 to 4. 3. A recombinant DNA in which the mutant β-lactoglobulin gene according to claim 2 is incorporated. 4. A transformant transformed with the recombinant DNA according to claim 3. 5. A method for producing a mutant β-lactoglobulin, which comprises culturing the transformant according to claim 4 and separating the mutant β-lactoglobulin from the resulting culture.