JP4297519B2 - Fusion protein recognized by pemphigus vulgaris autoantibody, therapeutic agent, therapeutic instrument, and diagnostic agent for pemphigus vulgaris - Google Patents

Description

【００４０】
別に正常人の血清２００例を測定し、そのインデックス値の平均値＋３×標準偏差を正常範囲として、これを越えた場合ＰＶ抗体を持っていると判断する。
かかる診断法によれば、従来のように正常表皮切片を基質とする必要がないため簡便性、客観性に優れるという効果が得られ、また、特異性及び感度に優れるため尋常性天疱瘡の早期発見に役立つという効果も得られる。
【００４１】
また、ＰＶ抗体の濃度を測定する方法としては、高力価の患者血清を標準品として倍々希釈により検量線を作製し、診断対象となる患者血清中のＰＶ抗体の濃度を求めることもできる。このようにしてＰＶ抗体の濃度を測定することにより治療効果の判定、患者の臨床症状の推移を追うことができるという効果が得られる。
【００４２】
尚、PVAg-IgGを含む溶液を抗原溶液に用いると、ペルオキシダーゼ標識抗体はＰＶ抗体に結合するばかりでなく、PVAg-IgGのIgG部位にも結合してしまうため、ペルオキシダーゼ標識抗体を用いて上記ELISA法により診断することはできない。
【００４３】
【配列表】

【図面の簡単な説明】
【図１】 動物細胞（COS7）によって得られたPVAg-IgGと各種血清のイムノブロット解析の結果を表す説明図である。
【図２】 昆虫細胞（SF9）によって得られたPVAg-IgGと各種血清のイムノブロット解析の結果を表す説明図である。
【図３】 PVAg-IgGの分子構造を表す説明図である。[0001]
[Industrial application fields]
The present invention relates to a fusion protein recognized by a pemphigus vulgaris autoantibody, and a therapeutic agent, therapeutic instrument, diagnostic agent and diagnostic method for pemphigus vulgaris.
[0002]
[Prior art]
Pemphigus vulgaris (hereinafter referred to as PV) is a bullous autoimmune disease that causes blisters on the skin and mucous membranes and is sometimes fatal. Clinically, the initial blisters of PV are flaccid blisters, whose blisters are easily breached and exhibit a large erosion surface. The erosion surface is exudative and prone to bleeding. In severe cases, fluid loss from the erosion surface, secondary infection, and can be fatal. When the healthy part of a PV patient is rubbed with a finger, a so-called Nicolskie phenomenon is observed, in which the upper layer of the epidermis slips. In the histopathological image of the lesioned part, cell-cell connections of epidermal cells are indicated, and free epidermal cells are observed.
[0003]
Moreover, when the lesioned skin is examined by the fluorescent antibody method, IgG deposition is observed on the surface of the epidermal cell membrane. Furthermore, IgG binding to the surface of epidermal cell membrane is detected in the blood of PV patients.
When IgG in this patient's blood is injected into a newborn mouse, a blister having a tissue image characteristic of PV is induced in the mouse skin. Moreover, when the skin is organ-cultured in a state where PV patient IgG is placed in the culture solution, the skin is melted in the same way as PV, and thus IgG found in PV patients plays a pathogenic role in blister formation. It was thought to carry.
[0004]
Amaya et al. Screened a cDNA expression library prepared from normal human cultured epidermal cells using an anti-13OkD pemphigus antigen protein-recognizing IgG affinity-purified from sera of PV patients to obtain clones that react specifically. The cDNA sequence of pemphigus vulgaris antigen protein was determined (Cell, 1991, 67, 869-77).
[0005]
It was discovered that a protein consisting of amino acids based on the DNA sequence is a new type of cadherin molecule that is expressed in the stratified squamous epithelium. Similarly, the cDNA sequences of deciduous pemphigus and Brazilian pemphigus, which are autoimmune bullous diseases with other types of anti-epidermal cell membrane antibodies, have also been determined (European Journal of Cell Biology 1990, 53, 10 -12, Amagaya et al.).
[0006]
Thus, autoantibodies against pemphigus vulgaris antigen protein, a kind of cell adhesion factor force doherin in the epithelium, are produced in PV patients. When the antibody reacts with pemphigus vulgaris antigen protein, it inhibits the function of pemphigus vulgaris antigen protein as an adhesion factor, secondarily activates plasmin, complement, etc., leading to the release of the epidermis (Journal of Investigative Dermatology 1995, 104, 146-152, Amagaya).
[0007]
At present, the trial of treatment for PV patients is only symptomatic treatment that expects to reduce the primary symptoms by steroids and immunosuppressants, and there are strong side effects, there is nothing fundamental, and after considering the pathology of the disease There is no specific treatment.
On the other hand, pemphigus is diagnosed on the basis of clinical findings by specialists, histopathological findings of lesions, and immunohistologic findings by fluorescence antibody method using normal serum slices as a substrate using patient serum. Yes. These diagnostic methods lack simplicity and objectivity.
[0008]
Recently, pemphigus vulgaris antigen protein has been artificially synthesized by genetic recombination using an E. coli expression system, and attempts have been made for antigenic diagnostic agents using this protein, but the positive rate is about 50-60%. (Journal of Clinical Investigation, 1992, 90, 919-926, Amagaya et al.) And not practical.
[0009]
[Problems to be solved by the invention]
As described above, the current treatment with immunosuppressants has a problem that it suppresses all immune reactions uniformly, has strong side effects, and has a primary effect.
[0010]
Therefore, the present invention has been made in view of the problem of uniformly suppressing all conventional immune responses, and selectively suppresses only immune responses related to pemphigus vulgaris. That is, the pemphigus vulgaris autoantibody, which is the causative substance, is removed or autoantibody-producing cells are destroyed.
[0011]
In addition, pemphigus vulgaris, which has not had an effective diagnostic method in the past, has been a subject of development of a practical diagnostic agent that is excellent in specificity and sensitivity, is useful for early diagnosis, and correlates with disease state. Therefore, the present invention provides a fusion protein that is recognized by a pemphigus vulgaris autoantibody, provides a therapeutic agent and a therapeutic instrument for pemphigus vulgaris with less adverse effects, and is effective for pemphigus vulgaris It is an object to provide a diagnostic agent and a diagnostic method.
[0012]
[Means for solving the problems, functions and effects of the invention]
As a result of intensive studies in view of the above problems, the present inventors have completed the following invention.
The fusion protein of claim 1 is a fusion protein that is recognized by a pemphigus vulgaris patient autoantibody, and is located at the C-terminus of the protein represented by SEQ ID NO: 1, which is the extracellular region of pemphigus vulgaris antigen protein, The constant region of IgG is bound via the hinge part.
[0013]
Since the pemphigus vulgaris antigen protein is a transmembrane protein and autoantibodies cannot pass through the cell membrane in vivo, the present inventors consider the region recognized by the autoantibodies to be the extracellular region, and this extracellular region ( A gene encoding only SEQ ID NO: 1) was prepared (hereinafter, the extracellular region is simply referred to as PVAg unless otherwise specified). Since the amino acid sequence of PVAg, which is an extracellular region, was used in this way, the fusion protein expressed when the gene encoding the fusion protein of claim 1 was introduced into animal cells or insect cells to express the fusion protein. Has the effect of being reliably discharged outside the cell without being trapped inside the cell. In addition, since the fusion protein of claim 1 binds the hinge region of the IgG constant region to PVAg, the fusion protein can be produced stably and the higher order structure of PVAg is equivalent to that of the natural one. Furthermore, in the fusion protein of claim 1, since IgG was bound to the cleavage site on the C-terminal side, the extracellular protein that was cleaved on the way from the N-terminus to the C-terminus of the original protein (pemphigus vulgaris antigen protein) The region has the effect of being recognized by autoantibodies more reliably. Furthermore, since IgG was present in the fusion protein of claim 1, the presence or absence of expression after cell introduction can be easily detected by using a commercially available anti-IgG antibody, and in the purification of the fusion protein after expression. By performing Sepharose column chromatography having protein A having affinity for IgG as a ligand, there is an effect that purification can be easily performed. In addition, IgG used as the raw material of the fusion protein of claim 1 is not limited by the kind and animal species.
[0015]
As a method for producing the fusion protein of claim 1 (hereinafter referred to as the fusion protein of the present invention), for example, a gene encoding the fusion protein of the present invention is introduced into animal cells or insect cells to express the fusion protein. A method of collecting the culture supernatant and then purifying it by appropriate column chromatography can be mentioned. In this case, unlike an expression system using E. coli as a host, it is preferable to use an animal cell such as COS7 cell or an expression system using baculovirus as a vector and an insect cell as a host.
[0016]
The therapeutic agent for pemphigus vulgaris according to claim 2 contains the fusion protein of the present invention as an active ingredient. For example, selective destruction of autoantibody-producing cells can be performed by administering a conjugate of the fusion protein of the present invention and a toxin such as cholera toxin or mitomycin C covalently bound to a pemphigus vulgaris patient. Therefore, the conjugate can be used as a therapeutic agent for pemphigus vulgaris. At this time, the results of an acute toxicity experiment using mice showed that 50% lethal toxicity was observed in the administration group injected with 1000 mg / kg of the fusion protein into the peritoneal cavity. I couldn't. This fusion protein is a tissue-binding protein originally present in the living body and has extremely low toxicity. This LD ₅₀ value is about 100000 times the maximum dose for treatment per adult and is not a problem. In addition, even when a toxin such as cholera toxin or mitomycin C is bound to the fusion protein of the present invention, LD ₅₀ is 10 mg / kg, but this value is also about 10 times the maximum dose in adult daily treatment. There is no problem. Although the daily dose of such a therapeutic agent for pemphigus vulgaris varies depending on the administration method and the disease state, the usual dose is 3 × 10 ⁻⁹ M to 3 × 10 ^{−10 in} the amount of the fusion protein of the present invention. It is about M, and the administration route is preferably mainly intravenous injection or intramuscular injection. Specific examples of preparations include those containing 3 × 10 ⁻³ M to 3 × 10 ⁻⁶ M in physiological saline or citrate buffer. In addition to this, as other optional components, a pharmaceutically acceptable stabilizer, disintegrant, solubilizing agent, and the like may be added as appropriate.
[0017]
The therapeutic device for pemphigus vulgaris according to claim 3 has an adsorbent in which the fusion protein of the present invention is fixed to a carrier. For example, by immobilizing the fusion protein of the present invention on a water-insoluble carrier to obtain an antigen that selectively adsorbs pemphigus vulgaris patient IgG, and selectively removing pemphigus autoantibodies by plasma exchange, Can be used to treat pemphigus. Examples of the carrier used include organic polymers such as agarose, cellulose, polyacrylamide, polystyrene, and acrylate, natural organic polymers derived from living bodies such as collagen and chitosan, activated carbon, ceramics, and the like. An inorganic substance is exemplified, and various forms such as a flat form and a particulate form can be used as the form, but those capable of taking a large surface area are preferable. Immobilization of the PVAg-IgG fusion protein and the carrier is performed by a covalent bond. For example, it is carried out by directly bonding in an aqueous solution to a reactive functional group such as an epoxy group or an aldehyde group that the carrier has on its surface. Also, a functional group such as an amino group in the case of using a support having a surface, adding a compound having a reactive multifunctional dialdehyde or diepoxy compounds or the like in a mixed aqueous solution of PVAg-IgG fusion protein and carrier, It can also be carried out by reacting.
[0018]
The diagnostic agent for pemphigus vulgaris according to claim 4 uses the fusion protein of the present invention as an antigen for immunodiagnosis. For example, the fusion protein of the present invention can be used as an antigen protein in immunodiagnostic methods such as Western blotting, RIA and EIA, and can measure autoantibodies in patient blood. As a result, it is possible to provide a diagnostic agent that is excellent in specificity and sensitivity and useful for early diagnosis in pemphigus vulgaris for which there has been no effective diagnostic agent.
[0020]
【Example】
Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, this invention is not limited to the following Example at all, and as long as it belongs to the technical scope of this invention, it cannot be overemphasized that it can implement with a various aspect.
[Example 1] Fusion protein in which IgG is bound to PVAg (PVAg-IgG)
[1-1] Expression of PVAg-IgG by animal cells COS7 cells
PcDNA1-PVAg-IgG expression plasmid DNA containing PVAg-IgG protein expression region (signal peptide region-procedure region-extracellular protein region (SEQ ID NO: 1) -human IgG hinge region-human IgG CH2 region-human IgG CH3 region) (PCDNA1 is a trade name of Invitrogen). A method for preparing pcDNA1-PVAg-IgG expression plasmid DNA is described below.
[0021]
Basically, the required cDNA was amplified by PCR (polymerase chain reaction) using synthetic oligonucleotides complementary to sequences on both sides of the cDNA to be amplified. Oligonucleotides were designed to create restriction enzyme cleavage sites or added peptide coding regions. PCR was repeated 20 to 25 cycles of 94 ° C for 1 minute, 55 ° C for 2 minutes, and 72 ° C for 3 minutes.
[0022]
In order to construct an expression vector for COS7 cells, first, a cDNA encoding PVAg, a mammalian expression vector containing a chimeric protein consisting of the cytoplasmic region of PVAg and mouse E cadherin, pcDNA1-PVECIII (Journal of Investigative Dermatology) 1994, volume 102, pages 402-408, Amagaya et al.). The T7 primer in the vector sequence was used as the 5 ′ primer, and the following sequence was used as the 3 ′ primer.
[0023]
5'-CCTGCTCGAGCCTCCCTGAGTGCGGCCT-3 '(including antisense sequence and XhoI cleavage site immediately upstream of the end of the EC5 region of PVAg)
This PCR product had a 5 ′ non-coding region and a coding region covering the signal peptide, the sequence and PVAg (nucleotide 1-1929).
[0024]
On the other hand, cDNA encoding the constant region of human IgG1 was produced by PCR amplification on pTJ5 (Journal of Biotechnology 1988, Vol. 8, pp. 141-148, Nakamura et al.). Primers having the following sequences were used.
5 'primer: 5'-CCTGCTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA-3' (including all hinge region and XhoI site)
3 'primer: 5'-CCATCTAGATCATTTACCCGGGGACAG-3' (encodes antisense sequence and XhoI site immediately above the stop codon)
Expression of eukaryotes having cytomegalovirus (CMV) promoter digested with NotI-XhoI and XhoI-XbaI and cut with NotI-XbaI, respectively. The vector was combined with pcDNA1 / Amp (manufactured by Invitrogen). This was named pcDNA1-PVAg-IgG.
[0025]
This pcDNA1-PVAg-IgG expression plasmid DNA was temporarily transfected into COS7 cells using liposomes (trade name: Lipofectin, Gibco). That is, a plate in which COS7 cells having a diameter of 1OOmm were cultured was transfected with 10 μg of expression plasmid DNA, a mixture of serum-free DMEM 5 ml and lipofectin 5Oμ1, and incubated at 37 ° C. for 4 hours. 5 ml of DMEM containing 2% fetal bovine serum was added and incubated overnight.
[0026]
The next day, the culture supernatant was removed and replaced with 6 ml of DMEM without serum. Incubation was continued at 37 ° C. for 3 days, and the culture supernatant was collected and replaced with 6 ml of serum-free DMEM. After further incubation at 37 ° C. for 3 days, the supernatant was collected and the cells were discarded.
[0027]
The collected supernatant was centrifuged to remove non-adherent cells and debris, and two portions were collected in one place and stored at −70 ° C. until use. It was found by measurement with a protein measurement kit (protein assay kit, manufactured by BioRad) that, on average, the supernatant contained about 0.1 to 0.5 μg / ml of PVAg-IgG protein.
[0028]
FIG. 3 is an explanatory diagram showing the molecular structure of PVAg-IgG. PVAg-IgG includes a signal peptide (S), a prosequence (P), PVAg (EC1 to EC5), and a constant region of human IgG1 (including hinge (H), CH2, and CH3 regions). The lower part of FIG. 1 shows the amino acid sequence around the fusion position, but one leucine was introduced to construct a new restriction enzyme position.
[1-2] Expression of PVAg-IgG by insect cell Sf9
Similar to pcDNA-PVAg-IgG, the DNA of pEVmod-PVAg-IgG, a plasmid containing the PVAg-IgG protein expression region (pEVmod is a trade name of Pharmingen), and baculogold (Pharmingen) The recombinant baculovirus producing recombinant PVAg-IgG-Sf9 was obtained by co-transfecting both of the DNA of the product (manufactured) into Sf9 insect cells.
[0029]
In other words, approximately 3 × 10 ⁶ Sf9 cells are placed in a culture dish with a diameter of ⁶ Omm together with 1O ml of Grace's insect cell medium containing 1% fetal calf serum by a conventional method, and 27 ° C. so that the cells adhere. Incubated for 15 minutes.
Meanwhile, 2 μg of pEVmod-PVAg-IgG DNA, 0.5 μg of baculogold DNA, and 3O μl of liposome for DNA introduction (trade name: Lipofectin, Gibco) were dissolved in 1.5 ml of Grace's insect cell medium without serum at room temperature. A DNA-liposome mixture was prepared by incubation for 15 minutes.
[0030]
1.5 ml of Grace's medium containing the DNA-liposome mixture prepared previously and without serum was gradually added to the Sf9 cells incubated on the culture dish, and the transfected cells were incubated at 27 ° C. for 4 hours. After incubation, the cells were washed once with Grace's medium without serum and replaced with Grace's medium with 10% fetal calf serum and incubated at 27 ° C for 4 days.
[0031]
Thereafter, the culture supernatant was collected, infected with a semi-confluent Sf9 cell having a diameter of 1 OO mm, and incubated for 3 to 5 days. This operation was repeated three times to obtain a supernatant containing baculovirus that produces high-titer PVAg-IgG-Sf9. The supernatant obtained by culturing Sf9 cells infected with high-titer recombinant baculovirus for 5 days was centrifuged to remove cell debris, and then stored at -70 ° C. On average, the supernatant was found to contain about 5-10 μg / ml PVAg-IgG protein by quantification with a protein assay kit (Protein Assay Kit, Bio-Rad).
[1-3] Purification and concentration of PVAg-IgG fusion protein
To purify and concentrate the PVAg-IgG fusion protein, add 5 μl of Protein A Sepharose (Pharmacia) to 5 ml of COS7 or Sf9 cell supernatant as usual, and incubate at 4 ° C overnight and wash twice with PBS. Then, it was resuspended in 2 × SDS sample buffer (125 mM Tris-HCl pH 7.5, 2% SDS, 0.005% bromophenol blue, 2O% glycerol, 5% 2-mercaptoethanol) 5O-1OO μl.
[1-4] Confirmation of expression of PVAg-IgG fusion protein The concentrated PVAg-IgG fusion protein was separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, trade name: Immobilon). To detect PVAg-IgG fusion protein, blocking with 3% skim milk solution, PVDF membrane was 1OOO times diluted solution of goat anti-human IgG antibody (Zymed Labaratories lnc.) Conjugated with alkaline phosphatase, 1 at room temperature Incubated for hours. Thereafter, it was detected as a single band by an alkaline phosphatase coloring kit (BioRad), and its molecular weight was about 11 OkD.
[1-5] Immunoblot analysis In order to examine the reactivity of PVAg-IgG obtained by COS7 cells or Sf9 cells with the PVAg antibody in the serum of patients or healthy individuals, the PVDF membrane transcribed with PVAg-IgG was first treated with human serum. Incubate with a 5O diluted solution of mouse, then a 3O diluted solution of mouse anti-human IgG4 antibody (Oxoid), a 1000-fold dilution of goat anti-mouse antibody conjugated with alkaline phosphatase (Zymed Laboratories Inc.) The solution was incubated and the color development was examined. Blood used was 35 patients with pemphigus vulgaris (PV), 1O patients with pemphigoid (PF), 16 Brazilian patients with pemphigoid (BPF), 1O bullous pemphigoid Patient (BP), 5 healthy human sera (N). The serum of all 35 patients with pemphigus vulgaris tested positive for PVAg-IgG produced by COS7 cells, but was completely responsive to the control group of 36 patients with bullous disease or 5 healthy persons (Figure 1).
[0032]
PVAg-IgG produced with Sf9 cells also reacted positively in all 35 pemphigus patient sera, but was completely unreactive from the 36 bullous patients control group or 5 healthy individuals. It was not recognized (FIG. 2).
[1-6] Autoantibody absorption experiment by PVAg using newborn mouse Next, autoantibody absorption experiment by PVAg using newborn mouse was performed. The serum of pemphigus patients was incubated with 5 ml of Sf9 cell supernatant infected with PVAg-IgG recombinant baculovirus for 4 hours at room temperature, and centrifuged at 4 ° C. and 13000 × g for 3 minutes to remove precipitates. Thereafter, the IgG fraction was precipitated with 40% ammonium sulfate, dialyzed against a PBS-Ca solution, and concentrated to 1 ml (1 / O of the original volume) with a small concentrator CentriconlOO (Amicon). These enriched IgGs (100-15Oμ1 per mouse) can be found in the literature (N. Engl. J. Med., 1982, 3O6, pages 1189-1196, J. Clin. Lnvest., 1992, 9O, 919. -Page 926) was injected subcutaneously into newborn BALB / c mice (within 24 hours of birth). Newborn mice were biopsied for 18-24 hours after injection.
[0033]
As a control experiment, in the above-mentioned experimental system, Sf9 cells infected with PVAg-IgG recombinant baculovirus were replaced with Sf9 cells that were not infected with baculovirus. In the system in which erosion was observed but the supernatant of Sf9 cells infected with PVAg-IgG recombinant baculovirus was incubated, blister formation was not observed macroscopically or histopathologically. It was revealed that the pathogenic activity that induces blistering was lost.
[1-7] Treatment device for pemphigus vulgaris Based on the above findings, a treatment device for pemphigus was prepared. A spherical carrier (trade name: EAH Sepharose 4B, average particle size: 45 to 165 μm) covalently bonded to the surface via PVAg-IgG in a cylindrical polycarbonate housing with a liquid inlet and outlet at the upper and lower ends, respectively. (Manufactured by Pharmacia), and a 150 mesh polyester filter was fixed to each of the inlet and outlet.
[0034]
In order to confirm that this treatment device functions effectively, 1Oml of pemphigus patient serum was infused at a flow rate of about 1 ml / min using a peristaltic pump. Serum flowing out from the outlet was collected and immunoblot analysis was performed to check for the presence of autoantibodies. However, the presence of these antibodies could not be confirmed at all, and it is clear that this treatment device effectively absorbs pemphigus patients autoantibodies. became.
[0035]
Therefore, according to this treatment device, when using conventional immunosuppressive agents, all immune responses were suppressed and the side effects were strong, whereas only immune responses related to pemphigus vulgaris were selected. Therefore, the side effect can be reduced.
[ Experimental example 2] Fusion protein in which His tag is bound to PVAg (PVAg-His)
[2-1] Construction of PVAg-His expression plasmid Prepare primers encoding Xho1 cleavage site and E tag and primers encoding His tag and Kpn1 cleavage site as follows. We constructed a cDNA encoding.
[0036]
[Primers encoding Xho1 cleavage site and E tag]
5 'primer (DN516) GCCCTCGAGGGTGCGCCGGTGCCGTATA
[Primers encoding His tag and Kpn1 cleavage sites]
3 'primer (DN517) CGGGGTACCTCAATGATGATGATGATGGCC
PCR was treated at 94 ° C for 1 minute, then repeated 20 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, treated at 72 ° C for 7 minutes, chloroform was added and ethanol was added to precipitate the PCR product did. These PCR products were treated with restriction enzymes Xho1 and Kpn1 at 37 ° C for 2 hours, and then the DNA encoding the desired His tag and E tag was collected by in-gel electrophoresis and purified with QIAEX kit (Qiagen). did. The purified DNA was digested with restriction enzymes Xho1 and Kpn1 and ligated with pEVmod-PVAg-IgG excluding the region encoding IgG to prepare a PVAg-His expression plasmid pEVmod-PVAg-His. This pEVmod-PVAg-His includes a DNA sequence encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
[2-2] Expression of PVAg-His by insect cell Sf9
Recombinant PVAg-His-Sf9 is produced by co-transfecting both pEVmod-PVAg-His and baculogold (Pharmingen) DNA into Sf9 cells in the same manner as in [1-2] above. A supernatant containing baculovirus was obtained.
[2-3] Purification and concentration of PVAg-His fusion protein
To purify and concentrate the PVAg-His fusion protein, add the above culture supernatant filtered through a 0.22 μm filter to 1 ml of Ni ²⁺ -NTA-agarose (Qiagen), and add 8 ml of binding buffer (20 mM sodium phosphate). , 500 mM sodium chloride, pH 7.8) and further washed twice with a washing buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 6.3). Subsequently, elution was carried out with 0.6 ml of elution buffer (washing buffer containing 200 mM imidazole), and further elution was carried out twice with 1.2 ml of elution buffer.
[0037]
The fraction containing PVAg-His was confirmed by immunoblotting, sufficiently dialyzed against Ca-added physiological Tris-HCl buffer (TBS), and stored at 4 ° C.
The molecular structure of PVAg-His includes signal peptide (S), prosequence (P), PVAg (EC1 to EC5), and six histidines bound to EC5 of PVAg.
[2-4] Diagnosis as to whether or not the patient is a PV diagnosed pemphigus vulgaris by ELISA method can be performed as follows.
[0038]
Add 50 μl of PVAg-His solution to each well of a 96-well flat-bottom microtiter plate (manufactured by Titertech), incubate overnight at 4 ° C, remove the PVAg-His solution, and add 1% BSA, 0.005% tween20. Add 200 μl of Ca-added TBS (blocking buffer), block at room temperature for 1 hour, and wash with Ca-added TBS (wash buffer) containing 0.005% Tween-20. To this, 50 μl of patient serum diluted 100-fold with blocking buffer is added and reacted at room temperature for 1 hour. Wash 4 times with washing buffer, add 50 μl of peroxidase-labeled antibody, react at room temperature for 1 hour, wash again, then 100 μl of chromogenic substrate (0.1 mg citric acid / 0.2 containing 4 mg of orthophenylenediamine, 6 μl of 31% hydrogen peroxide) After adding M disodium phosphate solution (pH 4.8-5.0) and reacting at room temperature for 30 minutes to develop color, the reaction is stopped by adding 50 μl of 4N sulfuric acid, and the absorbance at 492 nm is measured. At this time, serum of 50 patients with pemphigus vulgaris was measured, patient serum corresponding to the average value was collected and used as standard serum, and measurement was performed for the standard serum and patient serum. Find the value.
[0039]
[Expression 1]

[0040]
Separately, 200 normal human sera were measured, and the average value of the index value + 3 × standard deviation was taken as a normal range, and when exceeding this, it is judged that the antibody has PV antibody.
According to such a diagnostic method, it is not necessary to use a normal epidermal slice as a substrate as in the prior art, so that an effect of excellent simplicity and objectivity is obtained, and because of its specificity and sensitivity, the early stage of pemphigus vulgaris It is also useful for discovery.
[0041]
As a method for measuring the concentration of PV antibody, a calibration curve can be prepared by doubling dilution using high titer patient serum as a standard product, and the concentration of PV antibody in patient serum to be diagnosed can be obtained. By measuring the concentration of the PV antibody in this way, it is possible to obtain an effect that the therapeutic effect can be determined and the clinical symptoms of the patient can be followed.
[0042]
When a solution containing PVAg-IgG is used for the antigen solution, the peroxidase-labeled antibody not only binds to the PV antibody, but also binds to the IgG site of PVAg-IgG. It cannot be diagnosed by law.
[0043]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing the results of immunoblot analysis of PVAg-IgG obtained from animal cells (COS7) and various sera.
FIG. 2 is an explanatory diagram showing the results of immunoblot analysis of PVAg-IgG obtained from insect cells (SF9) and various sera.
FIG. 3 is an explanatory diagram showing the molecular structure of PVAg-IgG.

Claims (4)

A fusion protein that is recognized by autoantibodies in patients with pemphigus vulgaris,
IgG constant region is bound to the C-terminus of the protein represented by SEQ ID NO: 1 which is the extracellular region of pemphigus vulgaris antigen protein via a hinge part,
A fusion protein characterized by expressing an animal cell or insect cell as a host. Pemphigus vulgaris therapeutic agent containing claim 1 Symbol placement of the fusion protein as an active ingredient. Pemphigus vulgaris therapeutic instrument and having an adsorbent of claim 1 Symbol placement of the fusion protein was immobilized on a support. Diagnostic agent for pemphigus vulgaris which comprises using the claim 1 Symbol placement of the fusion protein as an antigen immunodiagnostic.

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