JP3007037B2 - Helicobacter pylori urease protein-derived artificial antigen and antibody induced by the artificial antigen - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an artificial antigen derived from Helicobacter pylori urease protein which can be used as a vaccine or diagnostic agent for Helicobacter pylori infection, which is said to contribute to gastrointestinal lesions, and ,
The present invention relates to an anti-Helicobacter pylori urease antibody that can be used as a therapeutic agent or a diagnostic agent for Helicobacter pylori infection.

[0002]

2. Description of the Related Art Since 1983, Warren and Marshall et al. Isolated and cultured Helicobacter pylori (hereinafter, referred to as "HP") from gastric mucosa, gastric and duodenal ulcers and gastric cancer. Attention has been paid to the involvement of HP in gastrointestinal lesions such as those described above, and research is currently being conducted by many researchers. At present, there is a growing recognition that HP infection contributes to gastrointestinal lesions, but the pathology from infection to onset of pathology remains largely unknown. Recently, a mouse model of HP infection has been developed (Science, Vol. 267, No. 1665-1658).
(Page (1995)), it is expected that the elucidation of the pathological condition will progress in the future.

[0003] Since gastrointestinal lesions caused by HP can be regarded as HP infections, studies on immunological treatments using vaccines, antibodies, etc. are being conducted in parallel with elucidation of the pathological conditions. In particular, H, one of the HP cell components,
Since P urease is considered to be an indispensable enzyme for both proliferation of HP and damage to digestive mucosa by HP, HP urease or its subunits (short A subunit and long B subunit, Have been used as immunogens.

[0004] Gastroenterology
gy), Vol. 107, pp. 1002-1011 (1994)
In mice, HP urease, A subunit or B
When subunits were orally administered and immunized, HP was inoculated with Helicobacter felis, which was closely related to HP, and an oral immunization group with HP urease or B subunits was found to have a protective effect on infection. No effect was reported in the unit group.

[0005] Infection and immunity, Vol. 60, No. 11, 482
6-4831 (1992), among the total of 10 monoclonal anti-HP urease antibodies that recognize either the long chain subunit (B subunit) or the short chain subunit (A subunit), That the L2 antibody recognizing L2 antibody and the S2 antibody recognizing the short chain have strong urease activity inhibitory activity, and that the antigenic determinant recognized by the L2 antibody is presumed to be a structure common to Helicobacter species. Have been reported. This document also reports that the polyclonal anti-HP urease antibody contained an antibody that abolished the HP urease inhibitory action of the anti-HP urease antibody.

[0006] The Journal of the Japanese Society of Gastroenterology 91:12
No. 2202-2213 (1994), there is a specific anti-HPI IgG antibody which reacts strongly with HP urease but does not show cross-reactivity with other ureases in the serum of gastric / duodenal lesion patients. It has been reported that this antibody recognized the B chain (B subunit).

[0007] These research reports suggest that HP urease, its B subunit, or antigenic determinants present in these proteins may be used as immunogens against HP infection.

[0008] However, it is dangerous to use HP urease itself, which has a damaging effect on the digestive mucosa, as a vaccine. Further, even if a B subunit having no damaging effect is used, it cannot immunize only a specific antigenic determinant because it is a protein having a large molecular weight like HP urease. Therefore, there is a problem that it is difficult to produce a high-titer antibody, and as described above, there is a possibility that an antibody that invalidates the HP urease inhibitory action may be produced at the same time.

[0009] Despite the above problems, the structure of the antigenic determinant in HP urease has not been studied at all.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide an HP
An artificial antigen that has only an antigenic determinant capable of producing an antibody that strongly and specifically inhibits the activity of urease, and is useful as a vaccine or diagnostic agent for HP infection, and an induced antigen induced by the artificial antigen, An object of the present invention is to provide an anti-HP urease antibody useful as a therapeutic agent, a diagnostic agent, and the like.

[0011]

Means for Solving the Problems In the course of conducting research on the antigenic determinant of HP urease, the present inventors have found that a large number of fragments corresponding to the amino acid sequence fragments (fragments) of the A subunit and the B subunit of HP urease. Were synthesized, and the immunogenicity of each peptide fragment was examined in detail. As a result, it was found that a certain amino acid sequence (UB-33) in the B subunit had extremely specific immunogenicity. The present invention has been completed based on such findings.

That is, the present invention provides a compound represented by the following general formula (1): Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-Ala-Asp-Ser-Arg- Ile ... (1) [where Cys is cysteine, His is histidine, Leu is leucine, Asp is aspartic acid, Lys is lysine, Ser.
Is serine, Ile is isoleucine, Glu is glutamic acid,
Val represents valine, Gln represents glutamine, Phe represents phenylalanine, Ala represents alanine, and Arg represents arginine. Selected from a peptide represented by the amino acid sequence of the above, or a fragment of the peptide, a modified product obtained by deletion, addition or substitution of the peptide, a salt of the peptide, and an ester of the peptide. It has the same immunological specificity and has the following general formula (4): Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe (4) The same], wherein the artificial antigen has a minimum antigenic determinant represented by the amino acid sequence of Helicobacter pylori urease. Here, “has the same immunological specificity as the peptide represented by the amino acid sequence of the general formula (1)” or “has the same immunological specificity as the peptide represented by the amino acid sequence of the general formula (4)” "Having specific specificity" means that an antibody derived from a peptide represented by the amino acid sequence of the general formula (1) or (4) has cross-reactivity with a certain antigen, and It refers to inducing only antibodies having cross-reactivity with the peptide represented by the amino acid sequence of formula (1) or (4).

[0013] The present invention also relates to the use of an artificial antigen comprising the peptide represented by the amino acid sequence of the above general formula (1) or the above mutant having the same immunological specificity as the peptide as an immunogen. Also provided is an anti-HP urease antibody (excluding the L2 antibody) induced by

The artificial antigen and anti-HP urease antibody of the present invention have various excellent properties as described below. (1) The anti-HP urease antibody induced by the artificial antigen of the present invention not only strongly inhibits the activity of HP urease but also has a cross-reactivity with other ureases such as Jack Bean. Since it is not shown, the specificity is high.

(2) Since the artificial antigen of the present invention does not contain unnecessary antigenic determinants, it does not induce harmful antibodies that neutralize the urease activity inhibitory action of unnecessary antibodies or anti-HP urease antibodies.

(3) Particularly when the peptide has the structure of the general formula (1), the N-terminus of the peptide is cysteine, so that a carrier protein for enhancing immunogenicity or a label protein for use as a diagnostic agent can be easily prepared. Can be combined.

(4) The artificial antigen of the present invention is considered to be an antigenic determinant common to Helicobacter species, and the antibody of the present invention induced by this artificial antigen is capable of reacting with urease of mutant HP (variant). It is considered effective.

Therefore, the artificial antigens and antibodies of the present invention can be used as vaccines or therapeutics with high titer and high specificity, or for determining whether or not effective antibody production against HP infection is performed in the body. It is extremely useful as a diagnostic agent and the like. From the viewpoint of therapeutic application, HP urease is considered to be an indispensable enzyme for both the growth of HP and gastrointestinal mucosal damage caused by HP. Therefore, the HP urease activity is strongly enhanced by the artificial antigen or antibody of the present invention. If suppressed, the fundamental treatment of inhibiting HP proliferation and the symptomatic treatment of alleviating gastrointestinal mucosal damage can be performed simultaneously, which is very effective.

In the present invention, the following general formula (4): Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe (4) wherein Ser is serine, Ile is isoleucine, and Lys is lysine , Glu is glutamic acid, Asp is aspartic acid, Va
l represents valine, Gln represents glutamine, and Phe represents phenylalanine. Selected from the peptide represented by the amino acid sequence of the above, or a modified product obtained by addition of the peptide, an acid of the peptide and an ester of the peptide, and having an immunological specificity equivalent to that of the peptide. And an artificial antigen of Helicobacter pylori urease, comprising a mutant having a minimum antigenic determinant represented by the amino acid sequence of the above general formula (4).

At the same time, induction is achieved by using an artificial antigen comprising the peptide represented by the amino acid sequence of the above general formula (4) or the above mutant having the same immunological specificity as the peptide as an immunogen. Also provided are anti-HP urease antibodies (excluding the L2 antibody).

The amino acid sequence represented by the general formula (4) is the minimum antigenic determinant contained in the amino acid sequence of the general formula (1), and is extremely useful for the peptide represented by the general formula (1). Is a fragment. Therefore, a peptide containing the minimum antigenic determinant can be used as an antigen that is equal to or better than a peptide having the amino acid sequence of the general formula (1).

In the present invention, a “functionally equivalent variant” is a modified product obtained by deletion, addition, substitution, or derivation of an amino acid residue, and is an immunologically or biologically modified product. It refers to one that exerts a similar effect. For example, a synthetic peptide in which a part of the amino acid sequence represented by the general formula (1) or (4) is deleted or substituted, or several peptide residues are added to the N-terminal or C-terminal of the amino acid sequence The synthetic peptide, a fragment corresponding to a part of the amino acid sequence thereof, and a derivative such as a salt or an ester may have the amino acid sequence represented by the general formula (1) or (4) unless the object of the present invention is violated. This corresponds to a variant that is functionally equivalent to the peptide possessed.

[0023]

Hereinafter, the present invention will be described in more detail with reference to experimental examples. Experimental Example 1 (Specification of antigenic determinant) The artificial antigen of the present invention is an antigenic determinant contained in the B subunit of HP urease. In order to identify this antigenic determinant, the present inventors first synthesized a large number of polypeptides corresponding to partial amino acid sequences in the A and B subunits of HP urease, and The reactivity with a known anti-HP urease antibody having the ability to neutralize urease activity was examined. Furthermore, the immunogenicity of the peptide fragment that showed specific reactivity in the reactivity test was examined.

1- (1): Preparation of Peptide Fragments As candidate antigenic determinants, peptide fragments derived from 23 types of A subunits and 56 types of peptide fragments derived from B subunits were synthesized.

The HP urease is composed of a short A subunit represented by the following general formula (2) and a long B subunit represented by the following general formula (3). [Urease A subunit sequence] MKLTP KELDK LMLHY AGELA KKRKE KGIKL NYVEA VALIS AHIME EARAG KKTAA ELMQE GRTLL KPDDV MDGVA SMIHE VGIEA MFPDG TKLVT VHTPI EANGK LVPGE LFLKN EDITI NEGKK AVSVK VKNVG DRPVQ IGSHF HFFEV NRCLD FDREK TFGKR LDIAS GTAVR FEPGE EKSVE LIDIG GNRRI FGFNA LVDRQ ADNES KKIAL HRAKE RGFHG AKSDD NYVKT IKE (2) wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G
Is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, and V is valine. , W represents tryptophan, and Y represents tyrosine. ] [Urease B subunit sequence] MKKIS RKEYV SMYGP TTGDK VRLGD TDLIA EVEHD YTIYG EELKF GGGKT LREGM SQSNN PSKEE LDLII TNALI VDYTG IYKAD IGIKD GKIAG IGKGG NKDMQ DGVKN NLSVG PATEA LAGEG LIVTA GGIDT HIHFI SPQQI PTAFA SGVTT MIGGG TGPAD GTNAT TITPG RRNLK WMLRA AEEYS MNLGF LAKGN ASNDA SLADQ IEAGA IGFKI HEDWG TTPSA INHAL DVADK YDVQV AIHTD TLNEA GCVED TMAAI AGRTM HTFHT EGAGG GHAPD IIKVA G HNI LPAST NPTIP FTVNT EAEHM DMLMV CHHLD KSIKE DVQFA DSRIR PQTIA AEDTL HDMGI FSITS SDSQA MGRVG EVITR TWQTA DKNKK EFGRL KEEKG DNDNF RIKRY LSKYT INPAI AHGIS EYVGS VEVGK VADLV LWSPA FFGVK PNMII KGGFI ALSQM GDANA SIPTP QPVYY REMFA HHGKA KYDAN ITFVS QAAYD KGIKE ELGLE RQVLP VKNCR NITKK DMQFN DTTAH IEVNP ETYHV FVDGK EVTSK PANKV SLAQL FSIF (3) [The symbols in the formula are the same as above. The peptide fragment derived from the A subunit is represented by the following UA-1
~ UA-23. These are in principle 2
It was synthesized so as to have a sequence consisting of 0 amino acids and shifted out from the N-terminus of the A subunit by shifting 10 amino acids at a time. Therefore, the amino acid sequences of adjacent peptide fragments overlap by 10 amino acids.

UA-1: MKLTP KELK LMLHY AGELA UA-2: LMLHY AGELA KKRKE KGIKL UA-3: KKRKKE KGIL NYVEA VAL AGE AUGA VAL AGE AHA AGE AGE AGE AGE AGE AGE AGE AGE AGE AGA KPDDV UA-7: GRTLL KPDDV MDGVA SMIHE UA-8: MDGVA SMIHE VGIEA MFPDG UA-9: VGIEA MFPDG TKLVT VHTPI UA-10: TKLVT VHTPI EANGK LVPGE UA-11: EANGK LVPGE LFLKN EDITI UA-12: LFLKN EDITI NEGKK AVSVK UA -13: NE GKK AVSVK VKNVG DRPVQ UA-14: VKNVG DRPVQ IGSHF HFFEV UA-15: IGSHF HFFEV VRCLD FDREK UA-16: NRCLD FDREK TFGKR LDIAS UA-17: TFGKR LDIAS GTAVR FEPGE UA-18: GTAVR FEPGE EKSVE LIDIG UA-19: EKSVE LIDIG GNRRI FGFNA UA-20: GNRRI FGFNA LVDRQ ADNES UA-21: LVDRQ ADNES KKIAL HRAKE UA-22: KKIAL HRAKE RGFHG AKSDD UA-23;
There were 56 types of B-1 to UB56. These were composed of 19 amino acids in principle, and were synthesized so as to have a sequence in which amino acids were cut out by shifting 10 amino acids from the N-terminal of the B subunit. Therefore, the amino acid sequences of adjacent peptide fragments overlap with each other by nine amino acids.

UB-1: MKKIS RKEYV SMYGP TTG UB-2: SMYGP TTGDK VRLGD TDLI UB-3: VRLGD TDLIA EVHD YTIG UBGE: KELGGEKBGELKGEKBEGELK LDLI UB-7: PSKEE LDLII TNALI VDYT UB-8: TNALI VDYTG IYKAD IGIK UB-9: IYKAD IGIKD GKIAG IGKG UB-10: GKIAG IGKGG NKDMQ DGVK UB-11: GNKDM QDGVK NNLSV GPAT UB-12: NLSVG PATEA LAGEG LIVT UB -13: LAGEG LIVTA GGIDT H IHF UB-14: GGIDT HIHFI SPQQI PTAF UB-15: SPQQI PTAFA SGVTT MIGG UB-16: SGVTT MIGGG TGPAD GTNA UB-17: TGPAD GTNAT TITPG RRNL UB-18: TITPG RRNLK WMLRA AEEY UB-19: WMLRA AEEYS MNLGF LAKG UB -20: SMNLG FLAG NASND ASLA UB-21: ASNDA SLADQ IEGA IGFK UB-22: EAGAI GFKIH EDWGT TPSA UB-23: IHEDW GTTPS AINHALDVA VALV UAV VALV A : TLNEA GC ED TMAAI AGRT UB-27: TMAAI AGRTM HTFHT EGAG UB-28: HTFHT EGAGG GHAPD IIKV UB-29: GGHAP DIIIKV AGEHN ILPA UB THP TMP TPF TNP TPF TNP TPF TNP TPF TNP TPF TNP TBP KSIK UB-33: CHHLD KSIKE DVQFA DSRI UB-34: DVQFA DSRIR PQTIA AEDT UB-35: PQTIA AEDTL HDMGI FSIT UB-36: HDMGI FSITS SDSQA MGRV UB-37: SDSQA MGRVG EVITR TWQT UB-38: EVITR TWQTA DKNKK EFGR UB −39 : ADKNK KEFGR LKEEK GDND UB-40: KEEKG DNDNF RIKRY LSKY UB-41: RIKRY LSKYT INPAI AHGI UB-42: INPAI AHGIS EYVGS VEVG UB-43: SEYVG SVEVG KVADL VLWS UB-44: VADLV LWSPA FFGVK PNMI UB-45: AFFGV KPNMI IKGGF IALS UB-46: KGGFI ALSQM GDANA SIPT UB-47: GDANA SIPTP QPVYY REMF UB-48: QPVYY REMFA HHGKA KYDA UB-49: HHGKA KYDAN ITFVS QAAY UB-50: ITFVS QAAYD KGIKE ELGL UB-51: KGIKE ELGLE RQVLP KNC UB-52: RQVLP VKNCR NITKK DMQF UB-53: RNITK KDMQF NDDTA HIEV UB-54: DTTAH IEVNP ETYHV FVDG UB-55: ETYHV FVVGKVKVASKV KVA PKSKVK EVA TKV KVA PKK PKTS According to the simple t-BOC method (t-Boc chemistry), a model 4
It was synthesized using a 30 multiple peptide synthesizer (Applied Biosystems, USA). Synthetic peptide separated from the resin with HF (hydrofluoric acid) solution is 0.1% by weight TFA (trifluoroacetic acid)
The purity and concentration were measured by reverse-phase HPLC using a C18 column utilizing a concentration gradient of an aqueous solution of AFA and an acetonitrile solution of TFA. Then, purification was further performed using a Biogel P4 column (manufactured by Bio-Rad, USA) using 9% formic acid, and the purity and concentration measured by reverse-phase HPLC as necessary were used in the experiment.

1- (2): Reactivity test between peptide fragment and anti-HP urease antibody As anti-HP urease antibodies, L2 antibody having strong neutralizing ability against HP urease activity and L2 antibody having no neutralizing ability S5 antibody was used. These antibodies are BALB / c
The mice were immunized intraperitoneally with a mixture of purified HP urease protein and Freund's complete adjuvant, followed by booster immunization with the same immunogen at 2 weeks and 4 weeks, and collection It is a monoclonal antibody produced by a hybridoma produced by fusing myeloma cells to the immunized spleen cells by the polyethylene glycol method, and it has been confirmed that the S5 antibody recognizes the A subunit and the L2 antibody recognizes the B subunit. (Infection and Immunity, Vol. 60, No. 11, pp. 4826-4831 (199)
2), page 4827, left column, line 43 to right column, line 3).

The reactivity between the peptide and the antibody was evaluated by ELISA. 50 μl of each peptide fragment prepared in PBS (phosphate buffer) was spread on a 96-well ELISA microplate (Immulon 2; manufactured by Dynatech, USA) and incubated at 4 ° C. overnight or at 37 ° C. for 60 minutes. After coating, it was washed three times with PBS. 200 μl of 4-fold diluted Block Ace solution (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to each well, and blocking was performed at 4 ° C. overnight, and then 0.05% by weight Tw.
3 with 10-fold diluted Block Ace solution
Washed twice. Then, add 10 times diluted Block Ace solution to 1
After adding the anti-HP urease antibody diluted to 1: 300 and incubating at 37 ° C. for 60 minutes,
After washing 3 times with a washing solution, a 500-fold diluted goat anti-human IgG (manufactured by CAPPEL, USA) to which alkaline phosphatase is bound as a secondary antibody is added.
Incubation was performed for 0 minutes. After washing four times with the washing solution, the color former (10 ^-1 m in 5 ml of carbonate buffer)
ol / l phosphatase was dissolved in plus concentration MgCl ₂ 50μl substrate (Sigma Chemical company
(U.S.A., U.S.A.)), and when the color developed, the absorbance at 405 nm was measured using a microplate reader.

The reactivity (absorbance) of the peptide fragment derived from the B subunit is shown in Table 1 and FIG. UB-1 to UB-5
6, only UB-33 (molecular weight: 2223.8) having the amino acid sequence represented by the general formula (1) specifically reacts with the L2 antibody, and its reactivity is different from that of HP urease itself (positive control). The strength was almost the same.

[0031]

[Table 1] Symbols in the table Positive: A sample to which HP urease was added instead of a peptide fragment Negative: A sample to which no peptide fragment was added Back: Control to which only a secondary antibody was added 1- (3): Immunogenicity test Reactivity test described above Since UB-33 showed specific reactivity, the immunostimulatory activity (immunogenicity) of this peptide was examined.

In order to enhance the immunogenicity of UB-33 synthesized and purified in the same manner as described above, KLH (Keyhole lympet hemocianin) was used as a carrier protein by the maleimide method.
e: Raven shell hemocyanin) was bound to cysteine (C) at the N-terminus of UB-33. This UB-33-
1 mg of KLH was dissolved in 1 ml of PBS, and an equal volume of Freund's complete adjuvant (CF)
A mixture of A) and the rabbit was inoculated subcutaneously and intraperitoneally in a total of 1 ml, and further boosted with the same antigen 16 days after the inoculation. Blood was collected immediately before inoculation (negative control), 10 days and 16 days after inoculation (immediately before booster immunization), 1 week, 2 weeks, and 3 weeks after booster immunization.
The antibody titer of the serum for No. 3 was measured over time by ELISA. The ELISA method was operated in the same manner as in the case of the reactivity test in Experimental Example 1- (2).

Table 2 shows the results of the ELISA method. UB
In all three sera of rabbits immunized with -33, a high antibody titer to UB-33 was recognized 7 days after the start of immunization, and the antibody titer increased thereafter.

[0034]

[Table 2] Furthermore, sera at the end of the immunization period (3 weeks after the booster immunization) were collected and examined for their ability to neutralize HP urease activity.

The neutralization ability for urease activity was measured by
This was carried out by a modification of the urea bouillon method. That is, to a 96-well ELISA plate, 25 μl of a predetermined amount of HP urease and 25 μl of 0 to 25 μg of serum in terms of IgG protein were added in advance, and overnight incubation was performed at 4 ° C., followed by urea medium (3% urea). Aqueous solution,
Medium adjusted to pH 6.0 by adding 0.02% phenol red to 0.025 M phosphate buffer; Eiken Chemical Co., Ltd.
Japan) was added, and the absorbance at 595 nm was measured using a microplate reader (Model 3550; Bio-Ra).
d).

As shown in FIG. 2, the serum of the UB-33 immunized group strongly inhibited HP urease activity, and the inhibition rate increased with an increase in the amount of serum added. According to the above experiment,
UB-33 is a specific recognition site for anti-HP urease antibody and has strong immunogenicity.
Immunization with -33 produces an antibody having a strong ability to neutralize HP urease activity.
3 is an excellent antigenic determinant having reactivity and immunogenicity, and was found to be a site for inducing a strong neutralizing antibody against HP urease.

EXPERIMENTAL EXAMPLE 2 ( Crossity- 1) It was examined whether or not the anti-UB-33 antibody induced by UB-33 has cross-reactivity with other types of urease.

2- (1): Antibody used and urease Anti-UB-33 antibody used was a rabbit anti-UB-33 serum obtained from an immunized rabbit in the immunogenicity test in Experimental Example 1- (3). Was. Serum of a healthy rabbit was used as a negative control, and L2 antibody known to recognize the HP urease B subunit and S2 antibody known to recognize the HP urease A subunit were used as the positive control. , 10
What was diluted 100 times with a double dilution block ace solution was used. The L2 antibody and the S2 antibody were used in the same manner as in the case of the reactivity test in Experimental Example 1- (2), in Infection and Immunity, Vol. 60, No. 11, Nos. 4826-4831.
It is a monoclonal antibody obtained by the method described on page 4827, left column, line 43 to right column, line 3 in page (1992).

Urease to be reacted with the sample antibody includes HP urease, jack bean urease (Jack-bean urease, manufactured by Wako Pure Chemical Industries, Ltd.) and thermophilic bacillus urease (Thermophilic Bacillus).
s sp. urease, manufactured by Wako Pure Chemical Industries, Ltd.). As the HP urease, the same urease as that used as the positive control in the reactivity test of Experimental Example 1 (2) was used. A background in which no urease was added was also set.

2- (2): Cross-reactivity test The reactivity of each sample antibody to various ureases was
It was examined by the LISA method. The ELISA method was performed in the same manner as in Experimental Example 1 except that urease was used instead of the peptide fragment and rabbit serum was used instead of the anti-HP urease antibody.
The operation was the same as in the reactivity test of (2).

Table 3 shows the results of the ELISA method. Rabbit anti-UB-33 serum reacted strongly with HP urease, but only very slightly with other ureases and was negligible compared to background. That is, the antibody induced by UB-33 did not show any cross-reactivity with other types of urease.

[0042]

[Table 3] Experimental Example 3 ( Immunization with HP Urease Protein ) Using the serum of a mouse immunized with the HP urease protein, a linear epitope recognized by an antibody present in the serum was identified, and the urease activity of the serum was determined. Investigated the harmony.

3- (1): Immunization of Mice with HP Urease Protein BALB / c mice were immunized subcutaneously by administering a mixture of purified HP urease protein and Freund's complete adjuvant, followed by immunization at 2 weeks and 4 weeks. Weeks were boosted with the same immunogen. Two weeks later, blood was collected from the mouse heart to obtain immune serum and immune spleen cells.

3- (2): Identification of Linear Epitope Recognized by Mouse Anti-HP Urease Protein Serum The linear epitope was identified in the same manner as in the ELISA method performed in the reactivity test of Experimental Example 1- (2). Identified. That is, the above UA-
Using the peptides 1 to UA-23 and UB-1 to UB-56, these peptides were spread on a microplate for ELISA, and operated in the same manner as in Experimental Example 1- (2).
The site recognized by the mouse anti-HP urease protein serum was searched.

As a result, as shown in Table 4 and FIG.
Almost no linear epitope was found in the subunit, but as shown in Table 5 and FIG. 4, in the B subunit, other than UB-33, UB-28 to UB-3
A linear epitope was found in the portion corresponding to 1.

[0046]

[Table 4]

[0047]

[Table 5] 3- (3): Neutralizing activity test of mouse anti-HP urease protein serum The mouse anti-HP urease protein serum and UB-33 were recognized in the same manner as in the neutralizing activity test of Experimental Example 1- (3). The ability of the monoclonal antibody (L2) to neutralize HP urease activity was examined. The amount of the serum or L2 antibody used at this time was 0 to 8 μl in terms of IgG protein.
g.

FIG. 5 shows the results. The L2 antibody, which is an anti-HP urease monoclonal antibody, was prepared in Experimental Example 1- (3).
As in the case of the anti-UB-33 antibody evaluated in the above, it had a strong neutralizing ability in a dose-dependent manner, but the mouse anti-HP urease protein serum hardly neutralized the HP urease activity.

From these results, it was found that the whole serum of the mouse alone could strongly neutralize HP urease, and that UB-33
It has been found that despite having an antibody against, there is almost no ability to neutralize HP urease.
This fact suggests that an antibody against an epitope other than UB-33 may suppress the specific activity of the antibody against UB-33.

Experimental Example 4 ( Crossity- 2) In Experimental Example 3, it was found that a linear epitope other than UB-33 was present in the B subunit.
Anti-UB-33 antibodies induced by B-33
Whether to recognize a linear epitope other than -33 was examined.

4- (1): Antibodies and Peptide Fragments Used As anti-UB-33 antibodies, rabbit anti-UB-33 serum obtained from rabbits immunized in the immunogenicity test of Experimental Example 1- (3) was used. Using. Serum from a healthy rabbit was used as a negative control.

The peptide fragments reacted with the sample
B-31 to UB-35. A background to which no peptide fragment was added was also set. 4- (2): Cross-reactivity test The reactivity of rabbit anti-UB-33 serum to the peptide fragment was examined by ELISA. The ELISA method is
The same operation as in the reactivity test of Experimental Example 1- (2) was performed, except that rabbit serum was used instead of the anti-HP urease antibody.

405 nm measured by the ELISA method
Table 6 and FIG. Rabbit anti-UB-33
Serum reacted strongly with UB-33, an immunogen,
Neighboring UB-32 or UB-34 that has overlap with B-33
And did not react at all with UB-31, another linear epitope within the B subunit. That is, the antibody induced by UB-33
It showed no cross-reactivity to other parts within the subunit.

[0054]

[Table 6] Experimental Example 5 (Specification of minimum antigenic determinant) Several fragments constituting a part of the amino acid sequence of UB-33 were synthesized, and the reactivity of each fragment with an anti-HP urease antibody was examined. ).

5- (1): Preparation of Fragments Eighteen kinds of fragments as candidates for minimal sites
That is, the following UB-33 (10R) to UB-33 (1
8R) and UB-33 (10 L) to UB-33 (18
L) was synthesized. The synthesis was performed according to a general t-BOC method as in the case of Experimental Example 1.

UB-33: CHHLD KSIKE DVQFA DSRI UB-33 (10R): EDVQFA DSRI UB-33 (11R): KE DVQFA DSRI UB-33 (12R): IKE DVQFA DSRI UB-KFERV DSRI UB-33 (14R): KSIKE DVQFA DSRI UB-33 (15R): D KSIKE DVQFA DSRI UB-33 (16R): LD KSIKE DVQFA DSRI UB-33 (17R): HLD KSIKBDVFA : HHLD KSIKE UD-33 (10L): CHHLD KSIKE UB-33 (11L): CHHLD KSIKE D UB-33 (12L): CHHLD K IKE DV UB-33 (13L): CHHLD KSI DVQ UB-33 (14L): CHHLD KSIKE DVQF UB-33 (15L): CHHLD KSIKE DVQFA UB-33 (16L): CHHLD KSIKBDFAQ CHHLD KSIKE DVQFA DS UB-33 (18L): CHHLD KSIKE DVQFA DSR 5- (2): Reactivity test between fragment and anti-HP urease antibody 33
ELISA was used to determine the reactivity of each fragment derived from
The evaluation was performed by the method A. The test was performed twice. A blank test was performed using UB-33 and HP urease as a positive control and no peptide as a negative control. The reactivity of a fragment corresponding to UB-33 in the amino acid sequence of jack bean urease was also evaluated.

The reactivity (absorbance) of each fragment was determined as the seventh.
It is shown in the table and FIG. FIG. 7 visualizes the average values in Table 7. As the number of amino acid residues was reduced one by one from the N-terminal side of UB-33, the reactivity rapidly decreased when the seventh S (serine) residue from the N-terminal side disappeared. On the other hand, when the number of amino acid residues was reduced one by one from the C-terminal side of UB-33, the reactivity rapidly decreased when the sixth F (phenylalanine) residue from the C-terminal side disappeared. From these results, it was determined that UB-33-F8, that is, the fragment having the amino acid sequence represented by the general formula (4) was the minimum antigenic determinant.

[0058]

[Table 7]

[0059]

As described above, the artificial antigen of the present invention is a specific recognition site of an anti-HP urease antibody and has strong immunogenicity. By immunizing an animal with this artificial antigen, it is possible to efficiently produce an anti-HP urease antibody that specifically and strongly inhibits the activity of HP urease, which is essential for both the growth of HP and the induction of gastrointestinal lesions. it can. In addition, immunization with this artificial antigen is different from immunization with HP urease protein, unlike anti-H
There is no byproduct of inconvenient antibodies such as antibodies that inactivate P urease antibodies.

Therefore, the artificial antigen of the present invention and the anti-HP urease antibody induced by the artificial antigen can be suitably used as excellent vaccines, antibodies, diagnostic agents and the like.

[0061]

[Sequence list]

SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Helicobacter pylor
i) Sequence: Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp 1 5 10 15 Ser Arg Ile SEQ ID NO: 2 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type : Peptide Origin Organism name: Helicobacter pylor
i)

[Brief description of the drawings]

FIG. 1 is a graph showing the results of evaluating the reactivity of a peptide fragment derived from HP urease B subunit with L2 antibody.

FIG. 2 is a graph showing the results of evaluating the neutralizing activity of rabbit anti-UB-33 serum on HP urease.

FIG. 3. A recognized by mouse anti-HP urease protein serum
It is a graph which shows the result of having searched the linear epitope in a subunit.

FIG. 4. B recognized by mouse anti-HP urease protein serum
It is a graph which shows the result of having searched the linear epitope in a subunit.

FIG. 5 is a graph showing the results of evaluating the neutralizing activity of mouse anti-HP urease protein serum on HP urease.

FIG. 6 is a graph showing the results of a search for the cross-reactivity of rabbit anti-UB-33 serum with a linear epitope in the B subunit other than UB-33.

FIG. 7 is a graph showing the results of evaluating the reactivity of a fragment derived from UB-33 with the L2 antibody.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C07K 7/08 ZNA C07K 7/08 ZNA 16/12 16/12 G01N 33/569 G01N 33/569 F 33/573 33/573 A // A61P 1/04 A61K 31/00 601C 31/04 631C (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 14/195 A61K 39/00 C07K 7/08 C07K 16/12 G01N 33 / 569 G01N 33/573 A61K 31/00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPIDS (STN)

Claims (4)

(57) [Claims] 1. The following general formula (1): Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-Ala-Asp-Ser-Arg-Ile 1) [where Cys is cysteine, His is histidine, Leu is leucine, Asp is aspartic acid, Lys is lysine, Ser
Is serine, Ile is isoleucine, Glu is glutamic acid,
Val represents valine, Gln represents glutamine, Phe represents phenylalanine, Ala represents alanine, and Arg represents arginine. Peptide represented by the amino acid sequence of, or, the
Peptide fragments and deletion / addition of the peptide
Or a modified product obtained by substitution and a salt of the peptide
And an ester of the peptide
Having the same immunological specificity as the peptide and the following general formula
(4) Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe (4) [Abbreviations in the formula are the same as in the general formula (1)]
Helicobacter pylori urease , comprising a variant having the minimum antigenic determinant represented by the sequence
Artificial antigen . 2. The following general formula (1): Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-Ala-Asp-Ser-Arg-Ile ... 1) [where Cys is cysteine, His is histidine, Leu is leucine, Asp is aspartic acid, Lys is lysine, Ser
Is serine, Ile is isoleucine, Glu is glutamic acid,
Val represents valine, Gln represents glutamine, Phe represents phenylalanine, Ala represents alanine, and Arg represents arginine. Peptide represented by the amino acid sequence of, or, the
Peptide fragments and deletion / addition of the peptide
Or a modified product obtained by substitution and a salt of the peptide
And an ester of the peptide
Having the same immunological specificity as the peptide and the following general formula
(4) Ser-Ile-Lys -Glu-Asp-Val-Gln-Phe ... (4) [ the abbreviations in the formula, the general formula (1) and the same] amino distribution of
Anti-Helicobacter pylori urease antibody (provided that L2 antibody is derived by using an artificial antigen consisting of a mutant having the minimum antigenic determinant represented by the column as an immunogen)
) . 3. The following general formula (4): Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe (4) wherein Ser is serine, Ile is isoleucine, Lys is lysine, Glu is glutamic acid, Asp is aspartic acid, Va
l represents valine, Gln represents glutamine, and Phe represents phenylalanine. ] Or a modified product obtained by addition of the peptide.
And the acid of the peptide and the ester of the peptide
And immunological specificity equivalent to the peptide
And having the amino acid sequence of the above general formula (4)
An artificial helicobacter pylori urease antigen , comprising a mutant having a small antigenic determinant . 4. The following general formula (4): Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe (4) wherein Ser is serine, Ile is isoleucine, Lys is lysine, Glu is glutamic acid, Asp is aspartic acid, Va
l represents valine, Gln represents glutamine, and Phe represents phenylalanine. ] Or a modified product obtained by addition of the peptide.
And the acid of the peptide and the ester of the peptide
And immunological specificity equivalent to the peptide
And having the amino acid sequence of the above general formula (4)
An anti-Helicobacter pylori urease antibody (excluding the L2 antibody) induced by using an artificial antigen consisting of a mutant having a small antigenic determinant as an immunogen.