WO2003072782A1

WO2003072782A1 - Echinococcus antigen, gene encoding the same and method of diagnosing human hydatidosis

Info

Publication number: WO2003072782A1
Application number: PCT/JP2002/005094
Authority: WO
Inventors: Osamu Fujita; Tomoyoshi Nozaki
Original assignee: Japan Science And Technology Agency; Japan As Represented By Director-General Of National Institute Of Infectious Diseases
Priority date: 2002-02-27
Filing date: 2002-05-27
Publication date: 2003-09-04
Also published as: JP2003250555A

Abstract

It is intended to provide an examination method, a diagnostic method, etc. relating to hydatidosis which are excellent in sensitivity and specificity and economically advantageous owing to the use of gene recombination techniques. A gene encoding an Echinococcus-origin actin-modular protein (AMP); an immunoassay method of detecting an antibody against a polypeptide comprising this antigen protein or a part thereof; and a method of diagnosing human hydatidosis.

Description

Description Multi-hydatid antigen, gene encoding the same, and method for diagnosing polycysticercosis

The present invention relates to a novel polyhydatid antigen protein and a gene encoding the same, an immunoassay for detecting an antibody against a polypeptide comprising the antigen protein or a part thereof, and a method for diagnosing human polycysticercosis . Background art

Polycysticercosis is also found in almost all areas of Hokkaido and in Aomori Prefecture in Japan below 40 degrees north latitude, and is a multivesicular tapeworm that has foxes, dogs, cats, raccoon dogs, etc.

(Ech i nococcus mu lti 1 ocu laris) is a disease caused by accidental infection of human eggs. The number of patients with polycysticercosis worldwide is estimated to be between 100,000 and 300,000. In Japan, the cumulative number of patients up to 1998 was 373.

The parasitic sites of polycystic worms are mainly the liver, sometimes the lungs and the brain. Polycysticercosis causes liver dysfunction, etc. It is a highly lethal disease classified as a class of infectious diseases.

The condition is asymptomatic for as long as 10 years, and blood tests are often considered to be normal.In advanced stages more than 10 years after infection, hepatomegaly and discomfort in the ribs are found. Hepatoma is remarkable at the end stage, hepatic dysfunction progresses, ascites and portal hypertension symptoms, jaundice, and cachexia are seen, and the patient becomes critically ill with hepatic coma.

There is no effective drug treatment for polycysticercosis, and there is currently no cure except surgical resection. Disclosure of the invention

Methods for diagnosing polycysticercosis include interviews, immunological diagnosis, simple abdominal imaging, ultrasonography, and CT scan.

In particular, the conventional immunological diagnosis is that There is an ELISA method using a mixed antigen of m2 (glycoprotein) and cytovillin, a recombinant protein. Although the diagnostic method is highly sensitive, cross-reaction with closely related species is considerable (approximately 25% with monocysticercosis), and is expensive due to the need for a protein with a higher degree of purification. Primary screening and confirmation of infected individuals. Exclusion Not suitable for diagnosis.

In addition, an ELISA method using a crude antigen of a polycompartum is also being studied. However, although this method is advantageous in terms of cost, it uses a crude antigen, and therefore has many nonspecific reactions. There was a problem in using it as a differential diagnosis.

The inventor of the present invention aimed at providing a test method and a diagnostic method for polycysticercosis, which are excellent in sensitivity and specificity, and which are advantageous in terms of cost by using genetic recombination technology. As a result of diligent research, the present inventors have found a novel protein derived from a polycaptor and a gene encoding the protein, and have completed the present invention. That is, the present invention relates to the following embodiments.

1. A DNA encoding the actin-modulator protein (AMP) derived from multicapsule.

2. DNA containing the nucleotide sequence encoding the following protein (a) or (b) or a partial sequence thereof:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,

(b) Amino acid sequence A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a), and which has a fungal actin-regulating activity.

3. DNA containing the following nucleotide sequence (a) or (b) or a partial sequence thereof:

(a) the nucleotide sequence of SEQ ID NO: 1,

(b) a nucleotide sequence which hybridizes with the DNA consisting of (a) under stringent conditions and encodes a protein having actin-regulating activity of polycapsule,

4. Recombinant expression vehicle containing the DNA described in any one of 1 to 3 above (Vehicle) The recombinant expression vehicle must be prepared by any genetic engineering technique known to those skilled in the art. The types are, for example, various plasmid better And expression vectors such as virus vectors, cosmids, and phages.

5. The recombinant expression vehicle according to the above 4, which is an expression vector.

6. The recombinant expression vehicle according to the above item 5, which is an expression vector derived from pBR322.

7. A transformant which is a prokaryotic cell such as a bacterium and various eukaryotic cells such as a yeast cell and a mammalian cell, which have been transformed with the expression vehicle according to 4, 5, or 6.

8. The transformant according to the above 7, which is a bacterium.

9. The transformant according to the above 8, which is Escherichia coli.

10. A multiacting insect-derived actin regulatory protein, particularly a recombinant protein.

1 1. An actin regulatory protein of the following (a) or (b), particularly a recombinant protein.

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2,

(b) an amino acid sequence A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in (a), and which has a fungal actin-regulating activity.

1 2. A polypeptide comprising a part of the protein according to the above 10 or 11.

1 3. A diagnostic reagent for polyhydatid disease comprising the protein of 10 or 11 above as an active ingredient.

1 4. A diagnostic reagent for polyhydatid disease comprising the polypeptide of 12 above as an active ingredient.

1 5. A kit containing the diagnostic reagent for polycysticercosis according to 13 or 14 above. This kit generally includes test kits such as reaction plates, various buffers, diluents, washing solutions, blocking solutions, secondary reagents such as various enzyme-linked sera, and reaction reagents (color-forming compounds). It can be appropriately selected from other optional components known to those skilled in the art based on the reaction principle used for diagnosis.

16. The kit for diagnosing polycysticercosis according to 15 above, further comprising another antigenic protein of the polycysticercium as a diagnostic reagent.

1 7. The kit for diagnosing polycysticercosis according to 16 above, wherein the other antigenic protein of the polycysticercium is cytopyrin.

1 8. Various immunological measurement methods known to those skilled in the art, characterized by using the protein described in 9 or 10 above as an antigen and detecting an antibody against the protein in the subject. Law.

1 9. The measurement method according to 18 above, wherein the measurement method is an ELISA method.

20. The measurement method according to 18 above, wherein the measurement method is an immunoblot method.

2 1. The method according to any one of the above items 18 to 20, wherein the sample is serum of a subject.

2 2. An immunological measurement method comprising detecting an antibody against the protein according to 9 or 10 in a subject using the polypeptide according to 11 or 12 as an antigen.

2 3. The measurement method according to 22 above, wherein the measurement method is an ELISA method.

24. The measuring method described in ± 22, which is characterized in that it is a simulated knob method.

25. The method according to any one of 22 to 24 above, wherein the sample is serum of a subject.

26. A method for diagnosing a polycysticercosis patient, comprising detecting an antibody against the protein in a body fluid of a subject using the protein according to 9 or 10 above as an antigen.

27. The diagnostic method according to the above item 26, wherein the body fluid is serum.

2 8. Diagnosis of polycysticercosis patients, characterized by using the polypeptide described in 11 or 12 above as an antigen and detecting an antibody against the protein described in 9 or 10 above in a body fluid of the subject. Method.

29. The diagnostic method according to the above item 28, wherein the body fluid is serum. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results obtained by a measurement system using the ELISA method for the antigen Em crude antigen.

FIG. 2 shows the results obtained with the ELISA-based assay system for antigen-recombinant cytovirin.

FIG. 3 shows the results obtained by the ELISA system for the antigen-recombinant daltathione S-transferase.

FIG. 4 shows the results obtained for the antigen-recombinant actin regulatory protein in a measurement system by the ELISA method. BEST MODE FOR CARRYING OUT THE INVENTION

The DNA, protein and the like according to the present invention can be obtained and prepared using techniques well known in the art.

Further, those skilled in the art can also easily produce the diagnostic reagent and kit of the present invention based on the description of the examples in the present specification.

In addition, the immunological measurement method of the present invention can also be performed based on various principles and techniques known to those skilled in the art.

The amino acid sequence (343) of the actin regulatory protein derived from the multicapsule of the present invention and the nucleotide sequence (1902 base pairs) of the DNA encoding the same are shown below. Example

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1 (Acquisition of DNA encoding actin regulatory protein derived from polycystic insects · Determination of base sequence)

(1) Extraction of total RNA from larval tissues

Multicapsule recovered from the intraperitoneal gerbil of the gerbil maintained at the National Institute of Infectious Diseases

(Larvae tissue) was minced, and total RNA (7. g / 370ζ1) was extracted using an RNAqueous Kit (Ambion, USA) according to the conventional method.

(2) cDNA synthesis and creation of an expression library

The cDNA was synthesized using the ZAP Express cDNA Gigapack III Gold Cloning Kit (Strata gene, USA) according to the conventional method. Synthesized from the previously extracted total RNA

The cDNA was introduced into the Xho I site of the ZAP Express vector (pBK-CMV phagemid vector) (Xho I; New England Biolabs, USA), and these were introduced into the E. coli XL1-B1ue MR F ' Conversion was made.

(3) Selection of reactive clones using sera of patients with polycysticercosis Plaques formed on the transformed Escherichia coli culture plate were recorded on Hybond-N; ositively charged nylon membrane (Amersham Pharmacia Biotech, UK), and these were used as antigens using serum of patients with polyhydatid disease. Blacks showing a positive reaction in the antibody reaction were selected.

(4) Determining the nucleotide sequence of antigen 遗 gene

Rasmid DNA was purified from plaques that showed a positive reaction using Plasmid Mini Kit (QIAGEN, Germany). The DNA sequence of the inserted fragment was determined using Applied Biosystems ABI Prism 300 (Applied Biosystems, USA). The obtained DNA sequence was analyzed using the Sequence Information Survey (BLAST) of the National Center for Biotechnology Information (NCBI). As a result, we were able to obtain a total of eight types of cDNA, including the three newly discovered types.

They are shown in Table 1 below. table 1

Major antigen genes acquired

Identity Cytovillin (= EmlO, antigen Π / 3) E. multilocularis 100% quartathione S-transf; c-se '(GST) E. multilocularis 100% actin-filament fragment protein ** Echinococcus granulosus * 97 % Go to Ji Tao Leto 'Xin. Roxita'-se 'E. granulosus 96¾ ta'in light chain Drosophi la melanogas ter 85¾ ata' mus AS (= antigen 6) E. granulosus 80% calcium binding protein Schistosoma spp. 48¾ actinin human alpha-actinin2 36¾

* Species with the highest partial amino acid sequence identity

** In this study, the novel antigen gene obtained from E. multilocularis is indicated by an underline. Experimental Example 2 (Preparation of recombinant actin-modulator protein (AMP))

(1) Preparation of expression vector and expression of recombinant protein using each vector The insert fragment obtained in Experimental Example 1 (4) was amplified by PCR, and the restriction enzyme (BamHI; New England Biolabs, USA) PGEX-2T vector (GST fusion protein expression vector; Amersham Pharmacia Biotech, UK) digested with Escherichia coli and further transformed with Escherichia coli (DH5a). -D (-)-Chogalact topiranoside; Nacalai, Japan) was added to produce a large amount of recombinant protein.

(2) Separation and purification of expressed protein

The E. coli cells synthesized with the GST fusion protein were sonicated and passed through a Gluathione Sepharose 4B column (Amersham Pharmacia Biotech, UK) to adsorb only the GST fusion protein onto the column. 0.1% Triton · After washing the column with PBS. Recombinant protein fraction eluted with Thrombin (Amersham Pharmacia Biotech, UK), Experimental example 3 (ELISA kit preparation and detection)

(1) Development of measurement system by ELISA method

Dilute the recombinant protein to a concentration of 5 ug / m1 with an alkaline solution (0.05 M bicarbonate buffer) and place it in a 96-well microplate (Immulon 2HB; Dynex Technologies, USA). Drop 0 u1 onto each well, and immobilize the protein on the plate at 37 ° C for 2 hours. Washed three times with 0.05% Teen 20-PBS (PBS / T) and stored at −30 ° C. until use.

(2) Measurement with each kit

In preliminary experiments, the dilution ratio of primary and secondary sera was determined. Each plate to which antigen was added was blocked with 1% BSA in PBS / T, then primary serum (each patient's serum) diluted 200-fold with 1% BSA in PB SZT, and then 2000-fold Diluted secondary sera (anti-human IgG peroxidase conjugate; Cappel, USA) were each reacted at 37 ° C for 40 minutes. After color development with ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium; Sigma, USA), 1.25% sodium fluoride (Sigma, USA) Stop. Thereafter, the absorbance was measured at 414 nm using an ELISA microplate reader (Model 550; BIO-RAD, USA). The types of antigens used in the above kit are shown in Table 2 below. Table 2

ELISA method

Antigen used

Em crude antigen

Recombinant cytovirin (major surface antigen)

Recombinant glutathion S-transferase-se (GST)

Recombinant actin regulatory protein (AMP) cross-reaction sera from each helminthiasis patient

Number of samples

AE multiple kinoxosis E. multilocularis 22

CE single package ;! : Ki,? E. granulosus 23

NCC Cysticercosis Teania solium 20

PW ゥ I Paragonimus paragonimus wes termani 10

FH Fasciola sp. 10

NC Neka 'Tif' Control 41

Figures 1 to 4 show the results obtained for each antigen.

The results are summarized in Table 3 below.

Table 3

Selectivity

Cross reaction positive (%)

Antigen threshold: 95% Threshold: 100%

Em crude antigen 2.4¾ (M + 7SD) 0% (M + 8SD)

2 (PW2 sample) Cytovirin 0% (+ 13SD) 0¾ (M + 12SD)

GST 28¾ (M + 7SD) 20¾ (M + 8SD)

24 (CE10; NCC3; PW4; FH7) 20 (CE8; NCC3; PW4; FH5)

AMP 3.5¾ (M + 7SD) 2.4% (M + 10SD)

3 (CE1; FH2) 2 (CE1; FH1) When each antigen was used, 95% or 100! Helminthiasis patient serum

Was regarded as a false positive. The ¾ and the number of each sample are shown.

Industrial applicability

According to the present invention, a test method, a diagnostic method, and the like, which are excellent in sensitivity and specificity and are economically excellent, are provided.

Claims

The scope of the claims

I. DNA encoding a actin regulatory protein derived from polycystic worms.

2. DNA containing a base sequence encoding the protein of the following (a) or (b) or a partial sequence thereof:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,

(a) the base sequence represented by SEQ ID NO: 1,

(b) Nucleotide sequence that hybridizes with DNA consisting of the nucleotide sequence (a) under stringent conditions and encodes a protein having the actin-regulating activity of polyphelminth <

4. A recombinant expression vehicle containing the DNA according to any one of claims 1 to 3.

5. The recombinant expression vehicle according to claim 4, which is an expression vector.

6. The recombinant expression vehicle according to claim 5, which is an expression vector derived from pBR322.

7. A transformant transformed by the expression vehicle according to claim 4, 5, or 6.

8. The transformant according to claim 7, which is a bacterium.

9. The transformant according to claim 8, wherein the transformant is Escherichia coli.

10 0. Actin-regulating protein derived from polycystic insects.

I I. The actin regulatory protein of (a) or (b) below.

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2,

(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in (a), and which has a fungal actin-regulating activity.

12. A polypeptide comprising a part of the protein according to claim 10 or 11.

13. A diagnostic reagent for polycysticercosis comprising the protein according to claim 10 or 11 as an active ingredient.

14 'A reagent for diagnosing polyhydatid disease, comprising the polypeptide according to claim 12 as an active ingredient.

15. A kit containing the diagnostic reagent for polycysticercosis according to claim 13 or claim 14; 16. A kit further comprising another antigenic protein of the polycysticercosis as a diagnostic reagent. Item 16. The kit for diagnosing polyhydatid disease according to Item 15 above.

17. The kit for diagnosing polycysticercosis according to claim 16, wherein the other antigenic protein of the polycysticercium is cytovirin.

18. An immunoassay method, comprising using the protein according to claim 9 or 10 as an antigen and detecting an antibody against the protein in a subject.

19. The measurement method according to claim 18, wherein the measurement method is an ELISA method.

20. The measurement method according to claim 18, wherein the measurement method is an immunoblot method.

21. The measurement method according to any one of claims 18 to 20, wherein the specimen is serum of a subject.

22. A method for detecting an antibody against the protein according to claim 9 or 10 in a subject using the polypeptide according to claim 11 or 12 as an antigen. The immunological measurement method.

23. The measurement method according to claim 22, wherein the measurement method is an ELISA method.

24. The measurement method according to claim 22, wherein the measurement method is an immunoblot method.

25. The measurement method according to any one of claims 22 to 24, wherein the sample is serum of a subject.

26. A method for diagnosing a polycysticercosis patient, comprising using the protein according to claim 9 or 10 as an antigen and detecting an antibody against the protein in a body fluid of a subject.

27. The diagnostic method according to claim 26, wherein the body fluid is serum, 28. The polypeptide according to claim 11 or 12, wherein the polypeptide is used as an antigen. A method for diagnosing a polycysticercosis patient, comprising detecting an antibody against the protein according to claim 9 or 10 in a body fluid of the subject.

29. The diagnostic method according to claim 28, wherein the body fluid is serum.