JPH03206885A - Dna coding mouse-derived bullous pemphigoid antigen protein - Google Patents

Abstract

PURPOSE:To provide the title DNA useful for giving manifestation vector and, as a transformant thereof, Escherichia coli, containing a base sequence coding a specific polypeptide containing antibody recognition site for mouse-derived bullous pemphigoid antigen. CONSTITUTION:The objective DNA containing a base sequence coding a polypeptide part of which consists of an amino acid sequence of the formula and which contains mouse-derived bullous pemphigoid antibody recognition site. As a result of the comparison of the DNA base sequence of said antigen and the amino acid sequence estimated therefrom with human-derivied ones, the human- derived polypeptide has been shown to have 664 amino acids, compared to 997 amino acids for the above-mentioned polypeptide; thus, the lengths of polypeptide apparently differ from each other; however, the antibody recognition sites for the antigen are mutually overlapped in high homology, and it has been demonstrated to be 78.6% and 77.0% for DNA level and amino acid level, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a DNA containing a base sequence containing a polypeptide containing an antibody recognition site (epitope) of a mouse-derived hydrophilic antigen.

The present invention also relates to an expression vector containing a DNA that includes a base sequence encoding this antibody recognition site and optionally has a codon encoding methionine at the 5' end.

The invention further relates to E. coli transformed with this expression vector.

[Prior art] Bullous peiphigoi
d) is a disease of the elderly, and there is almost no difference between men and women.
The average age is approximately 70 years old, and 80% of patients suffer from eczematous dermatitis, which first appears after the age of 60.1 There are basically two types of eruption, and the first type is on normal skin. Or, the first type of water IUI, in which blisters are first seen on the erythema, is small hydration at first, but soon develops large blisters. It causes erythema, wheal, papular erythema, etc., and is often annular erythema, sometimes iris-like or target-shaped, and is a first-onset type of erythema in which blisters appear on the erythema only after several weeks. The majority of cases are of the initial blistering type, but when erythema first appears, it is difficult to confirm that the blisters have formed, and there is a period during which the blisters almost disappear and the only symptom is multiannular erythema. Sometimes it is misdiagnosed.

Hydrophilic ml is found along the dermal-epidermal border of the lesion.
Deposition of type G immunoglobulin was observed, and the epidermis of normal skin was detected from the patient's serum. ) Basement m
It is considered an autoimmune disease because IgG class antibodies that react with the enbrain zone have been detected [E, H, Eeutner et al.
cnsitization in Pemphig
us and BullousPempbigoi
d J, Charles CTholas Co.
, 5prinafield, l1linOis (
1970)].

The antibody is found in the skin epidermal basement membrane and serum of patients with hydrophilic adenopathy, but it also binds widely to epithelial cells of not only patients but also normal people, and even cells of vertebrates. [1. A, otaz et al., 8cta,
Demato-Venereol, 58. 537
(1978)]. The antigenic substance of the antibody present in the epidermal basement membrane was determined by immunoprecipitation method using patient serum and extract of cultured human epidermal basal cells (kera lorocyte) or immunoplot method using normal human epithelial cells. It is considered to be a Dalton (kD) protein.

However, a 170 kD protein was also detected, and 1
It is not known whether the 70kD protein is part of the 230kD protein [J, flStanl
el/et al., Ce1l 24, 897 (1981);
J, R, 5tanley et al., J, fnvesL [lc
rmatol, 82. 108 (1984): J, R
3tanley et al., Br, J., Dcrnatol,
113.67 (1985) R.S. Labib et al.
J, Immunol, 136.1231 (t9
861].

Also, recently, J, R, 5tanley et al. [J, Cl1n,
Invest. Decided.

Furthermore, Masayuki Amaya [J, Invest, Dermato
l, 92395 (1989) 1 is Pam cel
l [S, 11. Yuspa et al., cancerlie
s, 40.4694 (1980)]
NA Library, Toshiyuki Sugi et al. [J, Cl1n, Inve
st.
It was discovered that the code encoded the D part.

[Problem to be Solved by the Invention 1 As mentioned above, hydrophormia is an autoimmune disease caused by the production of autoantibodies against the epidermal basement membrane.
The mechanism of its onset is unknown.

Regarding the antigenic substance of the antibody, although there are reports centered on 5tanl/etc., full+en
A cDNA clone of gth has not been collected, and the structural protein of this antigen has not been elucidated. Furthermore, the recognition site of autoantibodies on proteins that serve as antigens has not yet been determined.

For this reason, the clinical symptoms very similar to this disease have been reported to be
It is difficult to distinguish between the two, and the situation is difficult from a treatment perspective.

If the recognition site in the human [C-terminal 120 kl of the mouse-derived antigen protein of the tuklopuru antibody] coincides with the recognition site of the autoantibody, it would be of great significance in diagnosis and treatment.

An object of the present invention is to provide a DNA containing a base sequence encoding a polypeptide containing an antibody recognition site for the above-mentioned aquatic myeloid antigen derived from non-human mouse epithelial cells.

Another object of the present invention is an expression vector containing a nucleotide sequence encoding the antibody recognition site and optionally having a codon encoding methionine at its 5' end. The purpose of the present invention is to provide transformed E. coli.

[Means and effects for solving the problems] The present invention provides the following amino acid sequence containing an antibody recognition site for the murine Hypnosis vulgaris antigen: Leu Ala Gin Ser Gl
n Asn Leu Val Ser Glu Phe
Lys (iln Lys Cys^sp Gln
Gln Ser Met Ile lie Gin L
ys Thr Glu Lys Glu Vat Ar
gSer Leu Ser^la, Glu Leu S
er Ala Ser Lys Glu Glu LY
s Arc ArgQlu Glu OIn Lys
Ala Gin Leu Oln Arg Al
a (iln Val Gln Glu Leu^
sn Asp^rg Leu Lys Arg Val
Gln Asp Glu Leu Hli Leu
Lys Thr+1e Glu Glu Oln Me
(Thr Hlm Arg Lys Met Ile
Leu Leu Gln GluGlu Ser As
p Lys Phe, Lys^rg Ser Ala
Asp Glu Phe Arg Lys LysM
et Glu Lys Leu Met Glu Se
r Lys Vat Mal Thr Glu Thr
Asp LeuSer Gly rle LYs H
ls Asp Phe Val Ser Leu G
ln Arg Glu Asn Phe＾rg＾la
Gln Glu Asn Ala Lys Leu T
rp Glu Thr Asn Ile ArgGl
uLeu Glu Ar(Gin Leu Gln C
ys Tyr Arg Glu Lys Met Gl
n 01n GLyPro Pro '/al
Glu Ala Asn Hls Dingyr
Gln Lys Cys Arc Arg
Leu GluGlu Glu Leu Leu A
la Gln Art^rg Glu Vat Glu
Asn Leu Lys GlnLys Met A
sp Gln Gln lie Lys Glu Hl
s Glu Hls Gin Leu Leu krg
Leu Gin Cys Glu Ile Gin L
ys Lys! ! er Thr Thr Thr Gln A
sp Hls ThrPhe Ala Ser^la
Phe Asp Thr Ala Gly Arg G
lu Cym Hls Hls Pr.

Ala Glu lie Ser Pro Gly
Asn Ser Gly Hls Leu Asn L
eu Lys Thr^rg Leu Pro Leu
Ser Arg Trp Thr Gln Olu
Pro Hls Gln Thr 01uGly Ly
s Trp Pro Hls Arg Ala^la
Glu Gln Leu Pro Lys Glu V
alGin Phe Arg Oln Pro qly
Ala Pro Leu Asp Arg Glu
Ser Ser GlnPro Cys Tyr Se
r Glu Tyr Phe Ser Gin Thr
Ser Thr Glu Leu Gln+1e T
hr Phe Asp Asp Lys Asn Pr
o Ile Thr ^ “g Leu Ser Gl
u LeuGlu Thr Met Arg Glu
Gln Ala Leu Outs Pro Ser Arg
Pro Pro Val Thr Tyr Gln A
sp Asp Lys Leu (ilu Art G
lu Leu Val Lys Leu LeuThr
Pro Leu Glu Ile Ala Lys
Asn Ly,s Gln Cys Gly Met
Hls Thr Glu Mal Dinghr Thr
Leu Lys Gln Glu Lys
Arg Leu Gly! 3er Se
r AlaGly Gly Trp He(Leu
Glu Gly Cys Arg Thr Ser G
ly Gly Leu LysGly Asp Phe
Leu LYs Lys Ser Val
Glu Pro Glu Ala Ser Pro
5erLeu Asp Leu Ain Gln Al
a Cys! ier Val Arg Asp
Glu Glu Phe GlnPhe (iln G
ly Leu-Arg Hls Thr Val T
hr Gly Arg Gln Leu Mal G
lu^1a Lys Leu Leu Asp M
et Arg Thr Mal Glu C1n
Leu Art Leu GlyLeu Lys Th
r Val Glu Glu Val Gln A
rg Set Leu 3er Lym Phe Le
uThr Lys Ala Thr Ser lie
Ala Gly Leu Tyr Leu Glu S
er Ser Lys (ilu Lys Met Se
r Phe Thr Ser Ala Ala Gln
Lys Ile lie Ile AspLy
s Met Ile Ala Leu Ala Ph
e Leu Glu Ala Gln Ala Ala
Thr GlyPhe Ile Ile Asp
Pro Vat Ser Oly Gin Thr
TYr Cys Val Glu Asp^la V
al Leu Hls Gly Ile Va
l Asp Pro Glu Phe Ar(Se
r Arg LeuLeu Glu Ala Glu
Lys Ala Val Leu Gly Tyr
Ser Hllll Ala Ser Lysdinghr
Leu Ser Vat Pha Gin
Ala Met Glu Asn Arg
Met Leu AIIp ArgLys L
ys Gly Lys Hls lie Leu Gl
u^la Gln Ile Ala! 3er Oly
GlyVat Ile Asp Pro Mal
Art Gly Val Arg Vat Pro P
ro Glu Met AlaVal Gln Gln
Gly Leu Leu Asn Asn Ala
Val Leu Gln Pha Leu HlsGl
u Pro Ser Ser Asn Thr Arg
Val Pha Pro Asn Pro Asn^
sn LysGln Ala Leu Tyr Tyr
5ePGlu Leu Leu Gln Ile C
ys Val Phe AspVal Asp Cys
(iln Cys Phe Leu Leu Pro
Phe Gly Glu Arg Olu ll5S
er Asn Leu Asn lie Glu Ly
s Thr Hls Lys Ile^la Mal
Val AspThr Lys Thr any^I
a Glu Leu Thr^la Phe Gin
Ala Phe Oln Ar(Ain Leu li
e AspLys Gly lie Tyr Leu
Glu Leu Ser Gly Gln GlnTy
r Gln Trp Lys Glu Ala Thr
Phe Phe Asp Ser Tyr Gly
Hls Pr.

Ser Hls Met Leu Dinghr
Asp Thr Lys Thr Gly
Leu Gin Phe Asn rleSe
rGlu^IaValGluGlnGlyThrLeu
AspLysAlaLeuMalGlnLys Ty
r Gln Glu Oly Leu Dingh
r Thr Leu Thr Glu Le
u Ala Asp PheLeu Leu S
er Lys Mal Vat Pro Lys Ly
i Asp Leu Hlg Ser Pro 1ie
All Gly Tyr Trp Leu Thr
Ala Ser Gly Olu Arg lle S
Ar Leu LeuLys Ala Ser Arz
^r(Asn Leu Val Asp Arz Va
l Thr^Ia Leu Ar(Cys Leu
Glu Ala GIn Ile Cys
Thr Gly cry lie He
Asp Pro LeuThr Gly Ly
s Lys Tyr Arg Yal Al
a Glu Ala Leu Hls Ar
g Gly LeuVal Asp Glu Gl
y Phe Ala Gln Gln Leu Arg
Gln Cys Glu Leu Ma+11e T
hr Gly Ile Ser Hls Pro V)
I Ser' Asn Lys Met Met Se
r MalMml Glu^Ia Val Asn A
la Asn rle lie Ser Lys Gl
u Met Gly MetArtc Cy,s Le
u Glu Phe Gin Tyr Leu Thr
Gly Gly Leu rle Glu Pr.

Lys Val Phe Ser^rg Leu Th
r lie Glu Glu Ala Leu Hls
Val crylle Ile Asp Val L
eu lie Ala Thr Arg Leu Ly
s Asp Gin Lys 5erTyr Val
Arg Asprle Met Cys Pro G
in Thr Lym Arg Lys Leu Th
rTyr Lys Glu Ala Leu Glu
Lys Ala Asp Phe Asp Phe H
ls Thr anyLeu Lys Thr anyLeu
Glu Val Ser Glu Pro, Leu
Gly Thr Gly Ile Set^sn L
eu Tyr Ser Ser G
It relates to a DNA containing a base sequence encoding a polypeptide consisting of ln. Furthermore, as an example of the base sequence encoding the polypeptide, the following sequence: CTA GCCC
AA AGT CAA AACCTA GTA AGC
GAG TTCAAG CAA AAG TGTGAC
CAG CAG AGCATG ATCATCCAG
AAA ACG GAG AAG GAG GTG A
GGAGCCTG AGCGCA GAA CTG A
GCGCG TCC,AAA GAG GAG AAG
CGG CGGGAA GAG CAG AAG G
CT CAG CTG CAG CGA GCT CA
G GTG CAG GAG CTGAAT GACA
GG CTCAAG AGG GTG CAA GAC
GAG CTG CACCTG^^G ACCATCG
AA GAG CAG ATG ACCCACAGG
AAG ATG ATCCTG CTCCAG CAA
GAG TCG CAT AAG TTCAAA CG
CTCG GCA GAT GAG TTT CGG
AAG AAGATG GAG AAG CTA
ATG GAG TCCAAG GTCGT
CACT GAA ACCCAT CTCTCA
GGCATCAAG CACGACTTT, GTG T
CT-CTCCAG AGA GAA AACTTCC
GG GCG CAAGAG, AACGCG AAG
CTG TGG GAG ACCAACATT CGA
GAGCTT GAG CGG CAG C
1 CAG TGCTACCGT GAG A
AG ATG CAG CAA GGCCCG
CCCGTG GAA GCA AACCACTAT
CAG AAG TGCCGG AGA CTCGA
GGAG GAG CTG TT (i GCCCAG
AGA CGG GAA GTT GAA AACCT
CAAA CAAAAAA ATG GACCAG CA
G ATA AAG GAA CAT GAG CAC
CAG TTG CTT CGOCTCAG TGT
GAA ATCCAA AAG AAG AGCAC
A ACCCAA GACCACACCTTCGCG
TCG GCT TTT GAT AcGGCA GG
G AGA GAG TGCCACCACCCTGCA
GAG ATCTCCCCCT GGG AACTCT
GGG CACCTT AACCTA AAG AC
CAGA CTCCCA CTG TCCAGG TG
G ACT CAG GAG CCA CACCAG
ACA GAAGGA AAA TGG CCG CA
CAGG GCT GCT GAA CAA CTT
CCG AAG GAG GTTCAG TTCCGA
CAG CCA GQG GCT CCG CTG
GACAGG GAG -AGCAGCCAGCCA
TGCTACTCG GAG TAT TTT TCT
CAG ACA AGCACT GAA CTG C
AGATA ACT TTT GAT GAT AAA
AACCCA ATCACG CGA CTG TC
T GAA CTAGAG ACG ATG A
GG GAG CAA GCCCTCCAT
CCCTCCAGA CCG CCG GTGA
CG TAT CAG GAT GACAAA
CTT GAA AGG GAG TTC
GTG AAG C7 ττGACA CCC
TTA GAG ATA CCT AAG A
ACAAA CAG TGT GGCATG
CAT ACAGAA GTCACG ACCTTA
AAA CAA GAG AAG ACiG CTG
GGT TCCAGT GCTGGT GGA TG
G ATG CTG GAA GGG TGCAGA
ACA TCT GGT GGA CTCAAGGGA
GAT TTCCTT AAG AAA AGCGT
A GAG CCA GAG GCT TCCCCG
AGCCTT GACCTT AACCAG GC
G TGCTCT GTT AGG CAT
GAG GAG TTT CAGTTT CA
A GGG CTCAGG CACACCGTG AC
T GGCAGG CAG CTG GTC, GAAG
CCAAG CTCCTG GACATG AGG A
CA GTT GAG CAG CTG CGG CT
T GGTCTG AAG ACT GTCGAA G
AA GTT CAG AGA AGT CTT Aa
c AAG TTT CTGACCAAA GCT
ACC CCATT GCA GGA CTT
TAT CTA GAA TCT TCC
AAAGAA AAA ATG TCG TTCACT
TCG GCG GCCCAG AAA ATCAT
A ＾τ＾GAC＾AA ATG ATA GCT C
TA GCCTTT TTA GAA GCT CAG
GCT GCA ACA GGTTT Ding ATA
ATT CAT CC (i GTT TCT
GGT CACACT TACTGT C7 block
GAA GATGCA GTT CTT, CA
T GGCATCGTT GAT CCT GA
G TTCAGG AGCAGG CTCCTG
GAG GCA GAG AAG GCA GTT T
TG GGA TAT TCA CAT GCT TC
T ^^GACG CTG TCA GTG TTCC
AG GC^^TG GAA AAT AGA ATO
CTT CAT AGGAAG AAA GGT AA
A CAT ATCTTG GAG GCA CAA
ATT GCCAGT GGG GGCGTCATT
GACCCT GTG AGA GGCGTC
CGT GTCCCT CCA GAA ATG
GCTGTG CAG CAG GGCTTG C
TG AACAACGCCGTCCTA CAG TT
CCTG CATGAA CCG TCCAGCAAC
ACG AGA GTCTTT CCT AAT CC
CAACAACAAGCAG GCT CTG TAT
TAT TCG GAG TTA CTG CAG
ATCTGT GTG TTT GATGTA GA
T TGCCAG TGT TTT CTCTTG C
CG TTT GGG GAG AGG GAA AT
ATCCAAT CTCAACATA GAG AAA
ACT CAT AAA ATT GCCGTG G
TA GAT^CCAAA ACT GGG GCG
GAA CTG ACG GCA TTCGAG GC
T TTCCAG AGAAAACCTCATT GA
CAAG GGT ATT Ding^T CTCGAA
CTCTCA GGOCAG CAGTAT C
AG TGG AAG GAA GCT ACA TT
T TTT GACTCCTACGGG CAT CC
TTCT CACATG CTG ACT GAT A
CT AAA ACT GGT CTG CAG TT
CAAT ATTAGT GAA GCT CTCGA
G CAG GGA ACG cTA GACAAA
acc TTG GTG CAAAAG TAT CA
G GAA GGCCTA ACT ACA CT^^
CA GAA CTG GCT GACTTTTCTT
CTG AGCAAG GTA GTT CCCAAG
'^^G GAT TTG CACAGT CCCAT
TGCA GGCTAT TGG CTG ACCGC
T AGT GGG GAG CGA ATCTCCT
TG CTG^AA GCCTCCCGT AGA A
ACTTG GTT GAT AGG GTT ACA
GCCCTCAG＾DingGCCTT GAA GC
CCAA ATC70 ACA GGA GG
G ATCATCGAT CCCCTAACτ G
GG AAA AAG TACAGG GTG GCC
GAG GCT TTG CAT AGA GGG C
TGGTG GACGAG GGCTTCGCCC
AG CAG CTA CGCCAG TGT
,GAA TTA GTGATCACG GGG
ATCAGCCACCCCCGTCAGCAATAAA
ATG ATG TCA GTGGTG GAA G
CCGTG AAT GCA AAT ATCATCA
GT AAA GAA ATG GGCATGCGG
TGCCTG GAA TT:T CAG
TACCTG ACA GGG GGCCTG
ATA GAG CCGAAG GTT TTC
TCG AGG CTG ACCATA GAA GA
G GCCCTT CACGTCGGTATCATT
GAT GTCCTCATCGCCACCAGA CT
CAAA GAT CAA AAG TCATAT
GTCAGG GAT ATA ATG TG
CCCCAG ACT AAG AGA A
AA TTG ACATAT AAA GAG G
CCCTG GAG AAA GCT GAT TTT
GAT TTCCACACA GGACTT AAG
CTG TTA GAA GTG TCT GAA
CCCTTG GGG ACA GGG ATA TC
CAACCTCTACTAT TCT TCCCAG, but is by no means limited thereto, and all base sequences that satisfy the above amino acid sequence are included within the scope of the present invention.

In order to achieve the object of the present invention, the isolation and cloning of DNA encoding an antigenic protein that produces a hydrated apothelial residue was carried out in paw cells derived from mouse epithelial cells.
[S, tl, Yuspa et al., Cancer Res,
40°4694 (1980)].

It has already been immunochemically demonstrated using serum from patients suffering from the disease that paw cells express the hydrophilic 1ffi antigen [S, Il, Yu
spa et al., Cancer Res, 40.4694
(1980)], but the details of the antibody recognition site for the antigen were unknown.

Paw cell c against Yamaki's aquatic acid antigen
A DNAΔ clone was created as follows.

RN from Pal cell by lithium chloride method
A was prepared using oligo-dT cellulose.
Prepare only ly(8) RNA. This poly(A)
A complementary strand of DNA is elongated using a reverse transcriptase using a random primer and a primer on the RNA, and then the RNA is separated using RNase and a double-stranded DNA is synthesized using DNA polymerase I.1! ECOR [linkers are connected to both ends of cDNΔ, introduced into λQt11, and further in vitro packaging is performed to construct phage particles. The phage particles are infected with λ phage-infected Escherichia coli Y1090, and plated on an agar medium. Cultivate to form lytic plaques of λ phage. A nitrocellulose membrane is brought into close contact with the agar medium on which the lytic plaques have formed, a part of λ phage is immobilized on the crotch, and isopropyl-β-thiogalactopyrano After inducing the target protein with the peptide, a 7-diplaque (in which DNA encoding the target antigen protein has been incorporated) is identified by immunoscreening. However, the antibodies used for identification were a human anti-epidermal basement membrane monoclonal antibody as a secondary antibody, and an anti-immunoglobulin antiserum labeled with alkaline phosphatase as a secondary antibody.

The identified phage plaques were found in 7 cases of hydrophilic 11 cases.
It reacted with all of M patient serum, and in Northern blotting with RNA in Pal11 cell using this cDNA as a probe, it reacted with approximately 9 kilobases (kb).
), this mR
It was determined that the NA was long enough to encode a 230 kD protein (data not shown).

The thus obtained phage DNA into which the DNA encoding the hydrophilic fire scar antigen protein has been incorporated is treated with restriction enzyme E.
The approximately 3.2 kb fragment obtained by digestion with CORI is subcloned into plasmid pUc9, which has similar restriction enzyme sites.

The thus obtained plasmid pBPM1 containing the DNA encoding the hydrophilic antigen protein was incorporated.
is digested with various restriction enzymes, and each fragment is subcloned into phage vectors M13ap18 and M13ap19. One-strand phage DNAt! from the obtained phage plaque! : was prepared, and the base sequence of each fragment was determined by the M13 dideoxy method.

As described above, a polypeptide containing an antibody recognition site for the hydrophilic macrophagous antigen found in mouse epithelial cells was obtained.
I) The base sequence of NA was determined (Figure 1).

Furthermore, an A-bundling reading frame was revealed by PCR analysis, indicating that this clone encodes 997 amino acids (Fig. 1).

DNA of the Mouse-derived Mouse Tenko Healing Antigen of the Present Invention
s=v sequence and the amino acid sequence deduced therefrom;
J, R, 5tanley et al. [J, Cl1n, Inv
est 821864 (198 g)] and the human-derived one. The polypeptide containing the antigenic site has a number of amino acids of 997, while that of human origin has 664 amino acids.
Although the length of the polypeptides of both proteins was clearly responsible, the antibody recognition sites for the antigens overlapped with the soybeans with high homology, with 78.6% overlap at the DNA level.
and 77.0% at the amino acid level (Figure 2).

Furthermore, the present inventors discovered that the recognition site of the mouse-derived hydrophilic macrophagia antigen by the human anti-epidermal basement membrane monoclonal antibody is Cys Val Phe Asp VatAs
p Cys Gln Cys PheLeu
Leu Pro Phe GlyGlu A
rg GILJ Ile 5erAsn L
eu Lys Thr Ala Va LVS Thr eulhr A la phe eulle 11eTV r 3er c+y G ln T r ρ Thr Phe Tyr Gly Has Met 1-hr Lys Gln Phe G lu A la Gl/ Thr Asn l1e 1-1 15LVs Vat ASO Gly Δ 1a Ala Phe Gln Arq ΔS p L, VS leu GIU Gln Gln LVS GIU Phe AsD 1. spr.

1-eu Thr 1'-hr Gly Asn l1e Val Qlu 1-eu ASD lu 1e Thr Qlu Qlu S n IV leu Tyr la er er ASD leu e r ln V 5 Ala Leu Val Gln L
It was revealed that the polypeptide has an amino acid sequence consisting of ysTyr Gln Glu Gly. The details will be explained below.

The above-mentioned cDNA of about 3.2 kb, which corresponds to about 120 kD of the C-terminal half of the mouse 230 kD hydrophilic protein [1 antigen], was inserted into the ECOR1 site of plasmid pUc9 to obtain the expression vector pBP (Fig. 4). Ta. This vector also contains the promoter (plac) and operator (Qlac) genes of the E. coli lactose operon and the ampicillin resistance gene (Amp). This vector was transferred to E. coli Pot) 213
After transfecting into 6 and transforming, an ampicillin-resistant strain was selected. This transformed bacterium 2136 (p[3PI)(
Deposited on February 20, 1990.

Microtechnology Research Institute No. 11292, FERM P-112
92). After this transformed bacterium was treated with lysozyme and sonicated, it was centrifuged and the precipitate was collected using NP40.
The insoluble fraction containing the 120 kD antigen protein was taken out and dissolved with urea.

After electrolytic method #J, a lysate containing a 120 kD antigen protein obtained by expressing the expression vector was transferred to Western
The 120 kD protein band was confirmed by blotting (Figure 6, lower diagram). At this time, hydrated epilepsy patient serum (lanes 1-11) was used as an anti-natant for detection. Serum from Tenakuka patients tested at the same time (Lane 12)
) and normal human serum (lane 13), this band was not detected, and therefore no immunological reaction occurred, indicating that the 120 kD protein is an antigen specific to Aquilinidae II. Do you get it.

By the way, from Western blotting analysis of the reaction between the above serum and the protein extracted from the skin section of a normal human forearm with SDS/β-mercaptoethanol/EDTA (Fig. 6, upper figure), it was found that the hydrophilic phthisis Each patient serum contains two 230kD and two 170kD proteins! one that recognizes only the 230kD protein;
and 170 kl) which recognize only proteins.

Experiments similar to those shown in FIG. 6 were conducted on 133 Akane sera from patients with hydrophilia, 20 sera from Ten 1fi patients, and 21 sera from normal individuals, and the results are summarized in Table 1.

Table 1 From Table 1, it was found that many of the sera containing antibodies that recognize the 230 kD antigen protein also recognized the 120 kD protein, while other sera did not recognize the 120 kD protein at all. This result was consistent with the result shown in FIG.

Next, the recognition site of the 120 kD protein by the human anti-epidermal basement membrane Nanatukurotaru antibody was determined.

Six types of restriction enzyme fragments (XhOIHindl[I
, Sac I-1-1indllll, Xba
IEcoR1, Xba I-Bc l I, Xba l
5tu I, Xba I-BgI II) [Figure 5 (Part 1)], and an expression vector (DA
Synthesized tryptoph?T-Trp) at C1al restriction site. Vector (pAT-Trp-1-rpE) obtained by ligating the 5' side nucleotide sequence of the protein synthase E gene (TrDE)
Each of the above-mentioned DNA fragments was integrated into 6 types of corresponding expression vectors, namely Boo7'D X H, D S
H, pXE, pXB.

pXS and pXBII were produced [Figure 5 (Part 2)]
.

Next, each constructed expression vector was used to infect E. coli 11B101.
The resulting transformed bacteria were cultured in LB medium in the presence of ampicillin to produce each fragment polypeptide of the 120 kD antigen protein.

These transformed bacteria were transformed into rs corresponding to each expression vector.
cherichia coli t-I B 1
01 (I) X H) (Deposited on February 20, 1990,
Wei ■ Hu Bing Qi No. 11298, F E R P -
11298), Eschcrichia colt I8 101 (DSI-1) (deposited on February 20, 1990).

Microorganisms No. 11293, FERM P-1129
3).

[5cherichia col i HB 1
01 (D

FFRM P-11297), Esche
richia coli+-IB 101 (pXB
) (February 20, 1990 B fNJ Miracle.

Microtechnology Research Institute No. 11295, FERM P-112
95).

Eschcrichia coli HBL 01
(pXs) (Deposited on February 20, 1990, Microtechnology Research Institute No. 11294.

FERM P-11294) and Escher
+chia coli-1B 1o1 (pXBI
[) (Deposited on February 20, 1990.

Microtechnology Research Institute No. 11296, FERM P-112
96).

Figure 7 shows the results of electrophoresis of the extruded product obtained by expressing the epidermal vector and analysis using t+ester blottinQ. In the figure, the expression vectors used are as follows: Lanes 1 and 9: pBP; Lanes 3.4.15 and 16: pSH; Lanes 5 and 6: pXH: Lanes 7 and 8: pxE: Lanes 2 and 10. ＝Horizontal flea.

In addition, lanes 3.5.7, 11.13 and 15 are the cases where induction was applied by adding β-indole acrylic acid, while lanes 4, 6.8°12.14 and 16
is the case without addition of β-indole acrylic acid. A human anti-epidermal basement membrane monoclonal antibody was used as the antibody for polypeptide detection.

As a result, polypeptide bands were detected in lanes 1, 7, 8, 9, 11, 12, 15, and 16 (the reason why the band was not single was probably due to degradation by protease). That is, D encoding the target antibody recognition site.
Expression vectors containing NA 1jpBP, pXE and pX
Sr (Moraro A and pXI3 also contain this DNA), whereas p x s m does not contain this DNA, so the DNA encoding the polypeptide of the antibody recognition site is BgI ll-8tLJ I restricted. It became clear that it existed between the parts.

The 132-base sequence of this DNA is shown in Figure 1.
TTT GΔD...AAG TAT CAG
It corresponds to GAA GGC (Fig. 8), and when it is incorporated into a vector and expressed, it may contain an initiation codon (ATG) encoding methionine at its 5' end.

Therefore, the amino acid sequence of the polypeptide representing the antibody recognition site was found to consist of 114 amino acids corresponding to the sequence between Cys at position 657 and Gly at position 770 in Figure 1 (Figure 9). . Furthermore, since this antibody recognition site also reacts with the serum of patients suffering from hydrophilic hyperplasia (FIG. 6), it is also the site recognized by autoantibodies in the patient's serum.

Therefore, the present invention also includes a base sequence encoding an antibody recognition site of the polypeptide encoded by the DNA of the present invention, and optionally has an initiation codon (ATG) encoding methionine at the 5' end of the polypeptide. An expression vector containing the DNA is provided. These expression vectors include the plasmids pXE, DXB, C)
Contains BP. Of course, it is also possible to use viral vectors as vectors.

The present invention further provides E. coli transformed with the above expression vector. These E. coli include.

Therefore, by culturing the above-mentioned transformed bacteria, it is possible to produce polypeptides in a stable manner, thereby stably supplying them as specific antigens for diagnosing hydrophilic hypertrophy.
Kotoka Hikiru.

The present invention will be explained in more detail with reference to the following examples.

[Example] 1) Preparation of a human anti-epidermal basement membrane monoclonal antibody: A human anti-epidermal basement membrane antibody was prepared using the serum of a 72-year-old male aquatic amphibians patient. 20af from the patient by venipuncture! Peripheral blood was collected and heparinized Fl! 11,
Iyiphoprep the core cells (Nycomed
A.S.

0slo, Norway) by centrifugation.

The cells were P B S (NaCj! 8.0 g,
PO40,29.

The cells were washed three times with C11O,29/R) and rosettes were formed with neuraminidase-treated sheep red blood cells.

The rosette-forming cells were centrifuged using lya+phoprep, and the resulting B lymphocyte fraction was
Washed with S three times.

EB virus was RPH11 containing 10% fetal bovine serum.
640 medium (obtained from the culture supernatant of B95-8 cells cultured in GIBCO1 at 37°C under 5% CO2 conditions for 8 days.

This culture supernatant 11R1 and 8 lymphocytes were incubated for 2 hours at 37'C with occasional shaking.

Cells were then washed three times with PBS and suspended in 20d of RPM I 1640 medium containing 20% fetal bovine serum.

This was dispensed into a 96-well plate in an amount of 0.2 ate per well, and cultured at 37°C under 5% CO2 conditions.

A screening assay was performed on the supernatants of wells in which colony formation was observed.

The assay was performed according to the method of R, E., Jordon et al. [Arch
, Dermatol.

103, 486. (1971)], an indirect fluorescent antibody method was used.

A normal human skin section was used as the substrate, and a fluorescent isothiocyanate 1-labeled anti-human IQG (γ chain) anti-human serum (manufactured by DAKO) was used as the secondary antibody. The obtained antibody-producing positive cells were cloned by limiting dilution method,
An anti-epidermal basement membrane monoclonal antibody was obtained.

According to westcrnb+ott1ng analysis, this monoclonal antibody specifically recognized the 230KO protein.

2) cD encoding mouse-derived BP antigen protein
Cloning of NA: Pal ce cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 31°C and 5% CO2.
11 was collected and diluted with Li CAI buffer (38 LiCl2
．． 6H urea, 50+eHTris-HCρpH7.5,
5 mHEDTA, 0. The suspension was suspended in IHβ-mercaptoethanol, pulverized, and then left in water overnight. Total RNA was precipitated by centrifugation (13,000 g, 45 min) and transferred to TESPM.
Li solution (10a+H Tris-11cI pH 7.5
, 1 mH EDTA.

0.5% SOS, 100 mg/-brotinase K). This was mixed with chloroform:1-butanol (4:1).
), and ethanol was added to the aqueous layer to obtain RNA as a precipitate. TE this! llii solution (10mHTris-
11ci) pH 7.5, 1 mH EDTA) and isolated by oligo dT cellulose column chromatography.

cDNA was prepared using random hexamer p(N)6 (Boehringer Mannheim) as a primer according to the manual of the CD DNA synthesis kit (Boehringer Mannheim).

The 1q cDNA was purified by Sepharose 4B column chromatography. Furthermore, using EC0RI medilese and S-adenosylmethionine, EC was added into the DNA.
After methylation for the case where oRl site is present, T
4 Use DNA ligase to attach [=C0
A RI linker (dGGA^TTCC) was ligated. Furthermore, this was cut with EC0RI, and Sepha [1-s CL-4
It was purified by B column chromatography and introduced into the ECoRl site of )7-divector λ (Jtll using T4 DNA ligase.

The resulting DNA was packaged using Gigapack Gold (Stratagene @l) according to the manual.

Using E. coli Y1090 as the indicator bacteria, the culture solution 10Ili
! When the OD reaches 3, collect the bacteria and 10nH
14 (suspended in lso45M1. 100 IJ of this solution
TMG buffer (0, IHNaCj
), 50nHTr+5-HCj+ pH7,5,10
A solution of about 10 4 phages suspended in nHHo504 (0.01% geladin) was added to the tube, and the tube was left at 37° C. for 20 minutes to carry out phage infection.

Spread this solution onto a soft agar medium (1% Bactodributon, 0.5% yeast extract, 05% NaCρ) on a 1B plate (1% Bactodributon, 0.5% yeast extract, 05% NaCρ).
10 nHHg5O, 0.4% maltose, 3 d of ampicillin/LB) was added to form a phage plaque.

3) Immunoscreening: After incubating the above plate at 42° C. for 3 to 4 hours, a nitrocellulose strip soaked with 30 mH isopropyl-β-thia galactopyranoside was placed on top of the plaque.
Incubate overnight at 7°C.

T T B S (10 nHTris-1
1cj) pH 8, (1,150nHMacl, 0.
The cells were washed twice with 05% Tween 20) and treated with 7Cl for 1 hour with 20% calf serum (C8)/TTBS. Next, human anti-epidermal basement membrane monoclonal antibody 10
After reacting with %C3-TTBS for 1 hour,
Washed twice. Next, alkaline phosphatase-labeled anti-human IQ (G+M+A>goat antiserum (Zymed,
San rrancisco.

After reacting with CA) for 30 minutes, the cells were washed three times with TTBS.

0.33■/7 for chromogenic substrate! Nitro blue tetrazolium, 0.17/1d5-bromo-4-chloro-3-
After giving indolyl phosphate and incubating for 10 to 30 minutes, the cells were washed with water.

Clones that developed color were considered to be expressing hydrophilic ml antigen (Fig. 3). In the figure, the left figure is clone 5
F was immunostained using a pile epidermal basement membrane monoclonal antibody, and the right image (control) was immunostained using normal human serum.

4) Determination of base sequence: The selected clones were determined using the Davis method (B
asic methods in molecules
Phage DNA was prepared according to the following method (Biology, 143 (1986)). After digesting this with EC0RI and purifying the resulting 3 to 2 kb DNA fragment by agarose gel electrophoresis, plasmid I)U
Subcloning was performed into the ECoRl site of O3.

The resulting recombinant plasmid IIBPMI was transformed with restriction protein E
coRI, PstI, XhoI, Hindm, T
aq■, XhoI, Haell, BglI[,5SI
II, 5tuI, 5phI, EC0RV and cloning vector M13+ap18 and mp19
(manufactured by Takara). Using this, E. coli JM103 or JM107 was transformed to form plaques (calcium chloride method). The single plaque was again infected with JM103 or JM107 culture solution, and single-stranded DNA was recovered from the resulting phage particles.

To prepare single-stranded DNA, bacterial cells were precipitated from the culture solution, polyethylene glycol (8w6000) was added to the supernatant, and phages were precipitated by centrifugation.

I'? The obtained fuji particles were treated with phenol,
Heavy chain DNAΔ was obtained by ethanol precipitation. Using this, the base sequence was determined by the M13 dideo:1-dide method.

5) Search for 'A-bread reading frame 2) The open reading frame in l) NA obtained as above was searched.

This search was performed by using a computer to confirm the existence of [terminal]ton for each frame.

As a result, an open reading frame encoding 997 amino acids was confirmed.

6) Preparation of the C-terminal region of the mouse 120 kD varicella-like antigen protein: From phage particles containing cDNA corresponding to the 120 kD protein in the gene, the oav r s method [Bas
icmethods in molecular bi
143 (1986)], Phage D
NA was prepared. This was digested with EcoRI, and the resulting 3 to 2 kb DNA fragment was purified by agarose gel electrophoresis.
After ribbing at the Rl site, E. coli pop213
6 was transformed.

1 g of this bacterium was added to B medium (1% bactodribusin, 0
The cells were cultured with shaking in 5% yeast extract, 1% NaC41) in the presence of ampicillin (501/d>) overnight at 37°C, and centrifuged (6000xo, 15 minutes) to obtain bacterial cells.

The bacterial cells were soaked in buffer ■ (50 nHTris-tlci p
It was suspended in H8, 0°, 25% 5ucrose, 1 mH [DT8) 10-, and 201 Rg of lysodeme was added.

After leaving the mixture at O'C for 20 minutes, PMSF was added to a concentration of 1 mM, and the mixture was subjected to ultrasound treatment on ice for 15 minutes. Centrifuge this (2
0000 x g for 30 minutes), and buffer solution 5i was added to the resulting precipitate to suspend it. Similarly super? After A-wave treatment and centrifugation, precipitate! ! Throat sap (20mHTris-I
IC4) ptl 7.5.2n+HEDTA, 0
．． After suspending in 2HNaCρ, 1% dioxycholate, 1% NP40) 51d and stirring well,
It was centrifuged (5oooxo, 10 minutes). Sedimentation! l
! ! i-liquid ■ (20mHTris-tlci) pH7
, 5,98 urea).

7) Identification of the protein recognized by autoantibodies contained in the serum of a patient with 1fi adipose disease: A skin section of a normal human forearm was diluted with buffer solution (iomN phosphate a).
ll 7.0.0.13HNaCl, 2mM [DT
A, 2mM PH3F) was left at 4°C for 2 days, and the epidermis was removed. This epidermis was soaked in a buffer solution (10IIIHTr).
is-11cj pit 6.8 1% 5OS5%
The mixture was crushed using a glass homogenizer while cooling in β-mercaptoethanol (2mM PHSF, 5m3/ρ each (leupeptin, antipain, chymostatin, pepstatin)) on ice. This crushed material was boiled for 5 minutes and centrifuged to obtain a supernatant.

This is U. K. The method of Laemmli et al. [Nat
It. ure, 227680-685 (1970)]. The method of Towbin et al. [Proc. N
atl. Acad. Sci. (U.S.^.),
4350-4354 (1979)], We
Stern blotting analysis was performed. Patient serum was used as the primary antibody, and peroxidase-labeled anti-human IqG rabbit serum was used as the secondary antibody (Figure 6).

8) Recognition of the 120 kD portion on the C-terminal side of the 230k[) antigen protein by an antibody contained in the serum of a 11N patient with erythematous disease: Except that the sample to be electrophoresed was obtained in 6), 7)
(Figure 6).

9) Human 1-L-Tuclor 1 antibody MAb-5E 120
Gene construction for determination of recognition site for kD protein Expression vector p△-'Tr
p was reported by Urakami Ken et al. [Cancer Society, 1987].

The present invention further proposes that the 5'-side nucleotide sequence of CM Butsu of Escherichia coli trib]-71in-synthesized M chord: CG8TATGAAAG
CrATC 1'CGTTCTGTAT8CT T T
To CG■GAAGCAAGΔC^AAGGTTCT
To C Ding GGACCG Ding GACGGAGTTTTCC^GA
GACCTGGCACTGCCTCCTCTCGAG GAGAGCTCTTAA was synthesized and ligated to pAT-Trp digested with C1aI (pAT-Trp-TrpE). Seed fragments were also used.

The gene corresponding to the 120k D protein was extracted with two restriction enzymes (XhoI, l-1indm, 5acI
and Hind m, XbaI and EcoRI, Xb
al and BclI.

The fragments were digested with Xbal and B(III>), and each fragment was purified by agarose electrophoresis.

For example, the fragment digested with
After digestion with [I[, ligation was performed. I would like to connect other fragments in the same way (Fig. 5, Part 2).

10) Expression of 120 kD antigenic protein fragment and presence or absence of its antigenicity: After transforming Escherichia coli H8101 with the expression vector constructed in 9), it was transformed into LB medium Fampicillin 50I19/
-) was cultured overnight at 37°C. This 20 years old M9C
A medium (6.4m) 4 phosphorus MpH 7.4.0.05%.

NaC1! , 0.1% NHci! , 2mHH
g5O, 0.2% glucose, 0.1 mHCaCj!
, 0.2% Casamino M) 2d plus OD 60
5 was cultured until 0 became 0.3 and dissolved in ethanol.
trty/ld β indole acrylic M2O9f
After adding 1, the cells were further cultured day and night. Centrifuge these 50 pieces and add U to the precipitate obtained. 1. aellllll l
Add sample buffer prepared according to the method of L et al.
After boiling for minutes, electrophoresis was performed on a 10-20% polyacrylamide gradient gel. After electrophoresis, QinYu
The method of Xu et al. [Analytical Biochem
istry170, 19-30 (1988)] and H.
, Western bl according to the method of Towbin et al.
otting analysis was performed.

The primary antibody was MAb-5E, and the secondary antibody was alkaline phosphatase-labeled anti-human IOA and IgG.

It was an IgM rabbit antibody (Figure 7).

[Effect of the invention] The present invention produces effects as described below.

In the present invention, we have isolated a DNA encoding a polypeptide containing an antibody recognition site for a mouse-derived Aquarium spp. antigen, and clarified its amino acid sequence.

It was found that the sequence had a high homology of 77.0% with the known amino acid sequence of the human-derived polypeptide.

In addition, it was found that the antibody recognition site is a polypeptide consisting of 114 amino acids, and this polypeptide]
- By culturing Escherichia coli transformed with an expression vector that incorporates a DNA containing a base sequence containing
It is possible to produce human products with specific antigens that can be used to diagnose the disease, and which can be distinguished from those of A. tortoiseus and normal people.

In this way, by expressing a polypeptide containing the antibody recognition site for the Affi antigen derived from Ma. By experimentally causing hydrophilia in mice, it is possible to establish an animal model for the disease, which is thought to be useful in the development of Ft7 therapy.

[Brief explanation of drawings]

Figure 1 shows the mouse-derived hydrophiloid M1! The base sequence of the DN which contains the polypeptide containing the antibody recognition site for the antigen and the amino acid sequence deduced from it are shown. Figure 2 shows mouse and human l-(J, lt, 5tanle
A comparison of the amino acid sequences of the polypeptide containing the antibody recognition site of the aquatic hominin 111N antigen (see the above-mentioned publication by Y. et al.) is shown. FIG. 3 is a photograph showing E. coli Y1090 infected with λ phage, immobilized on a nitrocellulose membrane and immunostained. Figure 4 shows the EC of plasmid pUc9 containing DNA encoding a polypeptide containing the human anti-epidermal basal IIQ monoclonal antibody recognition site of mouse-derived varicella-like turtle antigen.
This is an expression vector inserted into the oRI site. Figure 5 is a diagram (Part 1) showing a gene fragment encoding a 120kD protein to be expressed to determine the recognition site of a human anti-epidermal basement membrane monoclonal antibody, and an expression vector for expressing the gene fragment. It is a figure (part 2) showing. In the figure, 8rTI D indicates an ampicillin resistance gene, and when a restriction enzyme is placed in parentheses, it means that the gene cannot be digested by that restriction enzyme. The upper part of Figure 6 shows SDS/
The reaction between β-mercaptoethanol-extracted protein and serum. The figure below shows the reaction between serum and a 120 kD protein expressed from an expression vector.
The photograph does not show the results of analysis by ttting. As for the serum, 1-11 is a serum from a patient with hydrophilic atrophy, 12 is a serum from a patient with macroacne, and 13 is a serum from a normal person. Figure 7 shows epitope mapping of a 120 kD protein using a human anti-epidermal basement membrane monoclonal antibody, and is a photograph showing the results of electrophoresis of the tube-sleeve extrudate obtained by expressing the expression vector and analysis using Western blottir + O. be. FIG. 8 shows the base sequence of the DNA encoding the mouse-derived hydrophilic Jt [antibody recognition site (polypeptide) of the antigen]. In the figure, n is O or 1. FIG. 9 shows the amino acid sequence of the antibody recognition site (polypeptide) of the mouse-derived hydrophilic 11N antigen. Engraving of drawings (no changes in content) 1st drawing (Zono 1) alu alu Leu Leu Ala
Gln Arg Arg Glu Vllll
Glu ASII Leu L7$ uln
iV5 generation J1 person J: Nol: “l, ; 翳山BUKA 11th edition (Part 2) Figure 1 (f of 4) Lys Tyr Gln Glu Gly L
eu Thr Thr Thr Leu Thr Glu
Leu Ala Asp rI'Ie 11th Sword Example 3) Notebook (¥5) Asn Leu Tyr Tyr Ser
Ser Gin EndGCCGTG TCG
GCT GCA TCCTGCCAT CTG
TTCAGA GCA GAT GGT
GTCTGCTAA ATG TGCAGCCTA
GGA ACT GTG TCA CCA
ACT CGA AGCGCTGTT AA
T TTCTTG TAG GCT AGA
AAT AGCTCCCTG CTCAGA
AAA CACGGCCAT TCT TAA
GTT GAA AGCAGA AAA C
GA TTG GCT GCCCCT GAC
AAA GTA ACT TAA AAA
AAA AAA TAT CTT ACT
AAA ATA AATl-4, b ~” J-J I Chair Po from Ward. Pri , / 1υ 11 Ptrp 0trp ba 1 Amp' [A T G 1n GTA ・TTT GG GG GA A, G TT GG AA AO AC GT TC AT CT Ding CC CT AT TC GT AG AA AG lol GT AT TC AG AT AA GG AA TC CT TC TT CA AG CA AC AC CT AG AA GA GG AT 8 times TC GG TC GG TC CT TC CT GG TC TT AT GG GG TTT GG TC AA TC CT GG TC A G TT AG GG AA AC AT GTA GG CA AG AC TC AG AG TT AT TC CT AT TC GTA TC AA AT GT TT △TA GTA AA AT GG TC GA AG AA AG AA AC CT CT GT TT AG AC AA GG yS S p eu S n VS L”/S eu la eu er hr Yr hr 1 n lu IV yr th a yS し eu rQ eu hr ■a hr hr he yr y r p he IV et し ys he hr eu wa he pr. S n ly la SD し eu VS he is su hr sn a eu a fig yS he S p he rO yS 5D pr.

Claims (10)

[Claims] (1) The following amino acid sequence containing the antibody recognition site of the mouse-derived bullous pemphigoid antigen: [Amino acid sequence is available] [Amino acid sequence is available] DNA containing a base sequence encoding a polypeptide consisting of:
A. (2) The DNA according to claim 1, wherein the base sequence encoding the polypeptide is [there is an amino acid sequence] [there is an amino acid sequence] [there is an amino acid sequence]. (3) Claim 1, wherein the antibody recognition site is a polypeptide consisting of the following amino acid sequence: [There is an amino acid sequence]
DNA described in. (4) Claim 1 or 3, wherein the antibody is a human anti-epidermal basement membrane monoclonal antibody that recognizes the mouse-derived bullous pemphigoid FAIR antigen or an autoantibody in the serum of a bullous pemphigoid patient. DNA described in. (5) The DNA according to claim 3, wherein the base sequence encoding the polypeptide consists of the following sequence: [There is an amino acid sequence] and n is 0 or 1. (6) A DNA comprising a base sequence encoding an antibody recognition site of the polypeptide encoded by the DNA according to claim 1 and optionally having a codon encoding methionine at the 5' end. expression vector. (7) The expression vector according to claim 6, wherein the expression vector is a plasmid. (8) The expression vector according to claim 7, which is selected from the group consisting of plasmids pXE, pXB, pXS and pBP. (9) E. coli transformed with the expression vector according to claim 6. (10)\Escherichia\\coli\HB
101 (pXE) (Choken Bacteria No. 11297), ¥E
scherichia￥￥coli￥HB101(pX
B) (Microtechnical Research Institute No. 11295), ￥Escheri
chia￥￥coli￥HB101(pXS) (Choken Bacterial Serial No. 11294), and ￥Esherichia￥
The E. coli according to claim 9, which is selected from the group consisting of \coli\Pop2136 (pBPI) (Feikoken Bibori No. 11292).