KR100321825B1 - A gene for coding a cystein proteinase isolated from Paragonimus westermani and a use of the cystein proteinase coded by the same - Google Patents

Abstract

PURPOSE: A gene encoding cysteine proteinase derived from Paragonimus westermani Brown and use of cysteine proteinase are provided, thereby reducing the cross-reaction with other bacteria, and increasing sensitivity of diagnosis when the cysteine proteinase is used for diagnosis of infection of Paragonimus westermani Brown, so that accuracy of the diagnosis can be improved. CONSTITUTION: The gene encoding cysteine proteinase derived from Paragonimus westermani Brown has the nucleotide sequence set forth in SEQ ID NO:1, wherein the cysteine proteinase gene is isolated and sequenced by the steps of: preparing primers, primer 1 and primer 2 using beta-cyanoethyl phosphoramidite in which the primer 1 has the nucleotide sequence of 5-ACA GAA TTC CAR GGI CAR TGY GGI TCI TGY TGG-3, and the primer 2 has the nucleotide sequence of 5-TTA AAG CTT CCA IGA RTT YTT IAC RAT CCA RTA-3, wherein I is inosine, R is A or C, and Y is T or C; reacting cDNA derived from mRNA of Paragonimus westermani Brown with the primers to isolate DNA fragments associated with the cysteine proteinase; and sequencing the DNA fragments. A composition for diagnosis of infection of Paragonimus westermani Brown comprises the cysteine proteinase as an effective component.

Description

A gene for coding a cystein proteinase isolated from Paragonimus westermani and a use of the cystein proteinase coded by the same}

The present invention relates to genes encoding cystine proteases from pulmonary worms and to the use of cystine proteases encoded by them.

Pulmonary pneumoconiosis, also known as lung dystomosis, is a disease caused by the ingestion of freshwater lobsters containing parasitic insects called paragonimus westermani brown (also known as paragonimus westermani Brown ). It is distributed in South America, but unlike soil-borne parasites such as roundworm, duodenum, and worm, it is a dull parasitic insect, despite many efforts.

Pulmonary pneumococcal infection occurs when the eggs in the eggplant (miracidum) grown in the field for 15 to 20 days breaks the eggshell and parasitises in mollusks such as the first host, Daschalgi, and is converted into cercaria. It invades crustaceans, such as crayfish, and converts it into metacercaria, whereby humans and animals eat these infected crustaceans, such as freshwater dogs and lobsters, by invading the lungs and the like through the intestinal mucosa. Parasitology of Human Parasites 著).

The symptoms of pneumonia are fever, shortness of breath, and pain, such as general pneumonia and pleurisy, and sputum of the same color as fishy fishy green tea. When a pulmonary vermin is injured in a pulmonary vessel, blood is mixed with phlegm. On the other hand, when the lungs invade the brain, epileptic symptoms such as vision impairment or general paralysis appear.

Therefore, in order to treat the above-mentioned symptoms, Emethine hydrochloride (trade name; Prazacantel), which is also used as a medicine for hepatic insufficiency, is administered, but this is a treatment selected without confirming the cause of the disease. Risk of lung disease and the presence of treatment may occur. Therefore, it is necessary to first diagnose an infection caused by pulmonary pneumoniae prior to treatment.

As a test method that has been used conventionally, there is a method of finding characteristic eggs of pulmonary pneumoniae in sputum or stool of a patient. In addition, there is an intradermal reaction test that was developed in the 1950s and has been widely used to date. However, detecting an egg from sputum or feces was inconvenient to collect a sample and a test method, and furthermore, the recovery rate of the egg was only 10 to 30%. In addition, the intradermal test also showed positive response in the past infected persons not infected with pulmonary repellent (Chung et al., 1995; Sandun et al., 1959; Walton and Ju, 1959; Kim Dong-chan et al., 1969; Hunter (Hunter et al. (1958) reported that 44.7% of the fact that also appear to be positive for patients with hepatitis, this method also has a problem of poor sensitivity and specificity.

Therefore, studies have been attempted to increase the sensitivity and specificity of the intradermal reaction test using purified pneumococcal antigens (Dong-Ik Choi, 1959; Sawada et al, 1964; Ahn and G., 1975). Crude carcass extracts extracted from the antigens are antigens, and monoclonal antibodies 2Z2B3 and A3D12D3 derived from these antigens (Zhang et al., Am. J. med. Ahy., 44, 108, 1991; ibid, 49, 329, Serological methods have been tried, but it is difficult to continuously obtain antigens, severely cross-react with other parasites, and provide information on whether the results are acute, chronic or treated. (Yong et al., Korean Journal of Parasitology, 29, 293, 1991).

Therefore, the present inventors have been studying the antigens that can solve the above problems in diagnosing pulmonary pneumoconiosis by intradermal reaction test or enzyme immunoassay, while cysteine proteinase (cystosomasis) ), And is used for the diagnosis of diseases such as trichomoniasis (Mckerrow, Exp. Parasitol., 68, 111, 1989; Cynthial et al., Am. J. Tropical Med. Hyg., 42, 3435, 1990; Alderete et al., Genitourin.Med., 67, 331, 1991) attempted to use cystine protease in lung pneumococcus for the diagnosis of pneumoconiosis. Discovered and completed the present invention.

Accordingly, it is an object of the present invention to provide a gene represented by SEQ ID NO: 1 that encodes a cysteine proteinase derived from pulmonary insect repellent.

Another object of the invention relates to the use of cystine proteases encoded by the genes described above.

1 shows the results of electrophoresis of recombinant cystine proteases according to the present invention.

Figure 2 is an antibody by using the recombinant protease according to the present invention as an antigen, serum and enzyme immunoassay of pulmonary pneumoconiosis (PW), hepatosiosis (Cs), Yokogawa hyperplasia (My), Toxoplasma (Toxo) patients It is a graph showing the results of the measurement.

Figure 3 shows the results of the intradermal reaction using the recombinant cystine protease according to the present invention.

Hereinafter, the present invention will be described in more detail.

Recombinant protein prepared from the gene encoding cystine protease according to the present invention is a skin test (injection of antigen into the skin, hypersensitivity reactions (rash or papules) appear in a matter of minutes, measuring the size of yin-yang It can be used as a specific antigen in the method of measuring the enzyme) and enzyme immunological measurement, and can be used for the preparation of DNA probes, monoclonal antibodies, vaccines, etc. Can be.

Cloning the gene encoding the cystine protease from the pulmonary respiratory tract first involves designing the primers by the β-cyanoethyl phosphormidite method to isolate the genes involved in the cystine protease. Since cystine protease preserves the active site of cystine and asparagine, primer 1 (5'-ACA GAA TTC CAR GGI CAR TGY GGI TCI TGY TGG-) with specificity cleaved by cystine active site and restriction enzyme EcoR I 3 ') and primer 2 (5'-TTA AAG CTT CCA IGA RTT YTT IAC RAT CCA RTA-3') with specificity cleaved by the asparagine active site and restriction enzyme Hind III are designed. In the sequence of the primer, I is inosine, R is A or C, and Y is T or C.

In the second process, the DNA-derived cDNA library extracted from the pulmonary insect repellent is reacted with the primers to separate DNA fragments related to cystine proteases.

The third step is to determine the DNA sequence.

For example, the procedure for determining the nucleotide sequence of the cystine protease gene of the present invention and a method for producing a recombinant protein using the same will be described in more detail.

Example 1

Pulmonary larvae (metacercariae) obtained Cambaroides similis from Bogildo, Jeollanam-do, extracted the larvae from lobsters, infected five dogs at 100 dogs, slaughtered the dogs after 3 months, and recovered the lung worms. Total RNA was isolated from the collected worms of the collected lung worms using the CsCl centrifugation method. That is, after homogenizing with guanidium thiocyanate, 5.7 M (pH 7.5) of CsCl or CsTFA cushions were placed on fresh pneumococcal extracts recovered from dogs, and SW28 swing bucket rotor; Beckman. CA. USA) were centrifuged at 25,000 × g for 24 hours. Then, RNA was separated by sodium acetate (0.3M, pH 5.2) and ethanol precipitation, followed by pure separation of poly (A) ⁺ -mRNA using an oligo (dT) affinity column (Sambrook J et al., 1989).

Example 2

Using a ZAP-cDNA synthesis kit (Lot No. UC118, Stratagene, La zolla, Calif.) (Stratagene Co, 1992), a primer having a poly (A) ⁺ -mRNA and a poly dT sequence prepared in Example 1 was used as a murine mierblade. The first strand cDNA was synthesized by treatment with the murine myeloblastic leukemia virus reverse transcriptase. The second strand cDNA was synthetically polymerized using DNA polymerase I. Again, TD DNA polymerase, T4 polynucleotide kinase and Xho I restriction enzymes were used to synthesize cDNA having EcoR I and Xho I smooth ends at both ends. Thereafter, a cDNA fraction having a size of 0.5 kb or more was recovered using a Sephacryl S-400 spun column, and a Uni-ZAP XR vector (Lot No. UC118, Stratagene, La zolla, CA) was used. ) And the Gigapack II Gold packaging extract was inserted into SURE cells (Lot No. UC118, Stratagene, La zolla, Calif.) Capable of checking the recombination frequency. The transformed SURE cells were cultured in an NZY plate (made by adding agar to NZY Broth containing yeast extract and casein hydrolysate) to obtain a Uni-ZAP XR library, which was named "cDNA library".

Example 3

10 µl of the cDNA library obtained in Example 2 and 10 µl of the primers 1 and 2 were respectively added to the GeneAmp PCR reagent Kit with AmpliTaq DNA Polymerase (Perkin-Elmer Cetus) system. -Elmer Cetus, model = 9600) was subjected to PCR amplification. PCR amplification was performed by denaturing DNA at 94 ° C. for 1.5 minutes; Annealing at 25 ° C. for 1.5 minutes; The synthesis of DNA at 72 ° C. for 3 minutes was repeated a total of 45 times. To confirm the amplified DNA fragments, 1 μl of sample buffer solution (6 ×, 0.25% bromophenol blue, 0.25% xylene cyano FF, 40% sucrose) was mixed with 10 μl of the PCR amplified reaction solution. It was electrophoresed with agarose gel, in which tris-acetic acid / ethylenediaminetetraacetic acid (TAE) electrophoresis buffer was electrophoresed at 100V for 30 minutes to separate a 1.4 kbp DNA fraction.

Example 4

The amplified DNA fraction obtained by electrophoresis in Example 3 was electrophoresed on a 1% agarose gel, and then cut and purified using a Geneclean II kit (Bio 101 Inc.) to amplify the DNA fragment (Wilson VG et al., 1988), purified DNA was cloned into pT7Blue T-vector (Novagen). The cloned plasmid DNA was purified using an alkalisis method (Birnboim HC et al., 1979; Ish-Horowicz O and Barke JF, 1981) and then purified using the Geneclean II kit.

Example 5

The DNA purified in Example 4 was subjected to a PCR reaction under the same conditions as in Example 3 by adding a T7 promoter primer and a T3 reverse primer (ABI cloning system), and containing 8M urea. Electrophoresis was performed for 12 hours at 2,500 V, 30 mA using a 4.75% polyacrylamide gel. Electrophoresis results were analyzed using a Microgene software program (ABI). The result is shown in SEQ ID NO: 1.

SEQ ID NO: 1 is a nucleotide sequence of a partial nucleotide sequence of a pneumococcal cystine protease deposited to GenBank with U69120 on October 3, 96, and is an amino acid sequence with several known protease enzymes. 49.6% of the cystine protease of the enzyme, 57.7% of the cystine protease of the Schistosoma mansoni , 41.5% of the cystine protease of the human cathepsin L , Fasciola hepatica It shows 40.4% similarity to the cystine protease of, and it can be seen that asparagine, cystine and histidine conserved sites exist in the amino acid sequence.

Example 6

A cystine protease encoded by the nucleotide sequence of the gene obtained in Example 5 was prepared by the following method. That is, primers AmPo'1 (5'-GCGCTCGAGAAAAGAATGCGCTCAACTCTGAG-3 ') and primers AmPo'3 (5'-CGCGTC GACCTGAATGATTGAGGTTGT-3') were designed based on the nucleotide sequence obtained in Example 5, and were made to order (Biosynthesis). ) Then, PCR amplification was performed using the ExTaq (manufactured by Takara) system for the cDNA library, primer AmPo # 1, and primer AmPo # 3 obtained in Example 2. PCR amplification is a step of denaturing DNA for 30 seconds at 94 ℃ using a DNA Thermal Cycler (Perkin-Elmer Cetus); Annealing at 55 ° C. for 30 seconds; The synthesis of DNA at 72 ° C. for 1 minute was repeated a total of 35 times. The amplified DNA fragments were electrophoresed with 1% agarose gel to confirm the expected size, cut out, and the pure PCR product PwCP1 was isolated using a QIAEXII Gel Extraction Kit (QIAGEN). The isolated PwCP1 was immediately reacted with the TOPO vector included in the TOPO TA Cloning Kit (Invitrogen) to synthesize TOPO-PwCP1, and transformed into E. coli TOP10 cells also included in the kit.

Transformed TOPO-PwCP1 / TOP10 were isolated as a single clone by incubating at a temperature of 37 ° C. in solid LB medium (containing 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 50 μg / ml empicillin). Each isolated clone was shaken (250 rpm) at a temperature of 37 ° C. in a liquid LB medium (containing 50 μg / ml empicillin), and then the inserted TOPO-PwCP1 was separated to sequence the base obtained in Example 2. In comparison with the sequence, clonies having the same sequence were selected. The TOPO-PwCP1 obtained from the selected clones was digested with restriction enzymes XhoI (Promega) and SalI (Promega) to separate PwCP1 from the TOPO vector, and then electrophoresed with 1% agarose gel to confirm the expected size. Pure PwCP1 was obtained using a QIAEXII Gel Extraction Kit (QIAGEN), which was named "XSPwCP1".

Meanwhile, pPICZαA / TOP10 transformed with the expression vector pPICZαA (Invitrogen) in Escherichia coli TOP10 cells was shaken at 37 ° C in a liquid LB medium (containing 50 µg / ml Zeosin (Zeocin ^™ ; Invitrogen)). After (250rpm), pPICZαA was isolated and digested with restriction enzymes XhoI and SalI. The cleaved product was electrophoresed with 1% agarose gel to confirm the expected size, cut and separated into pure pPICZαA using QIAEXII Gel Extraction Kit (QIAGEN), which was named "XSpPICZ".

XSPwCP1 and XSpPICZ separated above were mixed by the same mass number and then applied to a T4 DNA ligase (New England Biolabs) system for ligation. A portion of the reaction solution was then transformed into E. coli TOP10 cells, cultured in solid low salt LB medium (containing 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride and 50 μg / ml zeocin) into a single clone. Separated. Each isolated clone was shaken (250 rpm) at a temperature of 37 ° C. in liquid low-salt LB medium (containing 50 μg / ml zeocin), and then the inserted pPIC-PwCP1 was isolated and the PwCP1 portion was sequenced. The sequencing results were compared with the nucleotide sequence of Example 2 to have the same sequence as "pPIC-PwCP1" and E. coli clones transformed with pPIC-PwCP1 was named "pPIC-PwCP1 / TOP10".

The pPIC-PwCP1 / TOP10 was shaken (250 rpm) in a liquid low-salt LB medium (containing 50 µg / ml zeosin) at a temperature of 37 ° C, and then pPIC-PwCP1 was purified using Plasmid Midi Kit (QIAGEN) Separated.

The isolated pPIC-PwCP1 was cut with restriction enzyme SacI (Promega), and then mixed with Yeast KM71 cells (invitrogen) and transformed using Gene Pulser II (Bio-Rad). Transformed Yeast KM71 cells were inoculated in solid YPDS (25 g / ml zeocin, 1% yeast extract, 2% peptone, 2% dextrose, 1M sorbitol, and 2% agar) medium, incubated at 30 ° C, and monoclonal. Separated. Each isolated clone was then inoculated into a solid YPDS medium containing 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml zeocin and then grown well at high zeocine concentrations. The clone was isolated and named "pPIC-PwCP1 / KM71".

The isolated pPIC-PwCP1 / KM71 was inoculated in 10 ml of MGYH (1.34% YNB (Yeast nitrogen base; Difoco)), 1% glycerol, 4 × 10 ⁻⁵ % biotin and 0.004% histidydine) medium, followed by OD ₆₀₀ Shaking was carried out at 30 ° C. at 250 rpm to obtain a value of 2-6. The culture was then inoculated again in 1 L of MGYH and shaken at 30 ° C. until the OD ₆₀₀ value was 2-6. Was centrifuged for 5 minutes at 1500x g discard the culture supernatant, MMH (1.34% YNB, 4 x 10 -5% biotin, and 0.5% ethanol) to the culture medium 250㎖ suspension was incubated at 30 ℃. At this time, methanol was added at intervals of 24 hours to give a final concentration of 0.5%, which was continued for 96 hours.

Then, the supernatant was collected by centrifugation at 1500x g for 5 minutes, and sodium chloride solution was added to 0.3 M. Then, buffer A (20 mM sodium phosphate, 500 mM sodium chloride, pH 6.0) was added to pH 6.0. The solution was concentrated using a concentrator (Amicon). Meanwhile, after washing the ProBond coulum of the expression system (Invitrogen) with buffer A twice, the concentrated supernatant was added to the column to bind to the resin. The column is then washed three times with Buffer A, then with Buffer B (same as Buffer A and pH 5.5) to wash nonspecifically bound proteins and pass through Buffer C (same as Buffer A, pH 4.0). PwCP1 was eluted. Eluted PwCP1 was concentrated with a concentrator. As a result of electrophoresis on 1% agarose gel, the expressed PwCP1 showed a molecular weight of 45 kD (FIG. 1).

Example 7

Using the protein obtained in Example 6 (PwCP1) by the enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay) lung insects, liver insects, metagonimus yokogawai ( Toxoplasma gondii ) neutralizing antibodies as follows method Detected.

(1) After diluting the recombinant cystine protease isolated in Example 6 to a concentration of 1 to 5 µg / ml using 0.05 M carbonate buffer (pH 9.6), an immunoassay plate (Immunoplate, Nul) was dispensed in 100 μl, and then maintained at 4 ° C. for 18 hours to adsorb antigen.

(2) After the adsorption was completed, the plate was washed three times with ELISA washing solution (0.85% saline solution, 0.05% Tween 20) three times, and then 100 mL of the plate was plated with 3% Scheme Milk (Difico). The reaction was blocked for 2 hours at < RTI ID = 0.0 >

(3) Neutralizing antibody measurement of each parasite and protozoa was performed by separating serum from blood collected from a test animal, and then diluting them from 1: 200 to 1: 500 using 3% skim milk (Difico). Diluted in.

(4) After the blocking reaction of the plate to which the antigen was attached, the blocking solution was discarded, and 100 μl of each test serum diluted to the above concentration was dispensed.

(5) Serum-infused plates were incubated at 37 ° C. for 1 hour, and then each wash was washed three times with a wash solution for about 5 minutes.

(6) peroxidase conjugated rabbit anti-human immunoglobulin (cappel), peroxidase conjugate anti-, diluted to 1,4,000 in PBS / Tween 20 in each washed plate. 100 μl of peroxidase conjugated anti-dog immunoglobulin and peroxidase conjugated rabbit anti-cat immunoglobulin were aliquoted and incubated at 37 ° C. for 1 hour.

(7) The reaction was washed three times or more the respective plate in the wash liquid then substrate solution (0.01M phosphate citrate buffer solution (o to pH 5.0) to each of the plate-to-phenylenediamine (o -phenylenediamine) 0.05% ( After dissolving to W / N), the solution prepared by adding H ₂ O ₂ to 0.06% (W / V) was dispensed by 100 μl, and then reacted and developed for 30 minutes at room temperature, followed by 2M sulfuric acid (H ₂ SO ₄ ) was added to stop 50 μl.

(8) Absorbance was measured at 490 nm using an ELISA reader for the reaction in the plate where color development was stopped, and the results are shown in FIG. 2.

As shown in FIG. 2, enzyme immunoassay was carried out using a recombinant protein prepared from a gene encoding cystine protease derived from a pulmonary worm as an antigen. It can be clearly distinguished from.

Example 8

An intradermal reaction test was performed using the recombinant cystine protease obtained in Example 6 as an antigen. That is, after shaving the back of Spraqu-dowley rat infected with pulmonary worms, 20 μl of physiological saline, crude extract purified from adult pulmonary worms, and the recombinant cystine protease of Example 6 at 5 cm intervals (each, Papules were made by intradermal injection). After 20 minutes, 1% Evans blue (evans blue) was injected into the tail vein of the white paper, and then slaughtered and skinned to examine the inner reaction site, and the result is shown in FIG. 3.

3, it can be seen that only the portion treated with recombinant cystine protease is stained blue with Evans blue.

As described above, the present invention discloses a sequence of a gene encoding a cystine protease derived from a lung absorbing layer, thereby using a cystine protease encoded by the cystine protease as an antigen in the diagnosis of pneumoconiosis. Regarding the use of, cystine proteases can reduce cross-reaction with other parasites and increase sensitivity, thereby increasing diagnostic efficiency.

Claims (3)

A gene represented by SEQ ID NO: 1 and encoding a cystein proteinase derived from pulmonary insect repellent (肺吸蟲, Paragonimus westermani ). A cystine protease encoded by the gene of claim 1. The composition for diagnosing pulmonary pneumoconiosis, comprising the cystine protease of claim 2 as an active ingredient.