JP2005029558A - Antigen used for inspecting echinococcosis (alveolar hydatid disease) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inspecting echinococcosis (alveolar hydatid disease) having a sufficient sensitivity by using an antigen consisting of an amino acid sequence having a size capable of being synthesized. <P>SOLUTION: This specific amino acid sequence in an Em18 molecule (160 amino acid residues) used conventionally as the antigen for inspecting the alveolar hydatid disease, is found to have an equivalent inspection sensitivity to that of the Em18, and the inspection antigen for the alveolar hydatid disease consisting of the amino acid sequence from 389-391 to 418-428 of the Em18 is used. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antigen used for examination of echinococcosis (polycystematosis).

Echinococcosis is basically a chronic disease of the liver that occurs when an ectococcal egg that parasitizes a fox accidentally enters the human mouth via the excrement of the fox and the infection is established. (Non-Patent Documents 1 to 3).
Echinococcus is a parasitic disease that is classified as a type 4 infectious disease and is required to be reported in the Infectious Disease Prevention Law enacted on April 1, 1999.
In Hokkaido, it is reported that 50-60% of foxes are infected with Echinococcus. There are two types of echinococcosis: “polycystic disease”, which is known as an endemic disease in Hokkaido, and “monocystic disease”, which causes problems with imported cases.
“Polycystiasis” is a modern “death-causing disease” that, like a malignant tumor, continues to increase in foci and follows the death outcome within 15 years after some symptoms appear. Early diagnosis and early treatment are absolutely essential for polycystiasis, and surgical debridement is currently the only treatment. When surgical treatment is not possible, chemotherapy with albendazole is recommended, and at least an effect of suppressing the worsening of the disease state has been observed, and cases in which a curative effect can be obtained have been reported. The possibility of increasing the cure rate by continuous administration of the drug has begun to be pointed out.

Asahikawa Medical University has already established a serum test method using a protein Em18 having a molecular size of 18 kDa as a polycystic sera test antigen as a preoperative diagnostic method (Non-patent Documents 4 and 5). Em18 is a degradation product of EM10 (65 kDa) by cysteine protease. At present, the antigens of serologic tests for polycystic diseases are adjusted from those of parasite larvae. However, it is difficult to prepare a sufficient amount of parasite materials and to adjust antigens with stable quality. Is a problem. Therefore, a serum test method using a recombinant protein expressed by E. coli has been developed.
In addition to these methods, a method for diagnosing Echinococcus infections using the excretion and secretion antigens of Echinococcus as immunizing antigens (diagnostic methods for foxes, dogs, etc.) has been developed (Patent Document 1).

INFECTION CONTROL 2001 vol.10, No.7, 670-672 Current Concepts in Infections Diseases vol.20, No.4, 18-19 (2001) Gifu Medical Association Magazine Vol.14 No.1 35-46 (2001) Ito A et al., Am J Trop Med Hyg, 60: 188-192 (1999) Sako Y et al., Journal of Clinical Microbiology vol.40, No.8, 2760-2765 (2002) JP2001-292766

When producing a recombinant antigen using Escherichia coli, problems remain in standardization of vote items, such as the effect of non-specific crossing by a hybrid derived from Escherichia coli, the amount of produced protein, and the like. Therefore, using a chemically synthesized peptide as an antigen is ideal for overcoming this problem.
An object of the present invention is to provide a test method for echinococcosis having sufficient sensitivity, which is performed using an antigen having an amino acid sequence of a synthesizeable size.

  The inventors of the present invention have identified a site recognized by an antibody in patient serum in an antigen for testing polycystiasis. As a result, it has been clarified that a specific amino acid sequence in the Em18 molecule (160 amino acid residues) that has been used as an antigen for testing polycystiasis has test sensitivity equivalent to that of Em18.

That is, the present invention is an antigen for testing for polycystiasis (echinococcosis) comprising the amino acid sequence from the 389th to the 391th to the 418th to 428th positions of SEQ ID NO: 1. This antigen for testing for polycystiasis preferably comprises the amino acid sequence of SEQ ID NO: 3 (positions 390 to 424 of SEQ ID NO: 1).
Further, the present invention is a polycystic test agent containing any one of these antigens as a main component.
The present invention also relates to a method for testing polycystiasis comprising reacting a subject's serum with the antigen or the test agent.
Furthermore, the present invention relates to a polycystic insect comprising immobilizing the above-mentioned antigen on a support, reacting the subject's serum with this, reacting a probe with a label thereto, and detecting the label. This is a test method for the disease.
In addition, the present invention is a test kit for detecting an antibody against echinococcosis (polycystematosis) from the serum of a subject, the test kit comprising the above-described antigen fixed to a support.

The Em18 molecule (Non-patent Document 4, 160 amino acid residues, SEQ ID NO: 2 (positions 349 to 508 of SEQ ID NO: 1)) that has been used by the present inventors as an antigen for testing polycystic disease is an existing diagnostic antigen. It is a degradation product by cysteine protease of EM10 (Frosch PM et al., Molecular and Biochemical Parasitology vol. 48, 121-130 (1991), SEQ ID NO: 1).
The antigen of the present invention is a peptide fragment consisting of 35 to 40 amino acids from any of 389 to 391 to any of 418 to 428 in SEQ ID NO: 1. These are a part of Em18, and include one of the two antigenic regions found by the inventors (Antigenic region 1 & 2, SEQ ID NO: 1, 366-384th and 401-414th) in Em18. Among them, the amino acid sequence (KLAEMNRKLKEETAASAEERDRLMAQRDEVQREVE, SEQ ID NO: 3 (positions 390 to 424 of SEQ ID NO: 1)) is most preferable. These relationships are shown in FIG.

  By using the antigen of the present invention and reacting with an antibody produced in a subject infected with polycystic disease, echinococcosis (polycystic disease) can be examined. Examples of such a test object include a subject's serum, urine, and the like, and preferably serum is used. If the subject is infected with polycystiasis, since the antibody is produced and exists in the serum, the antibody reacts with the antigen. The method for detecting the antigen-antibody reaction is not particularly limited, and examples thereof include an immunoblot method, a dot blot method, an ELISA method, and the like, and it is preferable to use the ELISA method.

A preferred method for testing polycystiasis is that the antigen of the present invention is immobilized on a support, the subject's serum is reacted with this, the labeled probe is reacted with this, and this label is detected. Become. It is preferable to insert a washing step as appropriate between each step.
Although there is no restriction | limiting in particular in this support body, A polystyrene etc. can be used.
This antigen is reacted with an antibody present in the serum of a subject (when infected), and a probe that recognizes this antibody is further reacted. Examples of the probe include anti-human IgG antibody, protein G, protein A, and protein L. This probe is usually labeled. Examples of the label include a radioisotope ( ¹²⁵ I) and an enzyme (peroxidase, alkaline phosphatase). When an enzyme antibody is used, the change (coloring or the like) may be observed by reacting the substrate.

The antigen of the present invention can be synthesized, and therefore can be conveniently and stably supplied as a polycystic disease test antigen, and quality control is easy.
This antigen can be used for preoperative serodiagnosis and serological prognosis determination of echinococcosis (polycystematosis).

The following examples illustrate the invention but are not intended to limit the invention.

In this example, first, the gene encoding Em18 was adjusted.
The gene encoding Em18 was prepared by the RT-PCR method. That is, messenger RNA was purified from polycysts that were passaged using gerbils in the laboratory, and then cDNA was synthesized using oligo dT primer and reverse transcriptase. Using the synthesized cDNA as a template, Em18 was obtained by PCR using Em18-specific primers (Em18F; 5'-AAGGAGTCTGACTTAGCGGAT-3 '(SEQ ID NO: 4), Em18R; 5'-TTTGAGGTTGGCCAGCTTCGT-3' (SEQ ID NO: 5)). The encoding gene was adjusted.

Next, a peptide library was constructed.
The DNA fragment encoding Em18 was randomly cleaved with DNaseI, then a short fragment (50 bp to 100 bp) was purified, and adenine (dATP) was introduced into the 3 ′ end of the DNA using T4 DNA polymerase and Tth DNA polymerase. . The DNA fragment introduced with adenine is cleaved with the restriction enzyme EcoRV in advance and thymine (dTTP) is introduced into the 3 ′ end thereof. Plasmid pSCREEN-1b (+) for E. coli expression (a restriction enzyme map is shown in FIG. 2). The peptide library was constructed by injecting into E. coli. By using this plasmid, a random short peptide of Em18 is expressed in E. coli as a fusion protein with the T7 gene 10 protein, which is a phage head protein. The NovaTope System (Novagen) was used for the construction of this peptide library.

Next, the peptide library was screened.
LB agar medium (1% Bacto Tryptone, 0.5% Yeast Extract, 0.5% NaCl, 1.5% Agarose) Square petri dish (14cm × 10cm: Eiken Equipment Co., Ltd.) Incubated overnight. A nitrocellulose membrane was placed on the cultured square petri dish to adsorb E. coli colonies to the membrane. The membrane adsorbed with E. coli is placed on a filter paper containing 1% SDS, 0.5N NaOH, and 1M Tris-HCl (pH 7.5) for 10 minutes, 5 minutes, and 5 minutes, respectively, to destroy the E. coli and the fusion protein. Bonded on the membrane. This membrane was washed with phosphate buffered saline (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , pH 7.4) containing 0.05% surfactant Tween20. ) Twice at room temperature and blocked with a blocking buffer (20 mM Tris-HCl, 1% Casein, 150 mM NaCl, pH 7.4) for 1 hour. Thereafter, the reaction was carried out at room temperature for 1 hour with the serum of a polycystic infection patient diluted 200-fold with a blocking buffer. The membrane after the reaction was washed 4 times for 10 minutes with a washing solution, and then reacted with a peroxidase-labeled anti-human IgG antibody diluted 1000 times with a blocking buffer at room temperature for 1 hour. After the reaction, it is washed 5 times with a washing solution for 10 minutes, and then developed with a substrate solution for color development (3,3'-diaminobenzidene dissolved in phosphate buffered saline at 0.05% concentration and 0.018% hydrogen peroxide added). I let you. Positive E. coli colonies (those that react with antibodies in patient serum) were cultured overnight at 37 ° C. in liquid LB medium (1% Bacto Tryptone, 0.5% Yeast Extract, 0.5% NaCl). As a result of screening about 6000 E. coli colonies, 44 positive E. coli colonies could be obtained.

Next, the amino acid sequence was determined and aligned.
Plasmids were extracted from E. coli cultured in liquid, their base sequences were determined, and alignment was performed. The result is shown in FIG.
The results show only one in the case of the same amino acid sequence. As a result of this alignment, two antigenic regions were determined from the overlapping regions of each positive clone. Clones Em18C09, Em18C08, Em18C01, Em18C14 and Em18C07 are included in antigenic region 1, and the other clones are included in antigenic region 2.

In this example, four types of clones (Em18E21, Em18E20, Em18J05, Em18C09) were subjected to immunoblot analysis to determine whether these had amino acid sequences that recognize antibodies.
First, a fusion protein containing an Em18-derived peptide was prepared.
Each E. coli clone was inoculated into 250 ml of liquid LB medium and cultured overnight at 37 ° C. The cultured E. coli was recovered by centrifugation, suspended in an extraction buffer (50 mM sodium phosphate, 0.3 M NaCl, pH 8.0), and disrupted by sonication. After centrifugation, the insoluble fraction (containing the fusion protein) was washed twice with extraction buffer and dissolved in extraction buffer containing 6M urea. The dissolved protein solution was applied to a fixed metal affinity resin column to bind the fusion protein to the resin. Thereafter, the resin was washed with a washing solution containing 6 M urea (50 mM sodium phosphate, 0.3 M NaCl, pH 7.0) to remove non-specifically bound proteins. The fusion protein specifically bound to the resin was recovered by elution from the resin with an elution buffer (150 mM imidazole, 50 mM sodium phosphate, 0.3 M NaCl, pH 7.0) containing 6 M urea. In this test, TALON Metal Affinity Resins (Clontech) was used as a metal-fixed affinity resin. Urea contained in the recovered fusion protein solution was removed by dialysis against phosphate buffered saline. The fusion protein concentration was determined by BCA protein assay kit (Pierce).

  The purified fusion protein of each clone was separated under a constant current of 30 mA by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) containing 0.1% SDS. For the fusion protein sample, add an equal volume of SDS-PAGE sample processing buffer (4.0% SDS, 10% 2-mercaptoethanol, 20% Glycerol, 125 mM Tris-HCl pH 6.8), and subject it to protein denaturation at 100 ° C for 5 minutes. What was given was used. Proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride (PVDF) membrane by energization at a constant voltage of 30 V for 2 hours. The membrane was washed twice with a washing solution at room temperature and blocked with a blocking buffer for 1 hour. Thereafter, the reaction was carried out for 1 hour at room temperature with polycystic infection patient sera (A and B) diluted 200-fold with blocking buffer. The membrane after the reaction was washed 4 times for 10 minutes with a washing solution, and then reacted with a peroxidase-labeled anti-human IgG antibody diluted 1000 times with a blocking buffer at room temperature for 1 hour. After the reaction, it is washed 5 times with a washing solution for 10 minutes, and then colored with a substrate solution for color development (3,3'-diaminobenzidene dissolved in phosphate buffered saline at 0.05% concentration and 0.018% hydrogen peroxide added) I let you.

  The result of immunoblotting is shown in FIG. When patient serum A is used, all clones are positive (detected as black bands), and it is found that antibodies that recognize all these clones exist in patient serum A. . On the other hand, it can be seen that patient serum B does not have an antibody that recognizes Em18C09. That is, antigenic region 1 (FIG. 1) is not recognized by the sera of all patients, and it can be seen that a peptide fragment having antigenic region 2 (FIG. 1) is suitable as a test antigen.

In this example, clone Em18C09 was selected from antigenic region 1 and Em18E20, Em18E21 and Em18J05 were selected from antigenic region 2, and evaluation as antigens for serum testing was performed by ELISA. Serum positive for Em18 was used in this test.
First, a fusion protein containing an Em18-derived peptide was prepared by the method described in Example 2.
Next, it was evaluated which clone was suitable as a test antigen by ELISA. The test (n = 68) was performed according to the following procedure.
Each clone was adsorbed to a 96-well plastic plate (Nalge Nunc: MaxiSorpPlate) at 400 ng / hole. After discarding the antigen solution, it was washed with phosphate buffered saline (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , pH 7.4). After adding 300 μL of blocking buffer (20 mM Tris-HCl, 1% Casein, 150 mM NaCl, pH 7.4) to each hole, it was sealed again and allowed to stand at 37 ° C. for 1 hour. The blocking buffer was discarded, and 100 μL of test serum diluted 100-fold with blocking buffer was added to each well. The plate was gently shaken to mix the serum. Then, it was made to react at 37 degreeC for 1 hour, and test serum was thrown away. A washing solution (surfactant Tween 20 was added to phosphate buffered saline at a concentration of 0.05%) was added to each hole and washed. The above process was repeated 3 times.

After discarding the washing solution, 100 μL of peroxidase-labeled anti-human IgG antibody solution (Cappel, diluted 2000 times with blocking buffer) was added to each well. The plate was gently shaken to mix the peroxidase-labeled anti-human IgG antibody solution. Then, it was made to react at 37 degreeC for 1 hour. Thereafter, the peroxidase-labeled anti-human IgG antibody solution was discarded, and a washing solution was added to each hole for washing. The above was repeated 4 times.
A chromogenic substrate (2,2'-azino-di- (ethyl-benzthiazoline sulfonate)) dissolved in 100 mM citrate buffer (pH 4.7) at a concentration of 0.4 mM and 0.003% hydrogen peroxide added to each hole. ) Was added every 100 μL. After standing at room temperature (about 30 minutes), 1% SDS solution was added to each hole (100 μL / hole) to stop the reaction.
Absorbance (OD value) was measured with an ELISA reader and judged based on the OD value. In this study, sera that showed an OD value equal to or greater than the average of the OD values of 10 negative human sera plus 3 times their standard deviation were judged positive (polycystemic patients).

Em18E21に関しては、全ての血清を陽性と判断できた。つまりEm18とEm18E21の感度は同一であり、Em18E21（40個のアミノ酸）でEm18と同等の感度を得るために十分であることがわかった。また、Em18E20は、Em18E21より若干劣るが、これも検査用抗原として高い感度を有している。従って、Em18E20とEm18E21との間のアミノ酸配列を有するペプチド断片は検査用抗原として有用であるといえる。他のクローンに関しては、陽性率が減少している。 The results of ELISA for each clone of Em18 positive serum (n = 68) are shown in Table 1.

For Em18E21, all sera were judged positive. In other words, the sensitivity of Em18 and Em18E21 is the same, and it was found that Em18E21 (40 amino acids) is sufficient to obtain the same sensitivity as Em18. Em18E20 is slightly inferior to Em18E21, but it also has high sensitivity as a test antigen. Therefore, it can be said that a peptide fragment having an amino acid sequence between Em18E20 and Em18E21 is useful as a test antigen. For other clones, the positive rate is decreasing.

With regard to Em18E21, which showed good results in Example 3, five clones (Em18D01 (389 to 424 of SEQ ID NO: 1) in which the N- or C-terminal was deleted based on the amino acid sequence of Em18E21 to further narrow the diagnostic antigen region. ), Em18D02 (positions 390 to 424 of SEQ ID NO: 1, SEQ ID NO: 3), Em18D03 (positions 391 to 424 of SEQ ID NO: 1), Em18D04 (positions 389 to 423 of SEQ ID NO: 1), Em18D05 (389 of SEQ ID NO: 1) ˜422)), and usefulness as a diagnostic antigen using serum positive for Em18E21 and negative for Em18J05 (12 cases) or weakly positive (7 cases) (total 19 cases) Were examined using the ELISA method.
The deletion clone was prepared by performing PCR using primers designed to delete the N- or C-terminus of Em18E21, and ligating the resulting DNA fragment to pSCREEN-1b (+), an expression vector. And introduced into E. coli. Subsequent operations are the same as for Em18E21.

  For DNA amplification, primer F1: 5'-GACAAACTGGCGGAGATGAAC-3 '(SEQ ID NO: 6), primer F2: 5'-AAACTGGCGGAGATGAACAGA-3' (SEQ ID NO: 7), primer F3: 5'-CTGGCGGAGATGAACAGAAAG-3 '(SEQ ID NO: 6) 8), Primer R1: 5′-CTCAACTTCGCGTTGCACTTC-3 ′ (SEQ ID NO: 9), Primer R2: 5′-AACTTCGCGTTGCACTTCGTC-3 ′ (SEQ ID NO: 10), Primer R3: 5′-TTCGCGTTGCACTTCGTCACG-3 ′ (SEQ ID NO: 11) It was used. Em18D01 was prepared using primers F1 and R1, Em18D02 using primers F2 and R1, Em18D03 using primers F3 and R1, Em18D04 using primers F1 and R2, and Em18D05 using primers F1 and R3.

以上の結果より、アミノ酸配列（KLAEMNRKLKEETAASAEERDRLMAQRDEVQREVE、配列番号３）が多包虫の血清診断に最適であると考えられる。

The results are shown in Table 2.

From the above results, it is considered that the amino acid sequence (KLAEMNRKLKEETAASAEERDRLMAQRDEVQREVE, SEQ ID NO: 3) is optimal for serodiagnosis of polycystic insects.

It is a figure which shows the relationship of EM10 molecule | numerator, Em18 molecule | numerator, and the antigen (EM18D02) of this invention. The numbers in the figure indicate the positions in EM10 (SEQ ID NO: 1). FIG. 2 is a diagram showing a restriction enzyme map of plasmid pSCREEN-1b (+) used in Example 1. It is a figure which shows the amino acid sequence of a clone positive to the antibody in patient serum. It is a figure which shows the result of an immunoblot analysis about four clones (E21, E20, J05, C09).

Claims (6)

An antigen for testing for polycystiasis comprising an amino acid sequence from the 389th to 391th to the 418th to 428th positions of SEQ ID NO: 1. An antigen for testing for polycystiasis comprising the amino acid sequence of SEQ ID NO: 3 (positions 390 to 424 of SEQ ID NO: 1). An agent for testing polycystiasis comprising the antigen according to claim 1 or 2 as a main component. A method for testing for polycystiasis, comprising reacting a serum of a subject with the antigen according to claim 1 or 2 or the test drug according to claim 3. A polycyste comprising immobilizing the antigen according to claim 1 or 2 on a support, reacting a serum of a subject to the antigen, reacting a probe with a label thereto, and detecting the label. Test method. A test kit for detecting an antibody against echinococcosis (polycystematosis) from the serum of a subject, comprising the antigen according to claim 1 or 2 fixed to a support.

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