JP4726364B2 - Method for measuring simple Helps virus antibody - Google Patents

Description

【０１３４】
表1に示す通り、2gpG51-70のMAP抗原と2gpG58-66のMAP抗原に反応性を示したHSV-1感染患者血清#1、および2gpG51-70のMAP抗原と2gpG58-66のMAP抗原とgpG2リコンビナント蛋白質抗原とに反応性を示したHSV抗原陽性健常者血清#5においては、両抗原プレート共に反応性の阻害が確認された。従って、この血清検体#1および#5は、抗HSV-2抗体を有していると考えられる。
【０１３５】
しかしながら、その他の4検体においては、反応性の阻害はみられなかった。従って、2gpG51-70のMAP抗原で陽性とされたこれらの4検体が示した反応性は、抗HSV-2抗体との反応ではなくて非特異的反応であることが確認された。
【０１３６】
また、血清中抗HSV抗体が陽性であった各検体における上記ELISAの結果を下記表2に示す。
【０１３７】
【表２】

【０１３８】
表2において、「リコンビナント」は、前記gpG2リコンビナント蛋白質抗原を用いた市販キットによる測定結果である。
【０１３９】
なお、上記(7)の反応阻害試験により抗HSV-2抗体の存在が証明された2例（#1および#5）は、表2の解析では除外している。
【０１４０】
表2に示す結果より次のことが判る。
【０１４１】
即ち、比較とする2gpG51-70のMAP抗原における陽性率は、HSV-2感染患者血清で82%(31/38)、重複感染患者血清で67%(2/3)、HSV-1感染患者血清で4%(2/55)、抗HSV抗体陽性健常者血清で4%(2/54)、また、抗HSV抗体陰性健常者血清において2%(1/41)である。
【０１４２】
一方、本発明にかかる2gpG58-66のMAP抗原における陽性率は、HSV-2感染患者血清で74%(28/38)、重複感染患者血清で33%(1/3)であった。更に、本発明MAP抗原を利用したMAP抗原ELISAでは、HSV-1感染患者血清、抗HSV抗体陽性健常者血清および抗HSV抗体陰性健常者血清においては、1例の陽性例も検出されなかった。
【０１４３】
以上の結果より、2gpG51-70のMAP抗原は、陽性率は高いものの数%の非特異的反応性を含むものであり、擬陽性反応を全く示さない本発明の2gpG58-66のMAP抗原の優れた有用性が確認された。
【０１４４】
【実施例２】
(1) HSV-1ペプチド
実施例1において見出されたgpG2の558-563番のアミノ酸配列（配列番号5）情報を基に、これに対応するHSV-1グリコプロティンG(gpG1)の配列およびその近傍配列を選択し、それらのHSV-1ペプチドとしての利用可能性を解析した。
【０１４５】
解析したgpG1の領域は、その68-85番目の領域(アミノ酸配列を配列番号7に示す、以下「1gpG68-85」という)、およびその下流に位置する92-108番目の領域、113-132番目の領域(以下「1gpG113-132」という)、126-145番目の領域並びに146-165番目の領域の5領域である。
【０１４６】
上記各領域に相当するペプチドを、実施例1に従いMAPとして合成し、実施例1-(6)と同様にしてMAP抗原ELISAにより、それらの検体に対する反応性を試験した。
【０１４７】
上記ELISAによる測定の結果、1gpG68-85のMAP抗原および1gpG113-132のMAP抗原は、HSV-1感染患者血清と抗HSV抗体陽性健常者血清に高率に反応性を示した。他の3種のMAP抗原においては、特異的な反応性は確認されなかった。
【０１４８】
上記で最も優れた反応性を示した1gpG68-85領域の配列をN末端側およびC末端側より1アミノ酸ずつ短くした25種類のペプチドについて、同様にしてMAPを合成し、これらの各MAPを阻害物質として用いて、実施例1-(7)に準じて反応阻害試験を行い、その中心的エピトープを決定した。反応阻害試験は、一次反応緩衝液にて101倍希釈した血清検体と10μg(10μl)の上記各MAPを37℃で1時間反応させた後、1gpG68-85のMAP抗原固相化プレートに添加し、37℃で1時間反応させることにより実施した。
【０１４９】
抗原抗体複合体の検出は、実施例1-(7)と同様にした。
【０１５０】
血清検体としては、1gpG68-85のMAP抗原にて反応性を示した32例の健常者血清を用いた。
【０１５１】
得られた結果を下記表3に示す。表3において上欄は反応阻害物質として用いたMAP抗原のアミノ酸配列（一文字にて表示）を示しており、下欄は血清検体との反応性(B/B0 %)を下記基準により示している。

【０１５２】
【表３】

【０１５３】
上記表3に示す結果から次のことが明らかである。
【０１５４】
即ち、N末端側削除ペプチドのMAPを反応阻害抗原として用いた場合、1gpG68-85のMAP抗原(表3中、阻害剤No. 0)に対する血清検体の反応性は、2番目のヒスチジン(H)を削除することにより32例中3例において、3番目のトレオニン(T)を削除することにより32例中19例において、それぞれ70%(B/B0)以上の回復を示した。従って、N末端側は、2番目のH迄が必要であると判断された。
【０１５５】
一方、C末端側削除ペプチドのMAPを反応阻害抗原として用いた場合、1gpG68-85のMAP抗原に対する血清検体の反応性は、4番目のグルタミン酸(E)を削除することにより32例中1例において、50%(B/B0)以上の回復を示した。また、5番目のEを削除することにより32例中8例において、反応性が50%(B/B0)以上回復し且つ32例中7例において反応性が70%(B/B0)以上回復した。従って、C末端側は、4番目のE迄が必要であると決定した。
【０１５６】
以上より、1gpG68-85領域の最短エピトープは、その69-83番目の15アミノ酸(以下「1gpG69-83」という)と決定した。
【０１５７】
1gpG69-83の反応性を確認するため、1gpG69-83のMAPを前記と同様にして合成し、このMAP抗原を用いて実施例1-(6)と同様にしてMAP抗原ELISAによる測定を行った。
【０１５８】
得られた結果を、表2に準じて下記表4に示す。
【０１５９】
【表４】

【０１６０】
表4に示される結果より、1gpG69-83のMAP抗原は、1gpG68-85のMAP抗原と同等の反応性を示した。その陽性率は、HSV-1感染患者血清で92%(23/25)、重複感染患者血清で100%(3/3)、抗HSV抗体陽性健常者血清で89%(49/55)および抗HSV抗体陰性健常者血清で0%であった。この陽性率は前記gpG1リコンビナント蛋白質抗原を用いた場合のそれよりも優れていた。
【０１６１】
以上のことより、本発明のHSV-1ペプチドおよびHSV-2ペプチドを用いることによって、抗HSV-1抗体および抗HSV-2抗体の特異的な検出系が確立され、これらは臨床上有用であると考えられる。
【０１６２】
【発明の効果】
本発明によれば、HSV-1感染患者およびHSV-2感染患者にそれぞれ特有の抗体を特異的に認識するペプチドが提供される。これらのペプチドはHSV感染の診断試薬として、特にHSV-1感染およびHSV-2感染を区別して診断できる試薬として有用である。これらのペプチドおよび診断試薬を利用すれば、HSV-1感染およびHSV-2感染の有無を、それぞれ区別して精度よく診断することができる。
【０１６３】
【配列表】

【図面の簡単な説明】
【図１】検体中の抗HSV抗体(IgG)を、HSV培養抗原を用いるヘルペスIgG-EIAキットにより測定した結果を示す図である。
【図２】検体中の抗HSV-1抗体(IgG)を、gpG1リコンビナント蛋白質抗原を用いてELISA法により測定した結果を示す図である。
【図３】検体中の抗HSV-2抗体(IgG)を、gpG2リコンビナント蛋白質抗原を用いてELISA法により測定した結果を示す図である。
【図４】実施例1の(1)で選択したファージクローンHSV-2c5およびHSV-2g60.3のそれぞれと検体中の抗HSV抗体との反応性をELISA法により測定した結果を示す図である。
【図５】実施例1の(3)に従い、HSV-1の培養抗原またはHSV-2の培養抗原を用いた反応阻害試験の結果を示す図である。
【図６】実施例1の(6)に従うMAP抗原ELISAによる測定結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring an HSV antibody for diagnosis of herpes simplex virus (hereinafter referred to as “HSV”) infection. More specifically, the present invention distinguishes between type 1 herpes simplex virus (HSV type 1, hereinafter referred to as “HSV-1”) infection and type 2 herpes simplex virus (HSV type 2, hereinafter referred to as “HSV-2”) infection. It relates to improved methods that can be made, as well as to peptide and reagent compositions utilized in this method.
[0002]
[Prior art]
HSV has type 1 (HSV-1) and type 2 (HSV-2). Among them, HSV-2 is known as a virus causing genital herpes. In recent years, an increase in sexually transmitted diseases (STD) caused by HSV-1 has been reported, and development of a method for determining the type of both viral infections has been desired.
[0003]
HSV infection can be diagnosed by detecting an anti-HSV antibody possessed by the subject. However, each of the constituent proteins of HSV-1 and HSV-2 has high homology in their amino acid sequences, and about 80% or more is retained. Therefore, no matter which virus extract protein is used as an antigen, anti-HSV- A monospecific antibody and an anti-HSV-2 specific antibody cannot be detected separately.
[0004]
Recently, a reagent for distinguishing and detecting anti-HSV-1 antibody and anti-HSV-2 antibody using glycoprotein G (gpG), which is one of the above-mentioned constituent proteins, as an antigen has been developed. However, there is an amino acid sequence common to this gpG, so that the reagent is still not sufficient in terms of its specificity and sensitivity.
[0005]
In addition, by using several types of HSV-2 specific monoclonal antibodies, a peptide that reacts with the antibody is cloned by a biopanning method using a random peptide display library, and an epitope (antigenic determinant or antigenic determinant site) is cloned. A literature report has been made that suggests that this epitope exists in the amino acid sequence of 525-587 of gpG of HSV-2 (gpG2) (A. Grabowska, et al., Jaurnal of General Virology,80, pp.1789-1798 (1999)). In this report, an ELISA system was constructed using a synthetic peptide corresponding to amino acid sequences 551 to 570 of gpG2, and an anti-HSV-2 antibody-positive serum and a negative serum confirmed by Western blotting were used. As a result of measurement, it was described that the sensitivity was 77% and the specificity was 98%.
[0006]
[Problems to be solved by the invention]
The object of the present invention is to provide anti-HSV-specific antibody-binding peptides, in particular anti-HSV-1 specific antibody-binding peptides and anti-HSV-2 that react specifically with HSV-1 patient serum and HSV-2 patient serum, respectively. It is to provide a specific antibody-binding peptide.
[0007]
Another object of the present invention is to detect and distinguish HSV-1 specific antibodies and HSV-2 specific antibodies with high sensitivity and high specificity (diagnosis by distinguishing between HSV-1 infection and HSV-2 infection). Is to provide a method).
[0008]
[Means for Solving the Problems]
The above object is achieved by the present invention having the following gist.
[0009]
That is, the present invention relates to a peptide having the amino acid sequence represented by SEQ ID NO: 1, or a peptide having an amino acid sequence in which one or more amino acids are modified by substitution, deletion or addition in the amino acid sequence, and the anti-HSV A peptide having specific reactivity with the -1 antibody is provided.
[0010]
The present invention also relates to a peptide having the amino acid sequence represented by SEQ ID NO: 2, or a peptide having an amino acid sequence in which one or more amino acids are modified by substitution, deletion or addition in the amino acid sequence, and the anti-HSV A peptide having specific reactivity with the -2 antibody is provided.
[0011]
Furthermore, the present invention relates to a diagnostic reagent composition for HSV infection comprising each of the above peptides as an active ingredient, in particular, a diagnostic reagent composition for HSV infection in which the peptide is in a multi-antigenic peptide form, and both diagnostic reagents. A reagent composition for distinguishing and diagnosing HSV-1 infection and HSV-2 infection comprising the composition is provided.
[0012]
In addition, the present invention is a method for distinguishing between HSV-1 infection and HSV-2 infection in a specimen and diagnosing both peptides (a peptide having specific reactivity with an anti-HSV-1 antibody and an anti-HSV-1 antibody). A peptide having specific reactivity with the -2 antibody) and contacting the specimen to detect the presence or absence of anti-HSV-1 antibody and anti-HSV-2 antibody in the specimen.
[0013]
Indications by abbreviations such as amino acids, peptides, base sequences, and nucleic acids in this specification are defined by IUPAC, IUB regulations, “Guidelines for the creation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and Follow convention symbols in the field.
[0014]
In addition, the DNA and amino acid sequences of gpG of HSV-1 and HSV-2 are known, and the amino acid sequence numbers in this specification shall follow this known sequence (McGeoch, et al., J. Mol. Biol., (1985), 181: 1-13; McGeoch, et al., J. Gen. Virol., (1987), 68: 19-38).
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The peptide of the present invention is characterized in that it reacts only with either an anti-HSV-1 antibody of an HSV-1 infected patient or an anti-HSV-2 antibody of an HSV-2 patient and does not react with the other. Therefore, by using these peptides, it is possible to specifically detect the anti-HSV-1 antibody and anti-HSV-2 antibody in the specimen, and whether the specimen is infected with HSV-1 or not. Can be diagnosed by distinguishing whether or not -2 is infected. The present invention provides such a method for diagnosing HSV-1 infection and / or HSV-2 infection, in particular, a simple, highly accurate and efficient diagnostic method.
[0016]
(1) The peptide of the present invention
Hereinafter, the peptide of the present invention having specific reactivity with anti-HSV-1 antibody (hereinafter also referred to as “HSV-1 peptide”) and the peptide of the present invention having specific reactivity with anti-HSV-2 antibody (hereinafter referred to as “HSV-2”). (Also referred to as “peptide”).
[0017]
The HSV-1 peptide of the present invention is a peptide having the amino acid sequence represented by SEQ ID NO: 1, or a peptide having an amino acid sequence in which one or more amino acids are modified by substitution, deletion or addition in the amino acid sequence. Characterized by having specific binding properties to anti-HSV-1 antibodies.
[0018]
The HSV-2 peptide of the present invention is a peptide having the amino acid sequence shown in SEQ ID NO: 2, or a peptide having an amino acid sequence modified by substitution, deletion or addition of one or more amino acids in the amino acid sequence. And characterized by having specific binding properties to anti-HSV-1 antibodies.
[0019]
Here, the term antibody is used as a normal term in the art. Therefore, anti-HSV-1 antibody and anti-HSV-2 antibody are an antibody that recognizes HSV-1 (or an antibody that binds to HSV-1) and an antibody that recognizes HSV-2 (or an antibody that binds to HSV-2, respectively) ). These antibodies are preferably antibodies induced in HSV-1 infected patients (anti-HSV-1 antibodies) and antibodies induced in HSV-2 infected patients (anti-HSV-2 antibodies).
[0020]
The amino acid sequence shown in SEQ ID NO: 1 is the 69-83th amino acid sequence of HSV-1 gpG (gpG1), and the peptide comprising this amino acid sequence has specific binding properties to anti-HSV-1 antibodies. Therefore, it is a preferred specific example of the HSV-1 peptide of the present invention.
[0021]
The amino acid sequence shown in SEQ ID NO: 2 is the 558-566th amino acid sequence of HSV-2 gpG (gpG2), and the peptide consisting of this amino acid sequence has a specific binding property to the anti-HSV-2 antibody. And is a preferred specific example of the HSV-2 peptide of the present invention.
[0022]
The peptide of the present invention includes, in addition to the respective peptides having the above amino acid sequences, those having partial amino acids in these amino acid sequences or amino acid sequences modified from the amino acid sequences, that is, one or more amino acids in each amino acid sequence Those having an amino acid sequence in which a single amino acid is substituted, deleted or added are included.
[0023]
Here, the degree and position of amino acid sequence modification, that is, the degree of substitution, deletion or addition of amino acids and their positions are determined by the peptide (or protein) of the modified amino acid sequence having the amino acid sequence before modification. As long as it has the same properties as those described above, that is, as long as it retains specific binding properties to the anti-HSV-1 antibody or anti-HSV-2 antibody, it is not particularly limited.
[0024]
Confirmation that the same effect is obtained, that is, that the anti-HSV-1 antibody or the anti-HSV-2 antibody retains specific binding properties is obtained by confirming that the peptide of the present invention and the anti-HSV-1 antibody or anti-HSV- It can be carried out by testing the reactivity or cross-reactivity with the two antibodies according to a conventional method. One specific example of this test will be described in detail in Examples below.
[0025]
When modified by the addition of amino acids, the HSV-1 peptide of the present invention retains specific binding properties to the anti-HSV-1 antibody and avoids unwanted cross-reactivity with the anti-HSV-2 antibody. In the above, it is important to remove the amino acid sequence from the amino acid sequence of the gpG following the 68th to 85th amino acid sequence of the HSV-1 gpG, particularly preferably the 69th to 83rd amino acid sequence. .
[0026]
Also, in the case of the HSV-2 peptide of the present invention, this retains specific binding to the anti-HSV-2 antibody and avoids undesirable cross-reactivity with the anti-HSV-1 antibody. It is important to eliminate the addition of the amino acid sequence of gpG following the amino acid sequence of 558-566.
[0027]
The peptide of the present invention preferably has a length of at least 5 amino acid sequences considering its antigenicity.
[0028]
If desired, the peptide of the present invention can be made into a form with enhanced antigenicity, for example, a multiple antigen peptide (MAP) form.
[0029]
(2) Production of the peptide of the present invention
The HSV-binding peptide of the present invention can be produced by a general chemical synthesis method according to its amino acid sequence. The method includes peptide synthesis methods by a normal liquid phase method and a solid phase method.
[0030]
More specifically, this peptide synthesis method is based on the stepwise erosion method in which each amino acid is sequentially linked one by one to extend the chain based on the amino acid sequence information provided in the present invention, and a fragment consisting of several amino acids. And fragment condensation method in which each fragment is subjected to a coupling reaction. The peptide of the present invention can be synthesized by any of them.
[0031]
Condensation methods employed for peptide synthesis can also follow various known methods. Specific examples include the azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive (additives: 1-hydroxybenzotriazole, N- Examples thereof include hydroxysuccinamide, N-hydroxy-5-norbornene-2,3-carboximide), and the Woodward method. Solvents that can be used in each of these methods can be appropriately selected from general solvents that are well known to be used in this type of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate, and a mixed solvent thereof.
[0032]
In the above peptide synthesis reaction, amino acids not involved in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, and tertiary butyl ester, such as benzyl ester, p-methoxybenzyl. It can be protected as an aralkyl ester such as an ester or p-nitrobenzyl ester. An amino acid having a functional group in the side chain, such as a hydroxyl group of Tyr, may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like. There is no need to provide such protection. Further, for example, a guanidino group of Arg has an appropriate protection such as a nitro group, a tosyl group, a 2-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group, etc. It can be protected by a group.
[0033]
The deprotection reaction of these protecting groups in the amino acids having the above-mentioned protecting groups, peptides and finally obtained peptides of the present invention is also carried out by conventional methods such as catalytic reduction, liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide. , Hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.
[0034]
The peptide of the present invention obtained according to the above method is used in the field of peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method, etc., according to ordinary methods. The purification can be carried out as appropriate according to the methods widely used in (1).
[0035]
The peptide of the present invention can also be produced according to a genetic engineering technique using a DNA sequence encoding the peptide.
[0036]
This genetic engineering technique can follow conventional methods. For example, synthesis of DNA, production of an expression vector of the DNA, expression method of the vector in a host cell, etc. can all be performed according to or in accordance with general genetic engineering techniques (Molecular Cloning 2nd Ed., Cold Spring Harbor Lab. Press (1989); Secondary Biochemistry Experiment Course "Gene Research Methods I, II, III", Japan Biochemical Society (1986), etc.).
[0037]
For example, DNA encoding the peptide of the present invention can be prepared according to a conventional method based on the amino acid sequence information of the peptide of the present invention (for example, Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm. , 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)).
[0038]
More specifically, the synthesis of DNA can follow chemical synthesis methods such as phosphoramidite method and triester method. Moreover, it can also implement, for example using the commercially available automatic oligonucleotide synthesizer. Double-stranded fragments are obtained by annealing the chemically synthesized complementary strand to the chemically synthesized single-stranded product under appropriate conditions or adding the complementary strand using DNA polymerase together with an appropriate primer sequence. Can do.
[0039]
The DNA synthesized above can be modified in the amino acid sequence encoded by the DNA as desired. Modification of DNA for this modified amino acid sequence can be performed by, for example, site-directed mutagenesis using an oligonucleotide (Zoller, M., et al., Nucl. Acids Res., 10, 6487-6500 (1982)), It can be performed by a known method such as cassette mutagenesis (Well, J., et al., Gene, 34, 315-323 (1985)).
[0040]
Production and expression of the desired peptide using the above DNA can be performed according to a well-known and commonly used technique in this field (for example, Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985). Proc. Natl. Acad. Sci. USA., 80, 5990 (1983), etc.).
[0041]
In producing and expressing the peptide of the present invention as a fusion peptide or a fusion protein, for example, the method of Ohno et al. “Protein Experiment Protocol 1 Functional Analysis, Cell Engineering Separate Volume, Experiment Protocol Series, 1997, Shujunsha” etc. Can be helpful.
[0042]
The desired peptide can be obtained by various separation operations utilizing its physical properties, chemical properties, etc. (for example, “Biochemical Data Book II”, pages 1175-1259, first edition, first print, June 23, 1980). Published by Tokyo Chemical Co., Ltd .; see Biochemistry, 25 (25), 8274-8277 (1986); Eur. J. Biochem., 163, 313-321 (1987), etc.). Specific examples of the separation and purification operations include normal reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography. Examples thereof include various liquid chromatography such as chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis method, and combinations thereof.
[0043]
The peptide of the present invention can be in a form with enhanced immunogenicity, for example, a MAP form, if desired. This MAP form is characterized as a form in which a plurality of peptides of the present invention are presented in the basic molecule.
[0044]
The peptide of the present invention in the MAP form can be easily produced, for example, by using a dendrimer as a basic molecule or a backbone.
[0045]
Dendrimers are generally known as spherical or other structural molecules having a dendritic to star configuration. The molecule is also characterized by a branch (repeating unit) having a plurality of functional groups (for example, JP-A-60-500295; JP-A-63-99233; JP-A-3-263431; US) Patent No. 4507466; No. 4568737; Polymer Journal, 17, p.117 (1985); Angewandte Chem. Int. Engl., 29, 138-175 (1990); Macromolecures, 25, p.3247 (See, for example, (1992)).
[0046]
As one specific example of the peptide of the present invention that is in the MAP form, for example, —CH that binds to the nucleus portion with a nitrogen atom as the initiation nucleus portion₂CH₂CONHCH₂CH₂-A dendrimer having a repeating unit (branch) consisting of a structure, wherein a plurality of peptides of the present invention are bonded to the outermost terminal of each branch. As other specific examples, for example, any amino acid such as Lys, Arg, Glu, Asp is used as a starting nucleus portion, and each amino acid is used as a repeating unit directly bonded to the nucleus portion, and at each branch end. Similarly, the thing which couple | bonded several this invention peptide can be mentioned.
[0047]
The dendrimer having the nitrogen atom as the starting nucleus can be produced according to a conventional method. The structure (the dendrimer raw material to which the peptide of the present invention before fusion or ligation is to be bound) can also be obtained as a commercial product (Polysciences, Inc., 400 Vally Road, Warrington, PA, 18976 U.S.A.). A dendrimer having the other amino acid as an initiation nucleus can be produced, for example, according to the above-described chemical synthesis method of peptides. For example, Fmoc₈-Lys_Four-Lys₂It can also be produced using commercially available dendrimer raw materials such as -Lys-βAla-Alko resin (manufactured by Watanabe Chemical Co., Ltd.).
[0048]
More specifically, the dendrimer raw material can be produced as follows. In other words, a solid phase peptide synthesis resin is subjected to a condensation reaction of α, ω-diamino acids in which two amino groups are protected with the same or non-identical protecting groups, with or without spacers, and then protected. The group is removed, and the same condensation reaction of the protected α, ω-diamino acid and deprotection group reaction are repeated.
[0049]
Here, as the resin for solid phase peptide synthesis, any of those commonly used for normal peptide synthesis can be used. Examples thereof include, for example, chloromethyl group, 4- (hydroxymethyl) phenoxy group, 4-((α-2 ′, 4′-dimethoxyphenyl)-at the terminal of polystyrene resin, polyacrylamide resin, polystyrene polyethylene glycol resin and the like. And those having a 9-fluorenylmethoxycarbonylaminomethyl) phenoxy group. Spacers can include one or more amino acids. Examples of α, ω-diamino acids include lysine, ornithine, 1,4-diaminobutyric acid, 1,3-diaminopropionic acid, and the like.
[0050]
Examples of the protecting group are as shown in the above-mentioned section for producing the peptide of the present invention. The removal reaction of the protecting group can also follow the method shown in the section for producing the peptide of the present invention. The number of branches is 2n by repeating condensation of the repeating unit and removal of the protecting group n times. The number of branches is preferably in the range of 2 to 16.
[0051]
A desired MAP can be obtained by binding the peptide of the present invention to a functional group (amino group, carboxyl group or hydroxyl group) at each branch end of the resulting dendrimer raw material. This binding reaction can follow the peptide synthesis method described above.
[0052]
The peptide of the present invention in the form of MAP can be purified by a chromatography method using a resin such as Sephacreel S-300 (manufactured by Pharmacia) according to a conventional method.
[0053]
In the MAP, the HSV-1 peptide or HSV-2 peptide to be bound to each branch end does not need to be the same, and any combination belonging to the HSV-1 peptide of the present invention or the HSV-2 peptide of the present invention It can be any combination.
[0054]
(3) HSV Diagnosis of infection
The HSV-1 peptide of the present invention functions as an antigen that specifically binds to an anti-HSV-1 antibody, and the HSV-2 peptide of the present invention functions as an antigen that specifically binds to an anti-HSV-2 antibody. Therefore, by using the peptide of the present invention, anti-HSV-1 antibody and anti-HSV-2 antibody produced by HSV infection can be selectively (differentiated) detected.
[0055]
The present invention provides a method for diagnosing HSV infection comprising the step of detecting such an HSV antibody.
[0056]
The diagnostic method of the present invention collects various body fluids such as blood (serum, plasma), urine, sweat, saliva, semen, and cerebrospinal fluid, preferably serum, from a subject suspected of having HSV infection. It is essential to include the step of contacting the peptide with the sample to detect the presence or absence of the anti-HSV-1 antibody and / or anti-HSV-2 antibody in the sample.
[0057]
The antibody detection method is not particularly limited as long as the peptide of the present invention is used as an antigen for a measurement system. Various conventional methods can be widely employed as antibody detection methods.
[0058]
Specifically, standard electrophoresis techniques and immunodiagnostic techniques including immunoassay techniques such as competition methods, direct reaction methods, and solid-phase sandwich type assay methods can be employed. Such immunoassay techniques include, for example, Western blots, agglutination tests, enzyme labels such as ELISA and enzyme-mediated immunoassays, biotin / avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc. .
[0059]
For example, taking the solid phase sandwich method using human serum as a test sample, the target antibody can be measured by the following method. That is, first, the peptide of the present invention, which is an antigen to be measured, is solid-phased, and a serum sample as a test sample is added thereto. As a result, an antigen-antibody reaction occurs between the immobilized peptide and the antibody in the sample, and the target antibody present in the specimen binds to the immobilized peptide. Next, the target antibody present in the test sample can be detected and measured by detecting the bound target antibody using a normal human antibody detection reagent such as an anti-human IgG antibody.
[0060]
Selection of various means in these measurement methods and modification thereof are well known to those skilled in the art, and any of these methods can be adopted in the present invention ["Proposal for clinical laboratory test method", Kanehara Publishing Co., Ltd. See 1995).
[0061]
In the above antibody detection method, when the solid phase method is adopted, the antigen of the measurement system, the human antibody detection reagent and the like can be immobilized on the solid phase according to a conventional method. The solid phase is not particularly limited as long as it is an insoluble and inert carrier, and commonly used ones are widely used. For example, glass, cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid Sticks, beads, microplates, test tubes, and the like made of various materials such as copolymers and ethylene-maleic acid copolymers are widely used.
[0062]
There is no particular limitation on the method for immobilizing an antigen or the like, and a method using both physical bonding and chemical bonding can be appropriately employed. For example, diazo method, peptide method (acid amide derivative, carboxychloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate derivative method as covalent bond methods Chemical reaction such as alkylation method, carrier binding method using a crosslinking reagent (using glutaraldehyde, hexamethylene isocyanate, etc. as a crosslinking reagent), carrier binding method using Ugi reaction, or the like: An ion binding method using a carrier such as an ion exchange resin: a physical adsorption method using porous glass such as glass beads as a carrier can be exemplified as a preferable example.
[0063]
In immobilizing an antigen on a carrier, the antigen can be first converted into a form having better binding characteristics according to a conventional method. Preferred examples of the antigen in this form include the peptide of the present invention in the form of MAP and the peptide of the present invention in the form of carrier binding.
[0064]
Examples of suitable carriers used for the production of the peptide in the carrier-bound form include various proteins such as serum albumin, keyhole limpet hemocyanin, immunoglobulin molecule, thyroglobulin, and ovalbumin. Other carriers include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and the like. Such carriers and methods for conjugating them to antigens are also well known to those skilled in the art.
[0065]
The labeling agent in each measurement system is not particularly limited, and any conventionally known one or those that can be used in the future can be used. Specifically, for example^ThreeH,¹⁴Radioisotopes such as C; enzymes such as alkaline phosphatase and peroxidase (POX); fluorescent substances such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC); and 1N- (2,2,6,6 -Tetramethyl-1-oxyl-4-piperidyl) -5N- (aspartate) -2,4-dinitrobenzene (TOPA); dye sol; metal sol; latex particles and the like. Immunoassays using these as labeling agents are called radioimmunoassay, enzyme immunoassay, fluoroimmunoassay, spin immunoassay, flow-through assay, and immunochromatographic assay, respectively. In the present invention, an enzyme immunoassay using an enzyme as a labeling agent is preferably employed from the viewpoint of convenience, safety, sensitivity, and the like.
[0066]
Examples of enzyme labeling substances for enzyme labeling include, in addition to the above, microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrase, amylase, phosphorylase, D-nase, P-nase Etc. can be illustrated. In addition, the labeling method with these labeling substances can be performed according to a method known per se (“monoclonal antibody” Ikuzaki Ikuo et al., Kodansha Scientific, 1984; “Enzyme Immunoassay” 2nd edition, Eiji Ishikawa See other books, Medical School, 1982, etc.).
[0067]
The enzyme activity can be measured according to a known method according to the type of enzyme used. For example, when peroxidase is used as a labeling enzyme, ABTSJ [2,2′-Adinobi (3′-ethylbenzthiazolinesulfonic acid)] is used as a substrate, and when alkaline phosphatase is used, p-nitrophenyl phosphate is used as a substrate. Incubate enzymes in these substrates, and measure the degradation of each substrate using a spectrophotometer, etc. ("Enzyme Immunoassay" 2nd edition, Eiji Ishikawa et al., Medical Shoin) 1982, etc.)
[0068]
Even when a label such as a radioisotope or a fluorescent substance is used in place of the enzyme label, the label can be measured according to a known method.
[0069]
The solvent used in each measurement system may be any solvent that does not adversely affect the reaction. Specific examples of commonly used solvents include buffers having a pH of about 5-9, such as citrate buffer, phosphate buffer, Tris-HCl buffer, and acetate buffer.
[0070]
The immune reaction (binding) conditions are not particularly limited, and usual conditions generally used in this type of measurement method are employed. In general, the reaction can be carried out under a temperature condition of about 45 ° C. or less, preferably about 4-40 ° C., taking several tens of minutes to several hours.
[0071]
Thus, detection of anti-HSV-1 antibody and anti-HSV-2 antibody in a subject sample can be performed. Detection (presence) of anti-HSV-1 antibody indicates that the subject is infected with HSV-1, and detection (presence) of anti-HSV-2 antibody indicates that the subject is infected with HSV-2 And the detection (presence) of both anti-HSV-1 and anti-HSV-2 antibodies indicates that the subject is infected (superinfection) with both HSV-1 and HSV-2.
[0072]
In carrying out the diagnosis of HSV infection of the present invention, it is easy to use the reagent composition (or reagent kit) for detecting or diagnosing the antibody, and the present invention provides a reagent composition that can be used for such HSV diagnosis. Also provide things.
[0073]
The reagent composition of the present invention comprises a diagnostic reagent composition for diagnosis of HSV-1 infection, comprising as an active ingredient at least one of the HSV-1 peptides of the present invention, which is an antigen reagent unique to an anti-HSV-1 antibody. is there.
[0074]
The reagent composition of the present invention comprises at least one HSV-2 peptide of the present invention, which is an antigen reagent peculiar to an anti-HSV-2 antibody, as an active ingredient. It is a thing.
[0075]
Furthermore, the reagent composition of the present invention is a diagnostic reagent composition for HSV infection comprising both the above-described diagnostic reagent composition for HSV-1 infection and the diagnostic reagent composition for HSV-2 infection.
[0076]
These reagent compositions of the present invention are a set of combinations of at least one selected from a solid phase, a labeling agent, and a substrate (detection reagent) corresponding to the labeling agent in addition to the active ingredient. May be.
[0077]
In addition, when a solid phase is included in the set, arbitrary components according to the measurement system may be immobilized on the solid phase in advance. Further, when a labeling agent is included in the set, similarly, an arbitrary component may be conjugated in advance with the labeling agent. Examples of the labeling agent include various compounds such as the aforementioned radioisotopes, enzymes and fluorescent substances, preferably enzymes.
[0078]
Furthermore, the reagent composition of the present invention may contain various diluents, standard antibodies, buffers, washing solutions, substrate lysis solutions, reaction stop solutions and the like suitable for the convenience of carrying out the measurement.
[0079]
【Example】
Hereinafter, in order to describe the present invention in more detail, first, a conventional HSV culture antigen, an HSV-1 (gpG1) recombinant protein antigen, and an antibody detection method using HSV-2 (gpG2) recombinant protein antigen will be described, and then Examples of the peptides of the present invention and antibody detection methods using them are given below.
[0080]
Specimen
Serum specimens (Dept. of Dermatology, Jikei University) were obtained from clinical diagnosis of 29 herpes sera diagnosed with HSV-1, 42 herpes sera diagnosed with HSV-2 infection, HSV-1 and HSV There are 3 herpes sera diagnosed with -2 superinfection and 96 healthy sera.
[0081]
Antibody detection
Anti-HSV antibodies (IgG) in these serum specimens were detected using a herpes IgG-EIA kit (Denka Seken) according to a conventional method using HSV culture antigen.
[0082]
A type 1 specific antibody (IgG) using HSV-1 glycoprotein G (gpG1) recombinant protein antigen for all cases of HSV patient samples and healthy subject samples that were found to be positive for serum anti-HSV antibody (IgG) above. ) And detection of type 2 specific antibody (IgG) using HSV-2 glycoprotein G (gpG2) recombinant protein antigen, respectively, commercially available kit (Primier Type specific HSV-1 or HSV-2 IgG ELISA Test; Meridian Diagnostics, Inc.).
[0083]
The obtained results are shown in FIGS.
[0084]
FIG. 1 is a graph showing the detection results of anti-HSV antibodies according to a conventional method using HSV culture antigens, and FIG. 2 is a graph showing the detection results of antibodies when using gpG1 recombinant protein antigens. These are graphs showing the detection results of antibodies when gpG2 recombinant protein antigen is used.
[0085]
In each figure, the horizontal axis indicates HSV-infected patients or healthy subjects, the vertical axis in FIG. 1 indicates absorbance (OD450-650 nm), and the vertical axes in FIGS. 2 and 3 indicate relative ELISA values (Rerative ELISA Volue: OD value of specimen divided by OD value of control serum).
[0086]
From the results shown in these figures, the positive rate of serum anti-HSV antibody (IgG) is 86% (25/29) in the serum of HSV-1 infected patients and 90% (38 in the serum of HSV-2 infected patients). It was revealed that 100% (3/3) of both co-infected patient sera and 57% (55/96) of healthy subject sera.
[0087]
Type 1 specific antibody (IgG) was highly reactive with sera of HSV-1 infected patients, but was also reactive with several anti-HSV antibody-negative healthy sera (see Figure 2) ). In addition, type 2 specific antibody (IgG) was highly reactive with sera of HSV-2 infected patients, but was also reactive with the sera of 2 healthy anti-HSV antibody positive patients (Fig. 3).
[0088]
[Example 1]
(1) HSV-2 peptide
Based on the phage vector pTV119N (Takara Shuzo), a library of random peptides (consisting of 9 amino acids) was created by the p8 protein display system of M13 phage. The obtained library was transformed into E. coli strain JM109 by electroporation to prepare a random peptide display phage library.
[0089]
Serum biopanning using the specimens (serums from healthy subjects, sera from HSV-1 infected patients, and sera from HSV-2 infected patients) showed reactivity to a plurality of HSV-2 infected patient sera, Two clones named “HSV-2c5” and “HSV-2g60.3” were selected as phage clones that showed no reactivity to the serum of healthy subjects.
[0090]
(2) Phage ELISA
A 96-well microtiter plate on which 1 μg / ml of an anti-M13 phage p8 protein antibody (Pharmacia) was immobilized was used. 90 μl of phage ELISA buffer (phosphate buffer containing 0.5% BSA, 10% normal goat serum and 0.05% Tween 20) and 10 μl of peptide display phage culture medium (prepared as follows) are added to the plate. After reacting at 37 ° C. for 1 hour (primary reaction), unreacted phages were washed four times and removed.
<Preparation of culture medium for peptide display phage>
A single colony was cultured at 37 ° C. for 8 hours in 50 μl of Luria-Bertani medium containing ampicillin, and then helper phage M13K07 solution (10¹²cfu) Peptide display phage cultures were prepared by adding 50 μl of infection and then adding 400 μl of liquid medium (Luria-Bertani medium containing ampicillin, kanamycin and IPTG) and culturing overnight at 37 ° C.
[0091]
Next, 100 μl of a serum sample diluted 101-fold with phage ELISA buffer was added to each well of the plate after the completion of the primary reaction, and allowed to react at 37 ° C. for 1 hour (secondary reaction), and then the unreacted antibody was washed 4 times. And removed.
[0092]
Furthermore, 100 μl of enzyme-labeled anti-human IgG (Fc region specific) antibody diluted 40,000 times in phage ELISA buffer was added to each well and reacted at 37 ° C. for 1 hour (tertiary reaction). Removed by washing 4 times.
[0093]
Finally, 100 μl of TMB solution was added to each well and allowed to stand at room temperature for 10 minutes to cause a color reaction, then 100 μl of 1N sulfuric acid solution was added to stop the color reaction, and then each well Absorbance (OD450-650nm) was measured.
[0094]
FIG. 4 shows the ELISA reactivity (phage ELISA) results obtained using both clones selected in (1) above.
[0095]
In FIG. 4, the upper row shows the results using HSV-2c5 clone, and the lower row shows the results using HSV-2g60.3 clone. In each figure, the horizontal axis represents each patient or healthy person, and the vertical axis represents the OD value (NET OD).
[0096]
From the results shown in FIG. 4, it is clear that both phage clones show similar reactivity with each patient serum and healthy subject serum.
[0097]
Hereinafter, the reaction inhibition test using the cultured antigens of HSV-1 and HSV-2 conducted to confirm that these two clones react with the antibody specific to HSV-2 infected patients will be described in detail.
[0098]
(3) Reaction inhibition test
HSV-1 or HSV-2 culture antigen was added to the secondary reaction solution of the phage ELISA shown in (2) above, and the reactivity between each clone and the antibody was measured in the same manner.
[0099]
The result is as shown in FIG.
[0100]
FIG. 5-A shows the results when HSV-2c5 is used, and FIG. 5-B shows the results when HSV-2g60.3 is used. In each figure, the horizontal axis represents the reaction-inhibiting antigen concentration (ng / mL), and the vertical axis represents B / B0 (%). In each figure, (1) is the result when E. coli culture antigen is added, (2) is the result when HSV-1 culture antigen is added, and (3) is the HSV-2 culture antigen. It is a result at the time of adding.
[0101]
From the results shown in FIG. 5, it is clear that the reactivity of both clones is inhibited only when HSV-2 culture antigen is added.
[0102]
This result indicates that HSV-2c5 and HSV-2g60.3 are peptides that mimic HSV-2 specific antigens.
[0103]
(Four) Determination of peptide sequence
In order to analyze the peptides displayed by the phage clones HSV-2c5 and HSV-2g60.3, the DNA sequences of both clones were determined and the peptide sequences were determined.
[0104]
As a result, the peptide sequence displayed by HSV-2c5 is as shown in SEQ ID NO: 3, and the peptide sequence displayed by HSV-2g60.3 is as shown in SEQ ID NO: 4. confirmed. When these amino acid sequences were compared, 7 of 9 amino acids matched. Since the reactivity of both clones was equivalent as described above, each of these clones was considered to mimic the same region of HSV-2.
[0105]
For the purpose of determining the mimicking region of both clones, homology analysis was performed focusing on the sequences retained between the clones.
[0106]
As a result, homology was confirmed with the 6 amino acid sequence (SEQ ID NO: 5) of 558-563 of gpG2. The C-terminal 3 amino acids of both clones showed no homology downstream of the above 6 amino acid sequence of gpG2, but this 9 amino acid sequence (the 558-566th position of gpG2, the sequence shown in SEQ ID NO: 2) Considering that this is a sequence retained between the sequences and an important sequence for binding to patient antibodies, this was assumed to be a central epitope.
[0107]
(5) MAP In the form of a peptide of the invention
The peptide of the present invention having the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “2gpG58-66”) and the peptide described in the prior art for comparison (the amino acid sequence shown in SEQ ID NO: 6, hereinafter referred to as “2gpG51-70”) Was synthesized as follows.
[0108]
That is, using a fully automatic peptide synthesizer (ACT357, manufactured by Advanced Chemtech), according to the company's program, Fmoc / NMP, HOBt method [Fmoc: 9-fluorenylmethoxycarbonyl group, NMP: N-methylpyrrolidone, Solid phase synthesis of peptides with HOBt: 1-hydroxybenzotriazole] was performed.
[0109]
According to the amino acid sequence information, Fmoc-amino acid-Alko resin corresponding to the C-terminal amino acid was subjected to chain extension by sequentially condensing Fmoc-amino acid corresponding to each of the second and subsequent amino acids from the C-terminal according to the synthesis program. .
[0110]
After completion of each reaction, the N-terminal Fmoc group was deprotected according to the program.
[0111]
Each peptide-resin obtained was collected in a polypropylene minicolumn (manufactured by Assist), washed with methanol, dried in vacuum, and subjected to the following operation to cleave the peptide from the resin and perform side chain deprotection reaction. went.
[0112]
That is, Reagent K (Reagent K: 82.5% TFA, 5% phenol, 5% H₂2 ml of O, 5% thioanisole and 2.5% ethane di-thiol was added and allowed to react for 60 minutes in a minicolumn.
[0113]
Subsequently, the reaction solution was dropped into 8 ml of cold diethyl ether to stop the reaction, and the peptide was precipitated. The minicolumn was washed with 2 ml of TFA, 5 ml of cold diethyl ether was added and centrifuged, and the precipitate was washed 4 times with 10 ml of diethyl ether, and the peptide was solubilized with about 5 ml of 50% acetonitrile and lyophilized. Further, solubilization and freeze-drying operations were repeated twice to obtain a desired crude freeze-dried product.
[0114]
This crude dry product was fractionated by reverse phase high performance liquid chromatography (HPLC) using an octadecyl column (diameter 20 × 250 mm, YMC) to isolate the desired peptide.
[0115]
The resin and amino acid derivative used above are both manufactured by Watanabe Chemical Industries.
[0116]
Each peptide thus isolated was identified by amino acid sequence analysis and molecular weight determination by mass spectrometry.
[0117]
A multi-antigenic peptide (MAP) was synthesized using Fmoc-MAP-Alko resin (manufactured by Watanabe Chemical Industries) using the peptide of the present invention and the comparative peptide obtained above.
[0118]
Fmoc-MAP-Alko resin (Fmoc₈-Lys_Four-Lys₂-Lys-βAla-Alko resin) and the starting peptide were carried out in the same manner as in the solid phase synthesis method.
[0119]
The structure of the obtained MAP is as follows.
[0120]
(2gpG58-66)₈-Lys_Four-Lys₂-Lys-βAla
(2gpG51-70)₈-Lys_Four-Lys₂-Lys-βAla
(6) MAP antigen ELISA
100 μl of a MAP solution prepared to 1 μg / ml with a phosphate buffer was dispensed into each well of a 96-well microtiter plate and reacted at 4 ° C. overnight to immobilize.
[0121]
The obtained solid-phased plate was blocked in a phosphate buffer containing 300 μl of 1% BSA after removing the MAP solution from each well.
[0122]
To each well, 100 μl of a serum sample diluted 101-fold with a primary reaction buffer was added and reacted at 37 ° C. for 1 hour (primary reaction), and then unreacted antibody was washed and removed four times.
[0123]
Next, 100 μl of enzyme-labeled anti-human IgG (Fc region specific) antibody diluted 5000 times with secondary reaction buffer was added to each well and reacted at 37 ° C. for 1 hour (secondary reaction), then unreacted The antibody was removed by washing 4 times.
[0124]
Finally, 100 μl of TMB solution was added to each well and allowed to stand at room temperature for 10 minutes to cause a color reaction, then 100 μl of 1N sulfuric acid solution was added to stop the color reaction, and then each well Absorbance (OD450-650nm) was measured.
[0125]
The above MAP antigen ELISA was performed on 41 specimens determined to be anti-HSV antibody negative by a test using a herpes IgG-EIA kit.
[0126]
The obtained results are shown in FIG. 6 in the same manner as FIG. The average OD value +0.4 of the measurement result is taken as the cut-off value.
[0127]
From the results shown in Fig. 6, both MAP antigens using 2gpG58-66 (Fig. 6-A) and 2gpG51-70 (Fig. 6-B) are highly reactive to the sera of HSV-2 infected patients. It turns out that it shows.
[0128]
However, as shown in FIG. 6-B, the MAP antigen of 2gpG51-70 showed reactivity in 6 serum samples other than HSV-2 infected patients. Six cases that showed this reactivity were 2 cases in HSV-1 infected patients' sera (shown as # 1 and # 2 in Fig. 6-B) and 3 cases in healthy HSV antibody positive (# 3 in Fig. 6-B). , # 4 and # 5) and 1 case (shown as # 6 in FIG. 6-B) in healthy HSV antibody-negative subjects.
[0129]
The serum of the HSV-1 infected patient shown as # 1 in FIG. 6-B also showed reactivity with the MAP antigen of 2gpG58-66 (see # 1 in FIG. 6-A). In addition, the HSV antibody-positive healthy serum indicated by # 5 in FIG. 6-B was also reactive with the MAP antigen of 2gpG58-66 (see # 5 in FIG. 6-A) and the gpG2 recombinant protein antigen described above. .
[0130]
(7) Reaction inhibition test
The reactivity of the above 6 cases was confirmed by the following reaction inhibition test using HSV-2 culture antigen.
[0131]
That is, after reacting a mixture of 25 μg HSV-2 culture antigen to each diluted serum sample for 1 hour at 37 ° C., add the reaction solution to the 2gpG51-70 MAP antigen plate or 2gpG58-66 MAP antigen plate The measurement was performed according to the MAP antigen ELISA method described in (6) above.
[0132]
The results are shown in Table 1 below.
[0133]
[Table 1]

[0134]
As shown in Table 1, sera # 1 of HSV-1 infected patients who showed reactivity to MAP antigen of 2gpG51-70 and MAP antigen of 2gpG58-66, and MAP antigen of 2gpG51-70, MAP antigen of 2gpG58-66 and gpG2 In the HSV antigen positive healthy subject serum # 5 that showed reactivity to the recombinant protein antigen, inhibition of reactivity was confirmed in both antigen plates. Therefore, serum samples # 1 and # 5 are considered to have anti-HSV-2 antibodies.
[0135]
However, no reactivity inhibition was observed in the other 4 specimens. Therefore, it was confirmed that the reactivity exhibited by these 4 specimens positive for the MAP antigen of 2gpG51-70 was not a reaction with anti-HSV-2 antibody but a non-specific reaction.
[0136]
In addition, Table 2 below shows the results of the ELISA for each sample that was positive for serum anti-HSV antibody.
[0137]
[Table 2]

[0138]
In Table 2, “recombinant” is a measurement result using a commercially available kit using the gpG2 recombinant protein antigen.
[0139]
Two cases (# 1 and # 5) where the presence of the anti-HSV-2 antibody was proved by the reaction inhibition test in (7) above were excluded from the analysis in Table 2.
[0140]
From the results shown in Table 2, the following can be understood.
[0141]
That is, the positive rate of MAP antigen of 2gpG51-70 to be compared was 82% (31/38) in the HSV-2 infected patient serum, 67% (2/3) in the superinfected patient serum, HSV-1 infected patient serum 4% (2/55), 4% (2/54) of anti-HSV antibody positive healthy serum, and 2% (1/41) of anti-HSV antibody negative healthy serum.
[0142]
On the other hand, the positive rate of MAP antigen of 2gpG58-66 according to the present invention was 74% (28/38) in the serum of HSV-2 infected patients and 33% (1/3) in the serum of superinfected patients. Furthermore, in the MAP antigen ELISA using the MAP antigen of the present invention, one positive case was not detected in the serum of HSV-1 infected patients, the serum of anti-HSV antibody positive healthy subjects and the anti-HSV antibody negative healthy subjects.
[0143]
From the above results, the MAP antigen of 2gpG51-70 has a high positive rate but includes several percent non-specific reactivity, which is superior to the MAP antigen of 2gpG58-66 of the present invention which does not show any false positive reaction. The usefulness was confirmed.
[0144]
[Example 2]
(1) HSV-1 peptide
Based on the information of the amino acid sequence of 558 to 563 (SEQ ID NO: 5) of gpG2 found in Example 1, the corresponding sequence of HSV-1 glycoprotein G (gpG1) and its neighboring sequences are selected, Their availability as HSV-1 peptides was analyzed.
[0145]
The analyzed gpG1 region is the 68-85th region (the amino acid sequence is shown in SEQ ID NO: 7, hereinafter referred to as “1gpG68-85”), and the 92-108th region, 113-132th position located downstream thereof. Region (hereinafter referred to as “1gpG113-132”), 126-145th region and 146-165th region.
[0146]
Peptides corresponding to each of the above regions were synthesized as MAPs according to Example 1 and tested for reactivity to these specimens by MAP antigen ELISA in the same manner as in Example 1- (6).
[0147]
As a result of the measurement by ELISA, the MAP antigen of 1gpG68-85 and the MAP antigen of 1gpG113-132 showed high reactivity to HSV-1-infected patient sera and anti-HSV antibody positive healthy sera. Specific reactivity was not confirmed in the other three MAP antigens.
[0148]
In the same way, MAP was synthesized for 25 types of peptides in which the sequence of the 1gpG68-85 region showing the most excellent reactivity was shortened by one amino acid from the N-terminal and C-terminal, and each of these MAPs was inhibited. Using the substance as a substance, a reaction inhibition test was performed according to Example 1- (7), and its central epitope was determined. In the reaction inhibition test, a serum sample diluted 101-fold with a primary reaction buffer and 10 μg (10 μl) of each MAP were reacted at 37 ° C. for 1 hour, and then added to a 1 gpG68-85 MAP antigen-immobilized plate. The reaction was performed at 37 ° C. for 1 hour.
[0149]
Detection of the antigen-antibody complex was performed in the same manner as in Example 1- (7).
[0150]
As serum samples, sera of 32 healthy subjects who showed reactivity with the MAP antigen of 1gpG68-85 were used.
[0151]
The results obtained are shown in Table 3 below. In Table 3, the upper column shows the amino acid sequence (indicated by one letter) of the MAP antigen used as a reaction inhibitor, and the lower column shows the reactivity with serum samples (B / B 0%) according to the following criteria: .

[0152]
[Table 3]

[0153]
The following is clear from the results shown in Table 3 above.
[0154]
That is, when the N-terminal deleted peptide MAP is used as a reaction inhibitory antigen, the reactivity of the serum sample to the MAP antigen of 1gpG68-85 (inhibitor No. 0 in Table 3) is the second histidine (H) In 3 cases out of 32 cases by deleting the threonine, and in 19 cases out of 32 cases by removing the third threonine (T), recovery of 70% (B / B0) or more was shown. Therefore, it was judged that the N-terminal side is required up to the second H.
[0155]
On the other hand, when MAP of the C-terminal deleted peptide was used as a reaction-inhibiting antigen, the reactivity of the serum sample to the MAP antigen of 1gpG68-85 was reduced in 1 out of 32 cases by deleting the 4th glutamic acid (E). 50% (B / B0) recovery was observed. In addition, by removing the 5th E, in 8 of 32 cases, the reactivity recovered 50% (B / B0) or more, and in 7 of 32 cases, the reactivity recovered 70% (B / B0) or more. did. Therefore, it was determined that the 4th E was necessary on the C-terminal side.
[0156]
Based on the above, the shortest epitope of the 1gpG68-85 region was determined to be its 69th to 83rd 15 amino acids (hereinafter referred to as “1gpG69-83”).
[0157]
In order to confirm the reactivity of 1gpG69-83, MAP of 1gpG69-83 was synthesized in the same manner as described above, and this MAP antigen was used and measured by MAP antigen ELISA in the same manner as in Example 1- (6). .
[0158]
The obtained results are shown in Table 4 below according to Table 2.
[0159]
[Table 4]

[0160]
From the results shown in Table 4, the MAP antigen of 1gpG69-83 showed the same reactivity as the MAP antigen of 1gpG68-85. The positive rates were 92% (23/25) for sera from HSV-1-infected patients, 100% (3/3) for sera from coinfected patients, 89% (49/55) for anti-HSV antibody-positive healthy sera, and anti- It was 0% in the serum of HSV antibody negative healthy subjects. This positive rate was superior to that when the gpG1 recombinant protein antigen was used.
[0161]
From the above, by using the HSV-1 peptide and HSV-2 peptide of the present invention, a specific detection system for anti-HSV-1 antibody and anti-HSV-2 antibody is established, and these are clinically useful. it is conceivable that.
[0162]
【The invention's effect】
According to the present invention, peptides that specifically recognize antibodies specific to HSV-1 infected patients and HSV-2 infected patients are provided. These peptides are useful as diagnostic reagents for HSV infection, particularly as reagents capable of distinguishing between HSV-1 infection and HSV-2 infection. By using these peptides and diagnostic reagents, the presence or absence of HSV-1 infection and HSV-2 infection can be distinguished and accurately diagnosed.
[0163]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of measuring anti-HSV antibody (IgG) in a specimen using a herpes IgG-EIA kit using HSV culture antigen.
FIG. 2 is a diagram showing the results of measuring anti-HSV-1 antibody (IgG) in a specimen by ELISA using gpG1 recombinant protein antigen.
FIG. 3 is a view showing the results of measuring anti-HSV-2 antibody (IgG) in a specimen by ELISA using gpG2 recombinant protein antigen.
FIG. 4 is a view showing the results of measuring the reactivity of each of the phage clones HSV-2c5 and HSV-2g60.3 selected in Example 1 (1) with an anti-HSV antibody in a sample by ELISA. .
FIG. 5 is a view showing the results of a reaction inhibition test using a culture antigen of HSV-1 or a culture antigen of HSV-2 according to (3) of Example 1.
FIG. 6 is a view showing a measurement result by MAP antigen ELISA according to (1) of Example 1.

Claims (7)

A peptide comprising the amino acid sequence shown in SEQ ID NO: 1, or a peptide comprising an amino acid sequence in which 1 to 3 amino acids are modified by substitution, deletion or addition in the amino acid sequence, and an anti- type 1 herpes simplex virus antibody; A peptide having specific reactivity. Peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence one amino acid substitution in, a peptide consisting of the modified amino acid sequence by deletion or addition anti herpes simplex virus type 2 antibodies specifically reactive, A peptide having sex.  A reagent composition for diagnosing herpes simplex virus infection comprising the peptide according to claim 1 as an active ingredient.  A reagent composition for diagnosing herpes simplex virus infection comprising the peptide according to claim 2 as an active ingredient.  5. The diagnostic reagent composition according to claim 3 or 4, wherein the peptide is in a multi-antigenic peptide form.  A diagnostic reagent composition for distinguishing between type 1 herpes simplex virus infection and type 2 herpes simplex virus infection, comprising the reagent composition according to claim 3 and the reagent composition according to claim 4. A method for distinguishing and diagnosing type 1 herpes simplex virus infection and type 2 herpes simplex virus infection in a specimen, comprising contacting the specimen with the peptide according to claim 1 and the peptide according to claim 2 A method comprising a step of detecting the presence or absence of an anti- type 1 herpes simplex virus antibody and an anti- type 2 herpes simplex virus antibody in the specimen.

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