JP2003180349A - MONOCLONAL ANTIBODY RECOGNIZING SECRETORY COMPONENT OF SECRETORY IgA AND HYBRIDOMA PRODUCING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a monoclonal antibody specific for mouse pIgR/SC by establishing a stable hybridoma in order to analyze an expression mechanism or a structure of a polymeric immunoglobulin antibody receptor/secretory component (pIgR/SC) molecule. <P>SOLUTION: This monoclonal antibody is capable of recognizing the secretory component of a secretory IgA which binds to a fusion protein (GST-SC fusion protein) of a glutathione transferase (GST) to the secretory component of the secretory IgA without binding to the GST. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that recognizes a secretory factor of secretory IgA and a hybridoma that produces this antibody.

[0002]

2. Description of the Related Art The main body of antibodies secreted into the mucosa is IgA (i
mmunoglobulin A), and it is estimated that humans produce about 66 mg IgA / kg body weight daily. The secretory IgA secreted into the intestinal lumen is the sum of those secreted via bile and secreted through the intestinal epithelial cells. Dimer IgA (dimer IgA; hereinafter referred to as dIgA) produced by plasma cells present in the lamina propria of the intestinal tract is a multimeric antibody receptor (poly) retained by intestinal epithelial cells.
meric immunoglobulin receptor; hereinafter referred to as pIgR).

The dIgA / pIgR complex is transferred inside the intestinal epithelial cells and then cleaved by protease at the extracellular portion of the membrane-bound protein pIgR. As a result, a secretory factor (secret) corresponding to the extracellular part of pIgR
ory component (hereinafter referred to as SC), dIgA is released into the intestinal lumen as secretory IgA.

It has been considered that secretory IgA plays a role of preventing adhesion of pathogenic microorganisms to intestinal epithelial cells, neutralizing toxins, suppressing allergen absorption, and the like. on the other hand,
It was reported that IgA-deficient mice were as resistant to viral infection as wild-type mice, and it was suggested that the role of IgA in defense against mucosal infection could be replaced by other immune defense mechanisms. Moreover, many unclear points remain regarding the IgA response to foreign antigens in the intestinal lumen including allergens.

Furthermore, the present inventors established a pIgR-deficient mouse incapable of synthesizing pIgR by a gene targeting method in order to clarify the IgA response to various antigens and the physiological role of secretory IgA (The Journa).
l of Immunology, 1999, 163: 5367-5373).

[0006]

SUMMARY OF THE INVENTION In the present invention, pIgR is used.
Mouse / mouse pIgR / SC specific monoclonal antibody (hereinafter referred to as mA
b)) and a hybridoma producing the antibody. Specifically, the objective is to obtain a mAb that recognizes a fusion protein containing mouse SC and a hybridoma that produces the antibody.

[0007]

A mAb recognizing a secretory factor of secretory IgA according to the invention described in claim 1 is:
SC of glutathione transferase and secretory IgA
And a glutathione transferase (hereinafter referred to as GST), but not to a glutathione transferase (hereinafter referred to as GST).

Secretion type I according to the invention described in claim 2
The mAb that recognizes gA SC is the antibody according to claim 1 which is 10.46 mAb.

The hybridoma according to the third aspect of the invention binds to the GST-SC fusion protein, but the GS
It produces an antibody that does not bind to T.

The hybridoma according to the invention described in claim 4 is the hybridoma according to claim 3,
These are 10.46 hybridoma cells producing 46 mAb.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, secretory IgA
Is a mAb that recognizes the SC. Specifically, GST-S
MA that binds to the C fusion protein but not GST
b and a stable hybridoma producing the antibody,
It is to provide an effective means for analyzing the expression mechanism and structure of pIgR / SC molecule.

The mAb according to the present invention is GST-S.
Any antibody may be used as long as it binds to the C fusion protein but does not bind to GST. Specifically, two types of mAbs produced by the present inventors were mentioned, and they were named “2.1.1 mAb” and “10.46 mAb”, respectively. Incidentally, 10.46
The mAb has stronger binding activity than the 2.1.1 mAb.

The mAb according to the present invention is produced by a hybridoma, which is a fusion of single antibody-producing cells, according to a conventional method. As this hybridoma, GST-
Any antibody that produces an antibody that binds to the SC fusion protein but not GST may be used.

As specific hybridomas according to the present invention, the hybridoma clone producing 2.1.1 mAb and the hybridoma clone producing 10.46 mAb described above were established in the present invention. Incidentally, 10.46
The hybridoma producing the mAb was 10.46 hybridoma cells and was transferred to the Institute of Biotechnology in 1851.
No. (deposited on October 02, 2001) has already been deposited.

The hybridoma according to the present invention is GST-
Obtaining antibody-producing cells from an animal immunized with SC fusion protein as an antigen and cell fusion with myeloma cells, and then selecting a hybridoma that produces an antibody that binds to GST-SC fusion protein but does not bind to GST Obtained by

As a monoclonal antibody recognizing a secretory factor of secretory IgA, a hybridoma producing an antibody which binds to the above-mentioned GST-SC fusion protein but does not bind to GST is cultured by a conventional method, By purifying IgG, it can be easily obtained in a large amount.

When producing the hybridoma of the present invention, a GST-SC fusion protein obtained from a microorganism producing a GST-SC fusion protein is used as an antigen.
Specifically, E. coli produced a GST-SC fusion protein, which was purified and used. As a result, hybridomas can be produced even when the amount of antigen necessary for immunization cannot be obtained from the living body.

[0018]

EXAMPLES Example 1 (1) Procedure (1-1) Cloning of cDNA: For the cDNA library, the liver cDNA of DS mouse was λgt1 according to a conventional method.
The one prepared by incorporating it into the 0 phage cloning vector was used.

(1-2) Determination of nucleotide sequence: pIgR cDNA
The base sequences of A are various synthetic primers and cDNA.
Deletion mutants covering the full length of the clone were combined and determined using a sequencer (Applied Biosystem).

(1-3) Preparation and purification of GST-SC fusion protein: Expression vector pGEX-5x-3 (Pharmacia B
iotech Uppsala, Sweden) cloning site (5 '
The base sequence portion encoding the SC protein was amplified by PCR using the mouse SC cDNA as a template with a synthetic primer containing the base sequences of side BglII and 3'side NotI).

Next, the PCR product was treated with a restriction enzyme that cuts at the cloning site, then incorporated into an expression vector and transformed into Escherichia coli. By culturing this recombinant E. coli pGEX-5X-3 (SC) in the presence of 1 mM isopropylthio-β-D-galactopyranoside, a GST-SC fusion protein was produced and glutathione affinity gel chromatography was performed. The GST-SC fusion protein was purified by chromatography (Pharmacia Biotech). E. coli pGEX-5X-3 (SC) has already been deposited at the Institute of Biotechnology as No. 18568 (deposited on October 22, 2001).

(1-4) SDS-PAGE analysis: GST-S
The C fusion protein and GST were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the gel was stained with Coomassie Brilliant Blue to detect the protein band.

(2) Results and Discussion (2-1) Cloning of mouse pIgR cDNA: FIG.
FIG. 3 is an explanatory diagram showing an operation for cloning mouse pIgR cDNA. As shown in the figure, human, rabbit, and rat pIgR were used as the first screening probe.
A 1.8 kb clone was screened using a synthetic oligonucleotide of 40 bases having a homologous base sequence in the cDNA as a probe. Using the cloned DNA as a second screening probe, a clone of about 3.5 kb was screened and the nucleotide sequence was determined. As a result, it was found that this clone did not contain a start codon.

Then, 5'-Ampli was extracted from the mRNA of mouse liver.
FINDER RACE Kit (CLONTECH Laboratories, Inc. Palo A
5'end was amplified by PCR using
Combined with the clone of b by genetic engineering, the total length is 3,863 bp
A cDNA clone was obtained (Fig. 1). Mouse pIgR c
When the nucleotide sequence of DNA was determined, 1 was found upstream of the 5'end.
It was found that the open reading frame contained a non-translated portion of 78 bases and had a base sequence of 2,313 bp.
From the nucleotide sequence, mouse pIgR consisting of 771 amino acids
The primary structure of was estimated. In addition, total length 3,863 bp mouse p
The base sequence of the IgR cDNA is shown in SEQ ID NO: 1 in the sequence listing described later.

The mouse pIgR cDNA nucleotide sequences showed homology of about 72%, about 60% and about 87% with human, rabbit and rat pIgR cDNA, respectively.
The deduced amino acid sequence of mouse pIgR shows homology of about 65%, about 49%, and about 86% with the deduced amino acid sequences of human, rabbit, and rat pIgR, respectively, and mouse pIgR is very similar to rat pIgR. Were found to have high homology.

(2-2) Measurement of molecular size of GST-SC fusion protein: purified GST-SC fusion protein and GST
Were separated by SDS-PAGE and the molecular size was measured. FIG. 2 is an explanatory diagram showing the results. In the figure,
Lane 1 is GST-SC fusion protein, lane 2 is GS
T was shown and the gel was stained with Coomassie Brilliant Blue to detect protein bands. As shown in the figure, the molecular sizes of 91 kDa and 26 kDa are shown respectively, and the difference of 65 kDa corresponds to that of the mouse SCcDN
It corresponded to the molecular size of the polypeptide encoded by A.

Example 2 (1) Operation (1-1) Immunization Schedule: Three 8-week-old male Armenian hamsters were used. GST described in Example 1
-SC fusion protein dissolved in PBS at 1 mg / ml and complete Freund's adjuvant (Difco, Detroit,
(MI) was mixed with 0.5 ml of the same amount every two weeks, and 0.5 ml each was injected subcutaneously on the back at three sites and once at the base of the tail. Three days after the third immunization, test blood was collected and it was confirmed that the antibody titer against the GST-SC fusion protein was increased, and then a GST-SC fusion protein (1 mg / m 2
The mixture of 1) and mouse bile was injected into the abdominal cavity in an amount of 0.5 ml each, and 50 μl was injected into the left and right foot pads.

(1-2) Preparation of hybridoma: Spleen cells of immunized Armenian hamster and lymph node cells of inguinal region and popliteal region were fused with NS-1 or SP-2 myeloma cell line using polyethylene glycol. , HAT
Only fused cells were selected with the solution. The fusion ratio with myeloma was 10: 1 for spleen cells and 5: 1 for lymph node cells.

(1-3) Purification of antibodies: Protein G-Sepharose column (Pharmaci) from serum of immune hamster and culture supernatant of hybridoma
a Biotech)) was used to purify IgG.

(1-4) Detection of anti-SC antibody by ELISA: GST or GST-on a microtiter plate
The SC fusion protein was coated at 0.1 μg / well, an antibody purified from the serum of the immune hamster or the culture supernatant of the hybridoma was added, and the mixture was incubated at room temperature for 90 minutes. After washing each well, anti-hamster IgG labeled with peroxidase (Jackson ImmunoResearch Lab
oratories, Inc., West Grove, PA) 0.008 μg / we
ll each was added and incubated at room temperature for 60 minutes.
Finally, o-phenylenediamine was added to develop color, 492n
The absorbance at m was measured.

(1-5) Collection of Small Intestinal Epithelial Cells and Preparation of Soluble Fraction: To prepare mouse small intestinal epithelial cells (IEC), the small intestine was opened after washing and cut into 1-2 cm pieces,
mM EDTA / 25 mM Hepes (pH7.2) / Ca ²⁺ , Mg ^2
+ -Free HBSS (CMF-HBSS) / protease in
hibitors (1mM PMSF, 0.2U / ml aprotinin, 1
μg / ml soybean trypsin inbibitor, 1 μg / ml
Leupeptin, 1μg / ml benzamidin) 37
Shake at 60 ° C. for 60 minutes. The detached epithelial cells were washed by centrifugation and the pellet was washed with 1% Triton X-100 / 50mM EDTA / 25m.
The cells were suspended in M Hepes / CMF-HBSS / protease inhibitors and left standing on ice for 45 minutes. 30 at 12,000 rpm
The supernatant obtained by centrifuging for minutes was collected and used as the IEC soluble fraction.

(1-6) Western blot: The IEC soluble fraction was separated by SDS-PAGE and then transferred to a PVDF membrane (Millipore, Bedford, MA). PVDF membrane was incubated with 1% BSA / 0.05% Triton X-100 / PBS overnight for blocking and then 0.05
The cells were washed 3 times with% Triton X-100 / PBS. Immune hamster serum IgG (5 μg / ml) diluted with PVDF membrane in 1% BSA / 0.05% Triton X-100 / PBS, 1
0.46 mAb (25 μg / ml), or 2.1.1 mA
Incubation with b (25 μg / ml) for 90 minutes at room temperature followed by washing. As a secondary antibody, peroxidase-labeled anti-hamster IgG diluted to 0.008 μg / ml was used, and the mixture was reacted at room temperature for 90 minutes and then washed. ECL detect
ion reagents (Amersham, international plc, buckingh
After processing for 1 minute in Amshire, UK) Hyper film-ECL (Amers
Specific proteins were detected in Ham, UK).

(1-7) Immunohistological staining: Mouse jejunum 2 cm
Tissu Mou is opened vertically on the membrane after cutting it into several lengths.
It was embedded in nt (Chiba Medical. Shoka, Japan) and frozen, and a section with a thickness of 5 μm was prepared with a cryostat. After the specimen fixed in cold acetone for 10 minutes, fixed further 10 minutes with cold chloroform to inactivate endogenous peroxidase was treated with 0.1% H ₂ 0 _2. After blocking with 3% BSA, immune hamster serum IgG (10 μg
/ Ml) or 10.46 mAb (100 μg / ml) was added and incubated at room temperature for 90 minutes. After washing 0.
008 μg / ml peroxidase-labeled anti-hamster I
After adding gG and incubating at room temperature for 60 minutes
Color was developed with 3,3'-diaminobenzidine (Sigma, St. Louis, MO). Methyl green staining was performed as a counter stain.

(2) Results and Discussion (2-1) Establishment of anti-GST-SCmAb-producing hybridoma clone: The antibody activity in the culture supernatant of 2,440 wells in which the cells were seeded was measured.
The production of an antibody that binds to the T-SC fusion protein but not GST was observed. By growing them, two cell lines stably producing the anti-GST-SC antibody were obtained. Each cell line was cloned by the limiting dilution method, and each clone "2.
The clones "1.1" and "10.46" were established. Antibodies were purified from the culture supernatants and subjected to ELISA. FIG. 3 is an explanatory diagram showing the binding activity of the obtained antibody. As shown in the figure, all of them specifically bound to the GST-SC fusion protein.

(2-2) Western blot: 10.46 m
Western blots were performed using mouse IEC solubilizable fractions to see if Abs could recognize mouse pIgR. FIG. 4 shows the result of Western blotting.
In the figure, lane 1 is serum IgG of the immune hamster,
10.46 mAb in lane 2 and 2.1.1 m in lane 3.
Ab, lane 4 is a control with secondary antibody only.

As shown in FIG. 4, immune hamster serum I
Both gG and 10.46 mAb, 2.1.1 mAb are 130 kDa and 110 kDa of the mouse IEC soluble fraction.
The protein of Da could be specifically recognized. 1
The 0.46 mAb showed a strong binding activity similar to serum IgG of the immune hamster, but the binding activity of 2.1.1 mAb was 1.
It was weak compared to 0.46 mAb.

Further, in order to confirm the specificity of 10.46 mAb, IEC of wild type mice and pIgR deficient mice was confirmed.
A soluble fraction was prepared and Western blotting was performed.

FIG. 5 is an explanatory view showing the result. In the figure, M is a marker, lane 1 is a wild-type mouse, and lane 2 is a pIgR-deficient mouse. As shown in Fig. A, when the IEC proteins of both mice were stained with Coomassie Brilliant Blue, no significant difference was observed in the protein composition of both. On the other hand, as shown in Fig. B, which was blotted with 10.46 mAb, 10.46 mAb
Is 130 and 110 kDa in the IEC of wild type mice
2 bands were detected, whereas no band was detected in the IEC of pIgR-deficient mice. This result is
It has been shown that 10.46 mAb is an antibody that specifically recognizes mouse pIgR.

(2-3) Immunohistochemical staining: The pIgR molecule is strongly expressed in epithelial cells of the small intestinal crypto, and the expression becomes weaker toward the tip of the villi. Immunohistostaining was performed to confirm that the 10.46 mAb recognizes pIgR in small intestinal epithelial cells. Figure 6 shows a frozen section of mouse jejunum
It is explanatory drawing which shows the result of carrying out the immunohistological staining with 0.46 mAb. Immune hamster serum IgG strongly stained epithelial cells, and the staining intensity weakened from the crypto to the villus tip. Similarly, 10.46 mAb, although weakly stained, specifically stained crypt epithelial cells (FIG. 6).

From the above results, 10.46 m produced by the hybridoma established from Armenian hamster
It was confirmed that Ab specifically recognizes the SC portion of mouse pIgR.

Example 3 (1) Operation (1-1) Preparation of bile: Bile was collected by making a hole in the excised gallbladder with a 26G injection needle and then centrifuging at 12,000 rpm for 10 minutes. 1 in bile to cleave Asn-linked sugar chains
00mU / ml N-glycanase (Glyko, Inc., Novato, C
A) was added and the mixture was treated at 37 ° C. for 18 hours.

(1-2) Western blot analysis: Each sample was separated by SDS-PAGE and then transferred to a PVDF membrane. PVDF membrane with 1% BSA /
After soaking overnight in 0.05% Triton X-100 / PBS for blocking, serum IgG (5 μg / m) of immune hamster
1) 10.46 mAb (25 μg / ml) or 2.
1.1 mAb (25 μg / ml) was added, and the mixture was incubated at room temperature for 90 minutes. After washing the PVDF membrane, peroxidase-labeled anti-hamster IgG diluted to 0.008 μg / ml was added and incubated at room temperature for 90 minutes. After washing, EC the PVDF membrane
It was dipped in L detection reagents and exposed to Hyper film-ELC.

(2) Results and Discussion (2-1) Recognition of core protein of bile SC: 10.46 mA
SD of bile to confirm that b recognizes SC
Separation by S-PAGE and Western blotting were performed.
FIG. 7 is an explanatory diagram showing the results. Immune hamster serum IgG recognized two bands of 95 kDa and 86 kDa. On the other hand, 10.46 mAb and 2.1.1 mA
b detects only the 95 kDa band, 10.46 mA
b detected a band stronger than 2.1.1 mAb. That is, 10.46 mAb exists in SC of 95 kDa and is 86k.
It was considered to recognize an epitope not present in Da SC.

Further, in order to confirm that 10.46 mAb recognizes the core protein excluding the SC sugar chain of 95 kDa, the bile sample was treated with N-glycanase and the change in SC molecular size was examined. FIG. 8 is an explanatory view showing the results, in which lane A (-) shows untreated bile S.
C was blotted with serum IgG of immune hamster, lane A (+) was blotted with N-glycanase-treated bile SC with serum IgG of immune hamster, and lane B (-) was blotted with untreated bile SC at 10.46 mAb.
Lane B (+) shows N-glycanase treated bile SC 10.4
It is the result of blotting with 6 mAb.

As shown in FIG. 8, immune hamster serum I
gG is 95 kDa and 86 kDa as untreated bile SC
Band was detected and 8 as SC after N-glycanase treatment
Bands of 3 kDa, 74-79 kDa, and 69 kDa were detected. On the other hand, 10.46 mAb detected a band of 95 kDa as untreated bile SC, and bands of 83 kDa and 79 kDa as SC after N-glycanase treatment. From these results, it was considered that 10.46 mAb recognizes SC core proteins with molecular sizes of 83 kDa and 79 kDa.

As described above, it was revealed from the analysis using 10.46 mAb that mouse pIgR / SC exist as various molecules. Since 10.46 mAb recognizes SC of 95 kDa in bile but not SC of 86 kDa, it is presumed that a portion near the N-terminal or C-terminal of the 95-kDa SC protein may be recognized as an epitope. Since 10.46 mAb recognizes pIgR and SC by Western blotting and pIgR by immunohistochemistry, it is considered to be a useful tool for analyzing the expression and structure of pIgR and SC.

[0047]

As described above, the present invention provides anti-GST-
Establish stable hybridomas producing SC antibody and pI
This has the effect of enabling analysis of the expression mechanism and structure of the gR / SC molecule.

[0048]

[Sequence list] SEQUENCE LISTING  110 KABUSHIKI KAISHA YAKULT HONSHA  120 Monoclonal antibody and hybridoma recognizing secretory factor of secretory IgA  130 PJ23900  160 1  210 1  211 3,863  212 DNA  213 Mouse Polymeric Immunoglobulin Receptor  400 5 aacagtttca gttttgacaa tagtcaccag tagtgccttc ctggaagctt ctagaagggt 60 acatactggc ttgcaggctg tgcccgaaac tggatcacct ggagagaagg aagtagctaa 120 aacattctca tacaagaagc caacctgagc ggcacagccc ccctggaagc cacaagca 178 atg agg ctc tac ttg ttc acg ctc ttg gta act gtc ttt tca ggg gtc 226 Met Arg Leu Tyr Leu Phe Thr Leu Leu Val Thr Val Phe Ser Gly Val   1 5 10 15 tcc aca aaa agc ccc ata ttt ggt ccc cag gag gtg agt agt ata gaa 274 Ser Thr Lys Ser Pro Ile Phe Gly Pro Gln Glu Val Ser Ser Ile Glu              20 25 30 ggc gac tct gtt tcc atc acg tgc tac tac cca gac acc tct gtc aac 322 Gly Asp Ser Val Ser Ile Thr Cys Tyr Tyr Pro Asp Thr Ser Val Asn          35 40 45 cgg cac acc cgg aaa tac tgg tgc cga caa gga gcc agc ggc atg tgc 370 Arg His Thr Arg Lys Tyr Trp Cys Arg Gln Gly Ala Ser Gly Met Cys      50 55 60 aca acg ctc atc tct tca aat ggc tac ctc tcc aag gag tat tca ggc 418 Thr Thr Leu Ile Ser Ser Asn Gly Tyr Leu Ser Lys Glu Tyr Ser Gly  65 70 75 80 aga gcc aac ctc atc aac ttc cca gag aac aac aca ttt gtg att aac 466 Arg Ala Asn Leu Ile Asn Phe Pro Glu Asn Asn Thr Phe Val Ile Asn                  85 90 95 att gag cag ctc acc cag gac gac act ggg agc tac aag tgt ggc ctg 514 Ile Glu Gln Leu Thr Gln Asp Asp Thr Gly Ser Tyr Lys Cys Gly Leu             100 105 110 ggt acc agt aac cga ggc ctg tcc ttc gat gtc agc ctg gag gtc agc 562 Gly Thr Ser Asn Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser         115 120 125 cag gtt cct gag ttg ccg agt gac acc cac gtc tac aca aag gac ata 610 Gln Val Pro Glu Leu Pro Ser Asp Thr His Val Tyr Thr Lys Asp Ile     130 135 140 ggc aga aat gtg acc att gaa tgc cct ttc aaa agg gag aat gct ccc 658 Gly Arg Asn Val Thr Ile Glu Cys Pro Phe Lys Arg Glu Asn Ala Pro 145 150 155 160 agc aag aaa tcc ctg tgt aag aag aca aac cag tcc tgc gaa ctt gtc 706 Ser Lys Lys Ser Leu Cys Lys Lys Thr Asn Gln Ser Cys Glu Leu Val                 165 170 175 att gac tct act gag aag gtg aac ccc agc tat ata ggc aga gca aaa 754 Ile Asp Ser Thr Glu Lys Val Asn Pro Ser Tyr Ile Gly Arg Ala Lys             180 185 190 ctt ttt atg aaa ggg acc gac cta act gta ttc tat gtc aac att agt 802 Leu Phe Met Lys Gly Thr Asp Leu Thr Val Phe Tyr Val Asn Ile Ser         195 200 205 cac cta acg cac aat gat gct ggg ctg tac atc tgc caa gct gga gaa 850 His Leu Thr His Asn Asp Ala Gly Leu Tyr Ile Cys Gln Ala Gly Glu     210 215 220 ggt cct agt gct gat aag aag aat gtt gac ctc cag gtg cta gcg cct 898 Gly Pro Ser Ala Asp Lys Lys Asn Val Asp Leu Gln Val Leu Ala Pro 225 230 235 240 gag cca gag ctg ctt tat aaa gac ctg agg tcc tca gtg act ttt gaa 946 Glu Pro Glu Leu Leu Tyr Lys Asp Leu Arg Ser Ser Val Thr Phe Glu                 245 250 255 tgt gac ctg ggc cgt gag gtg gca aac gag gcc aaa tat ctg tgc cgg 994 Cys Asp Leu Gly Arg Glu Val Ala Asn Glu Ala Lys Tyr Leu Cys Arg             260 265 270 atg aat aag gaa acc tgt gat gtg atc att aac acc ctg ggg aag agg 1042 Met Asn Lys Glu Thr Cys Asp Val Ile Ile Asn Thr Leu Gly Lys Arg         275 280 285 gat cca gac ttt gag ggc agg atc ctg ata acc ccc aag gat gac aat 1090 Asp Pro Asp Phe Glu Gly Arg Ile Leu Ile Thr Pro Lys Asp Asp Asn     290 295 300 ggc cgc ttc agt gtg ttg atc aca ggc ctg agg aag gag gat gca ggg 1138 Gly Arg Phe Ser Val Leu Ile Thr Gly Leu Arg Lys Glu Asp Ala Gly 305 310 315 320 cac tac cag tgt gga gcc cac agt tct ggt ttg cct caa gaa ggc tgg 1186 His Tyr Gln Cys Gly Ala His Ser Ser Gly Leu Pro Gln Glu Gly Trp                 325 330 335 ccc atc cag act tgg caa ctc ttt gtc aat gaa gag tct acc att ccc 1234 Pro Ile Gln Thr Trp Gln Leu Phe Val Asn Glu Glu Ser Thr Ile Pro             340 345 350 aat cgt cgc tct gtt gtg aag gga gtc aca gga ggc tct gtg gcc atc 1282 Asn Arg Arg Ser Val Val Lys Gly Val Thr Gly Gly Ser Val Ala Ile         355 360 365 gcc tgt ccc tat aac ccc aag gaa agc agc agc ctc aag tac tgg tgt 1330 Ala Cys Pro Tyr Asn Pro Lys Glu Ser Ser Ser Leu Lys Tyr Trp Cys     370 375 380 cgc tgg gaa ggg gac gga aat gga cat tgc ccc gtg ctt gtg ggg acc 1378 Arg Trp Glu Gly Asp Gly Asn Gly His Cys Pro Val Leu Val Gly Thr 385 390 395 400 cag gcc cag gtg caa gaa gag tat gaa ggc cga ctg gca ctg ttt gat 1426 Gln Ala Gln Val Gln Glu Glu Tyr Glu Gly Arg Leu Ala Leu Phe Asp                 405 410 415 cag cca ggc aat ggt act tac act gtc atc ctc aac cag ctc acc acc 1474 Gln Pro Gly Asn Gly Thr Tyr Thr Val Ile Leu Asn Gln Leu Thr Thr             420 425 430 gag gat gct ggc ttc tat tgg tgt ctt acc aat ggt gac tct cgc tgg 1522 Glu Asp Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Ser Arg Trp         435 440 445 aga acc aca ata gaa ctc cag gtt gcc gaa gct aca agg gag cca aac 1570 Arg Thr Thr Ile Glu Leu Gln Val Ala Glu Ala Thr Arg Glu Pro Asn     450 455 460 ctt gag gtg acg cca cag aac gca aca gca gta cta gga gag acc ttc 1618 Leu Glu Val Thr Pro Gln Asn Ala Thr Ala Val Leu Gly Glu Thr Phe 465 470 475 480 acc gtt tcc tgc cac tat ccg tgc aaa ttc tac tcc cag gag aaa tac 1666 Thr Val Ser Cys His Tyr Pro Cys Lys Phe Tyr Ser Gln Glu Lys Tyr                 485 490 495 tgg tgc aag tgg agc aac aag ggt tgc cac atc ctg cca agc cat gac 1714 Trp Cys Lys Trp Ser Asn Lys Gly Cys His Ile Leu Pro Ser His Asp             500 505 510 gaa ggt gcc cgc caa tct tct gtg agc tgc gac cag agc agc cag ctg 1762 Glu Gly Ala Arg Gln Ser Ser Val Ser Cys Asp Gln Ser Ser Gln Leu         515 520 525 gtc tcc atg acc ctg aac ccg gtc agt aag gaa gat gaa ggc tgg tac 1810 Val Ser Met Thr Leu Asn Pro Val Ser Lys Glu Asp Glu Gly Trp Tyr     530 535 540 tgg tgt ggg gta aag caa ggc cag acc tat gga gaa act acc gcc atc 1858 Trp Cys Gly Val Lys Gln Gly Gln Thr Tyr Gly Glu Thr Thr Ala Ile 545 550 555 560 tat ata gca gtt gaa gag agg acc aga ggg tca tcc cat gtc aac cca 1906 Tyr Ile Ala Val Glu Glu Arg Thr Arg Gly Ser Ser His Val Asn Pro                 565 570 575 aca gat gca aat gca cgt gcc aaa gtc gct ctg gaa gaa gag gta gtg 1954 Thr Asp Ala Asn Ala Arg Ala Lys Val Ala Leu Glu Glu Glu Val Val             580 585 590 gac tcc tcc atc agt gaa aaa gag aac aaa gcc att cca aat ccc ggg 2002 Asp Ser Ser Ile Ser Glu Lys Glu Asn Lys Ala Ile Pro Asn Pro Gly         595 600 605 cct ttt gcc aac gaa aga gag ata cag aat gtg gga gac caa gct cag 2050 Pro Phe Ala Asn Glu Arg Glu Ile Gln Asn Val Gly Asp Gln Ala Gln     610 615 620 gag aac aga gca tct ggg gat gct ggc agt gct gat gga caa agc agg 2098 Glu Asn Arg Ala Ser Gly Asp Ala Gly Ser Ala Asp Gly Gln Ser Arg 625 630 635 640 agc tcc agc tcc aaa gtg ctg ttc tcc acc ctg gtg ccc ctg ggt ctg 2146 Ser Ser Ser Ser Lys Val Leu Phe Ser Thr Leu Val Pro Leu Gly Leu                 645 650 655 gtg ctg gca gtg ggt gct ata gct gtg tgg gtg gcc aga gtc cga cat 2194 Val Leu Ala Val Gly Ala Ile Ala Val Trp Val Ala Arg Val Arg His             660 665 670 cgg aag aat gta gac cgc atg tca atc agc agc tac agg aca gac att 2242 Arg Lys Asn Val Asp Arg Met Ser Ile Ser Ser Tyr Arg Thr Asp Ile         675 680 685 agc atg gca gac ttc aag aac tcc aga gat ttg gga ggc aat gac aac 2290 Ser Met Ala Asp Phe Lys Asn Ser Arg Asp Leu Gly Gly Asn Asp Asn     690 695 700 atg ggg gcc tct cca gac aca cag caa aca gtc atc gaa gga aaa gat 2338 Met Gly Ala Ser Pro Asp Thr Gln Gln Thr Val Ile Glu Gly Lys Asp 705 710 715 720 gaa atc gtg act acc acg gag tgc acc gct gag cca gaa gaa tcc aag 2386 Glu Ile Val Thr Thr Thr Glu Cys Thr Ala Glu Pro Glu Glu Ser Lys                 725 730 735 aaa gca aaa agg tca tcc aag gag gaa gct gac atg gcc tac tcg gca 2434 Lys Ala Lys Arg Ser Ser Lys Glu Glu Ala Asp Met Ala Tyr Ser Ala             740 745 750 ttc ctg ctt cag tcc agc acc ata gct gca cag ctc cac gat ggt ccc 2482 Phe Leu Leu Gln Ser Ser Thr Ile Ala Ala Gln Leu His Asp Gly Pro         755 760 765 cag gaa gcc tag 2494 Gln Glu Ala ***     770 771 gcagtgctga ccacccaccc ttgcctgtga caatcaactt gagaatcaca ctgatctgct 2554 cgcagcccac actcacccat cacctccgct cttccctcct gtcctcagag gtgtgctggt 2614 tccttcctcg gccatggaag cctggcctag ttacgcctgt ttaggagaga gtgtgaggcg 2674 ttcttttctc tatgaagaga gtgaggtgga aatgaggagg aggtgaacct gagagacatc 2734 tctggaggaa gagggttcac aataggggct cgtttcagga gaaaaggcca tttgaatctt 2794 ctttataacc atatgatagg atgtcagcgt aactcttctc tcctccatct ctcctttcct 2854 atcctcttga ttcaaacaac acatctgaga actcactagg cttcagtgcc tactaaatgc 2914 tgagagccag gccacaatct ttctataaat attactggaa gagatgccat ctcctcccag 2974 attctgtctt ttcattaaga taagacatca ttaccaggca tacctcctgc ctctgtgcct 3034 cataggcata cacaagccat aagggcatca tgattttcag atgagaagag atgtttctca 3094 agagtgccta gtgagataga ctagcgtcaa accagatgtg gcaactcctg gctcttggcc 3154 tacgatctgt cttcaagacc tctgctctta ttagagggag aactttagca tgggggaaag 3214 taagagaaaa tgagttaaag gagcatggtg gtgtgctcct gcaatcccaa tattaagagg 3274 taaaaatagg atcagaagtt caaggtaatc cttggctacc tagggagttt aaggccagcc 3334 tgggatacat tgagacttgg attcaaagaa gcaggaggag ggagcaggaa gaggaggagt 3394 agaaaggggg aagaaggaga aggaaaaaag aagaagagga ggaagaggag gaggaggagg 3454 aggaggagaa gaaggagaag aagaagaaga agaagaagaa gaagaagaag aagaagaaga 3514 agaagaagaa gaagaagaag aagaagaaga agaagaagaa gaagaagaag aagaagaaag 3574 acagacagac aaaaagacaa atggatcttt cctggtctca cctttcccct tcctgtcact 3634 catgtggacc tcatataagg gaaagatact ctcaacctct tgtatttgga gagttttgag 3694 cagacaggta gaagatggag cctgggagca gctgtttttc caatagtcaa attaggactg 3754 tttctctctg agctctgttc ttcaaccagc acctatagac cgaaaccaac aaatcagaaa 3814 tgtccattaa taattaaagt ctatgattgc caccaaaaaa aaaaaaaaa 3863

[0049]

[Brief description of drawings]

FIG. 1 is an explanatory view showing an operation of cloning a mouse pIgR cDNA.

FIG. 2 is an explanatory diagram showing the molecular size of GST-SC fusion protein and GST.

FIG. 3: Hybridoma clone 1 by ELISA
It is a diagram which shows the result of the antigen specificity of the mAb which 0.46 and 2.1.1 produce.

FIG. 4 is an explanatory drawing showing the results of Western blotting in which the pIgR molecule of the mouse small intestinal epithelial cell soluble fraction was detected at 10.46 mAb and 2.1.1 mAb.

FIG. 5 is an explanatory diagram showing the results of preparing small intestinal epithelial cell soluble fractions of wild type mice and pIgR deficient mice and performing Western blotting with 10.46 mAb.

FIG. 6 is an explanatory drawing showing the results of immunohistological staining of frozen sections of mouse jejunum with 10.46 mAb.

FIG. 7 is an explanatory diagram showing the results of Western blotting in which SC molecules of mouse bile are detected at 10.46 mAb and 2.1.1 mAb.

FIG. 8 is an explanatory diagram showing the results of examining changes in the molecular size of SC by treating a bile sample with N-glycanase.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Noriko Nagaoka             1-1-19 Higashishimbashi, Minato-ku, Tokyo Stock market             Company Yakult Head Office (72) Inventor Masanobu Minamino             1-1-19 Higashishimbashi, Minato-ku, Tokyo Stock market             Company Yakult Head Office F-term (reference) 4B024 AA11 BA43 CA01 DA02 GA05                       HA15                 4B064 AG27 CA10 CA20 CC24 DA13                       DA20                 4B065 AA91X AB05 AC14 BA08                       CA25 CA46                 4H045 AA11 BA41 CA40 DA76 EA50                       EA54 FA72 FA74

Claims (4)

[Claims] 1. A secretory IgA secretory factor, which binds to a fusion protein (GST-SC fusion protein) of glutathione transferase and a secretory IgA secretory factor, but does not bind to glutathione transferase (GST). A monoclonal antibody that recognizes. 2. The monoclonal antibody that recognizes a secretory factor of secretory IgA according to claim 1, wherein the antibody is a 10.46 antibody. 3. A hybridoma which produces an antibody that binds to a fusion protein (GST-SC fusion protein) of glutathione transferase and a secretory factor of secretory IgA, but does not bind to glutathione transferase (GST). 4. The hybridoma according to claim 3, wherein the hybridoma is a 10.46 hybridoma cell producing a 10.46 antibody.