KR0175523B1 - Gene coding for cysteine protease in paragonimus westermani and recombinant protein - Google Patents

Abstract

The present invention relates to a gene sequence coding for cysteine protease of the parasitic worm, and a protein expressed by this sequence. The gene sequences and protein sequences according to the present invention have been identified by using specific primers, and these primers can be usefully used to diagnose infection of the paragonimiasis.

Description

The gene sequence encoding the cervical protease of the parasitic worm virus and the recombinant protein

The present invention relates to a whole gene sequence encoding a cysteine protease (hereinafter referred to as cysteine protease) and an expressed protein, which play an important role in the pathogenesis of the paratuberculosis causing lung disease.

Paragonimus is distributed in Korea, Japan, Taiwan, and China, and can cause lung disease in humans (Yokogawa et al., Adv. Parasitol 3: 99-158, 1965). When the paragonimus is infected, the parasite larvae of the paragonimus encapsulate in the duodenum. The larva immediately penetrates into the abdominal cavity and penetrates through the organs of the abdominal cavity for about 1-3 weeks, invades the lung, which is the main parasite, and parasites for about 5 years (Im et al .., Radio Graphics, 3: 99-158, 1993). The action of proteases in the parasitic worm has long been studied. As a result, the larvae of the parasitic larvae have 28 kDa molecular weight, the most active thiol protease at pH 7.5, the molecular weight of 20 kDa, and the most active acidic cysteine protease at pH 5, (See Yamakami et al., Comp Biochem Physiol 37B: 643-648, 1987). It has been proved that the protease secreted by the parasite not only participates in the transplantation of the host, but also is involved in the avoidance of the host immune response and the occurrence of the host inflammatory reaction. That is, cysteine proteases that hydrolyze immunoglobulin G in insect classes such as twelve mansoni and epilepsy have been discovered (Auriault et al., Immunolgy Letter 2: 135-139, 1980; and Chapman et al., Vet. Parasitology 11: 160-167, 1982), enzyme proteins that degrade immunoglobulin G in nematodes and astrocytes have also been found or purified and some amino acid sequences have been reported (Tamashiro et al., J. Parasitology 73: 149- 154, 1987; and Kong et al., Parasitology 109: 611-621, 1994).

In the past, pulmonary syphilis was diagnosed by the method of egg test, reaction in the blood and antibody test. However, in the case of the diagnosis of infectious disease by the egg test method, the detection rate of the egg test is only 10-40% (Lee et al., Korean J, Intern Med 37: 71-80, 1989) A method of diagnosing and measuring a specific antibody level in a patient's serum by enzyme immunoassay is used. Currently, treatment is with Praziquantel (brand name: Shinpung Pharmaceutical), but the treatment period is long and the specific antibody in the serum of the patient is gradually decreased after the treatment and the antibody is not negative even though the treatment is successfully completed (See, for example, Cho et al., Korean J. Parasitiol, 27: 15-21, 1989). Thus, cysteine protease plays an important role in the parasitic worm, but the gene has not been cloned, and it has not fundamentally solved the problem of diagnosis and treatment.

Up to now, the gene sequence of cysteine protease has been identified in human (Cao et al., Gene 139: 163-169, 1994), mouse (Chan et al., Proc. Natl. Acad. Sci. 83: 7721-7725, 1992), Cruzwort gibbons (see Aaslund et al., Genebank X54414, 1990), epilepsy (Wijffels et al., Biochem. J. 299: 781-790, 1994) et al., Mol. Biochem. Parasitol. 62: 1-8, 1992). However, the gene sequence of cysteine protease has not yet been reported in the parasitic insects.

Therefore, an object of the present invention is to provide genetic information of cysteine protease of the parasitic worms for the diagnosis of pulmonary avulsion syndrome and therapeutic agent, and a synthetic probe for diagnosing it, in order to identify the problem in diagnosis or treatment of pulmonary infectious disease at present .

FIG. 1 is a systematic diagram showing the process for locating a gene for cysteine protease in a pneumoniae.

Figure 2 shows the synthesis of single and double strands during the construction of the cDNA library for mRNA in the paragonimus.

Figure 3 shows the result of size selection of the cDNA generated during the construction of cDNA library for mRNA.

Figure 4 shows the gene sequence of the partial cysteine protease of the parasitic worms determined by reverse transcription polymerase chain reaction (RT-PCR).

5 shows the gene sequence of the parasitic cysteine protease and its corresponding amino acid sequence.

FIG. 6 shows a gene restriction map of the parasitic cysteine protease.

7 shows the process of constructing pETPwCP using the plasmid pET23d (+) for expression of the organism of corynebacterium cystein protease.

Figure 8 shows the gene map of PCR and pETPwCP during the process of making the recombinant vector pETPwCP.

Figure 9 shows SDS-PAGE photographs used for the analysis of recombinant cysteine protease.

FIG. 10 is a Northern analysis photograph showing the degree of expression of cysteine protease in the parasitic worms.

The above object of the present invention is achieved by providing a recombinant cysteine protease produced in E. coli using a gene sequence encoding a cysteine protease in a parasitic organism and a gene recombination technique. Another object of the present invention is achieved by providing a synthetic probe which is used for the rapid diagnosis of an infection in a human body of a parasitic organism.

The present invention firstly establishes a plan such as that of the first line in order to search for the gene of the parasitic cysteine protease (see FIG. 1). Specifically, messenger RNA (hereinafter referred to as mRNA) is isolated and purified from cells producing the desired protein in order to find the gene sequence encoding the protein. The obtained mRNAs were converted into complementary DNAs (hereinafter referred to as cDNA) using reverse transcriptase. After annealing the resulting cDNA with (dC) n tail and (dG) n tailed vector, And the like. This strain has a cDNA corresponding to various mRNAs, and the bacteria of such a strain is called a cDNA-library. In order to select cells having the desired gene from among the transformed cells, a colony hybridization method (see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, ), 99. 312-319, incorporated herein by reference). As a probe, a portion having high homology in the gene sequence of cysteine protease already known in other species is selected and synthesized. Thereafter, reverse transcription polymerase chain reaction (hereinafter referred to as RT-PCR) is carried out using this to obtain a partial gene sequence (see FIG. 4). The obtained product was labeled with a radioactive isotope to obtain a positive clone having the gene sequence of cysteine protease. Subsequently, this sequence was subcloned into a suitable carrier such as a known plasmid pBluescript II (-), and then a recombinant plasmid The gene sequence of the obtained cysteine protease can be obtained by using the primers (T3 and T7), for example, by the method of terminating the dideoxynucleotide chain (Maniatis et al., 1982 Molecular Cloning) . In order to confirm that this sequence is cysteine protease, a blast search program and the like can be utilized, and the conversion to an enzyme cleavage site and an amino acid sequence can be performed by a DNA strider 1.0 analysis program. Then, in order to express the cysteine protease gene (PwCP) in E. coli, the expression vector pET23d (+) (obtained from Novagen Co., WI 53711, USA, Madison, Wis.) Is genetically modified to carry out recombination. In the present invention, first, the expression vector pET23d (+) was treated with SacI and Xhol, and purified and separated by electrophoresis on 1% agarose gel. In this process, the start codon of the cysteine protease gene (PwCP) is removed to use the start codon of the vector, and an oligonucleotide containing a suitable enzyme cleavage site is synthesized. The present inventors synthesized 5'-GAAGTCTCGAGTTfIGTGAATGATGGCGG-3 'to have 5'-ATCGAGCTCAAGAACAGGGCACGG-3' to have a restriction enzyme Sacl cleavage site on the 5 'side and a restriction enzyme Xtlol cleavage site to the 3' The product was digested with restriction enzymes SacI and Xhol, separated and purified on a 1% agarose electrophoresis gel, and annealed in a vector to construct a recombinant pETPwCP plasmid (see Fig. 8). The recombinant plasmid was transformed into a suitable host such as Escherichia coli, for example, E. coli BL2l (DE3, commercially available from Novagen Co. of WI 53711, Madison, WI) and IPTG was added at 1 mM to express the fusion protein 7).

The degree of expression of cysteine protease was confirmed by Northern analysis of cysteine protease gene (PwCP) labeled with radioactive isotope. As a result, it was confirmed that a gene of about 1 kb in size was expressed at adult time.

Hereinafter, the present invention will be described more specifically by way of the following examples, which do not limit the present invention in any way.

[Example]

[Example 1]

To obtain a gene for producing cysteine protease, a cDNA library was first prepared from an adult. Figure 1 is a systematic flow diagram for gene cloning. Specifically, 100 parasitic larvae were orally infected to the dogs and 9 months later, the parasites were collected from the lungs of the dogs. About 2 mg of total RNA was isolated from 50 adults. RNA zol B (available from Biotecx Co. of Huston, TX 77033) was used to isolate total RNA. The total RNA thus obtained was electrophoresed on a 1.1% agarose gel mixed with 1 M formaldehyde. As a result, the amount of 18S was much higher than that of 28S in the case of parasites. 15 [mu] g of mRNA was purified by using oligo (dT) substrate that specifically binds mRNA from total RNA. A single strand of 0.5 kb to 4.5 kb in size was prepared using oligo (dT) primer and reverse transcriptase (AMV-RT) with 8 ㎍ of the prepared mRNA as a template and 5 'end with restriction enzyme Xho I (See FIG. 2). A double strand of the size corresponding to a single strand was made using a DNA polymerase in a single strand (see FIG. 2; in FIG. 2, the numeral 1 represents the first strand and 2 represents the second strand). Here, a restriction enzyme EcoR I linker was attached to the vector for annealing, and the EcoR I end was phosphorylated with T4 polynucleotide kinase. An overhang was made at the Xho I site of the linker-attached cDNA. The cDNA was passed through a Sephacryl S-400 spun column (Pharmarcia Co.) to collect 0.5 kb or more. FIG. 3 shows that as a result of the size fractionation of this cDNA, numeral 1 is a size marker and numerals 2-8 represent fractions 1-7. Then, about 100 ng of the prepared cDNA was reacted and annealed with 1 운 of a carrier (Uni-Zap XR vector (obtained from Stratagene Co. of CA 92037 La Jolla, USA) and then in vitro packaging (Giga Pak (Purchased from Stratagene Co., La Jolla, CA, USA), to construct a cDNA-library.

[Example 2]

In order to search for the cysteine protease gene in the cDNA-library, four kinds of primers were synthesized by selecting high-homology regions among known cysteine protease gene sequences (see Table 1).

Note) R represents A or T.

Reverse transcription polymerase chain reaction was performed using a synthetic primer. Namely, 4 μl of dNTP (2.5 mM), 5 μl of 10 × PCR buffer (500 mM KCI, 100 mM TriS-HCl, 15 mM MgCl ₂ and 1 mg / ml BSA) and 2 μl of sense primer 0.2 μg / μl), 2 μl of antisense primer (0.2 μg / μl), 24 μl of DW (distilled water) and 0.5 μl of Taq polymerase (5 U / ml) at 95 ° C. for 30 seconds, 55 ° C. for 1 minute, The chain reaction was performed 30 times at a cycle of 30 seconds to obtain a partial cysteine gene sequence of 223 bp in the combination of the PwCPF3 sense primer and the PwCPRl antisense primer (see also FIG. 4B). 4A, numeral 1 is a 100 bp marker, 2 is PwCPF1 + PwCPR1, 2 is PwCPF2 + PwCPR1, 3 is a combination of PwCPF3 + PwCPR1, and the sequence shown is a partial sequence of the cysteine gene. The gene sequence thus obtained is partly identical but has a similarity of 90% or more with the cysteine gene sequence of the epilepsy. This product was labeled with a radioactive isotope ( ³² P) and cDNA-library was searched in three steps to obtain two positive clones. This positive clone was obtained commercially from a purified clone using a helper phage (commercially available from Stratagene Co.), a recombinant plasmid, pBluescript II (-) (commercially available from Stratagene Co. of La Jolla, CA 92037 USA ) Was isolated in a single strand and this plasmid was transformed into Escherichia coli SORL strain (commercially available from Stratagene Co.) to obtain 8 cells having the gene of cysteine protease. Plasmid DNA was isolated from the obtained cells and reacted with restriction enzymes EcoR I and Xho I at 37 ° C for 1 hour to identify the inserted DNA by electrophoresis on 1% agarose gel, followed by dideoxynucleotide chain termination (Maniatis et al., 1982 Molecular Cloning) using T3 and T7 primers to determine the gene sequence of 1030 bp cysteine protease in the parasite. Figure 5 shows the full sequence of the gene. In Figure 5, * is the stop codon, the underlined portion near the N-terminus is the active site of cysteine protease, and the underlined portion at the bottom is the predicted polyadenylation site. The middle underlined sequence is the RT-PCR product. This gene has an N-terminal sequence at the 5 'end and a 3'-noncoding region. It can be seen that the 3'-noncoding region has a poly (A) sequence. Blast analysis showed that the prokaryotic cysteine protease had 235 amino acids of pro-from and ribosome binding sites of 6 nucleotides from -15 to -10 of pro-from. The mature enzyme consisted of 16 N-terminal amino acids and 214 polyphedides, 35.6% with papain, 48.1% with parasitic larvae neutral thiol protease, and the cathepsin L L) were 40.0 and 41.1%, respectively.

[Example 3]

For expression of the cysteine protease gene (PwCP) in E. coli, the expression vector pET23d (+) (Novagen Co., Wis. 53711, USA, Madison, USA) First, the expression vector pET23d (+) was treated with restriction enzymes SacI and XhoI and then separated and purified by electrophoresis on 1% agarose gel (see FIG. 8; In Figure 8, the number 1 is the lambda / HindIII marker, and 2-4 is the PCR product.) To use the start codon of the vector, PwCP removes the start codon of the cysteine protease gene (PwCP) 5'-GAAGTCTCGAGTTAGTGAATGATGGCGGCGG-3 'was synthesized to have 5'-ATCGAGCTCAAGAACAGGGCACGG-3' to have a restriction enzyme SacI cleavage site and 3 'end to have an XhoI cleavage site, and 2 μl To PwCP, 4 [mu] l dNTP (2.5 mM), 5 (0.2 μg / μl), 2 μl of the antisense primer (0.2 μg / μl), 24 μl of the antisense primer (0.2 μg / μl), 10 μl of PCR buffer (500 mM KCl, 100 mM Tris-HCl, 15 mM MgCl ₂ , 1 mg / PCR was carried out 30 times at a cycle of 30 seconds at 95 DEG C, 30 seconds at 60 DEG C and 1 minute at 72 DEG C (see Fig. 8). The reaction product was treated with restriction enzymes SacI and XhoI, and purified and electrophoresed on an electrophoresis gel, annealed in a vector to construct a recombinant plasmid. The plasmid was designated pETwCP (see Fig. 8). , BL2l (DE3) (commercially available from Novagen Co. of WI 53711, Madison, Wis.), Placed in 3 ml of LB medium at 1 mM IPTG, and centrifuged at 12,000 rpm for 1 minute The E. coli was collected and then the supernatant was removed, and 50 μl of water and 2 × SDS sample buffer (1.25 ml of 0.5 M Tris-Cl, pH 6.8, 2 ml of 10% SDS, 1 ml of 100% glycerol, 500 μl of 2-mercaptoethanol, 100 μl of water and 150 μl of 0.1% BPB) The cells were sonicated and electrophoresed on SDS-polyacrylamide gels at 12%. As a result, it was confirmed that a protein of about 28 kDa was expressed. The result is shown in FIG. 9, and the ordinate Mr is a molecular weight marker (kDa), numeral 1 is a protein expressing only a gene-free vector, 2 is a gene encoding a mature form of a parasitic cysteine protease but not expressed 3 is a protein in the case of inserting and expressing a mature form gene of a parasitic infestation cysteine protease, 4 is a protein in which the entire gene of the parasitic cysteine protease is inserted but not expressed, It is a protein that inserts and expresses the whole gene of the parasitic cysteine protease.

[Example 4]

To determine the expression level of the cysteine protease gene (PwCP), total RNA (obtained in Example 1) was obtained from the parasitic insect adults using RNA zol B (obtained from Biotecx Co.) reagent and then analyzed using an ultraviolet spectrometer 20 의 of total RNA was denatured in water for 5 minutes and then electrophoresed on 1.1% agarose gel mixed with 1 M formalin. After staining with ethidium bromide, it was observed under ultraviolet light to confirm the position of the 23S and 16S ribs. This RNA was transferred to a nylon membrane and then subjected to hybridization. PwCP was labeled with ³² P-dcTP by a random hexamer method (Amersham Co.) followed by 50% formalin, 6X SSC, 10% dextrin sulfate, 1% SDS, and blocking agent The reaction was carried out at 42 ° C for 18 hours, washed three times with a solution of 1 × SSC and 0.1% SDS, and exposed to X-ray film for color development. As a result, it was confirmed that about 1 kb of the gene was expressed in the adult period as shown in FIG. 10.

The synthetic primer sequences used in the present invention can be used not only for the diagnosis of the infection with the parasitic infestation but also the gene sequence and the protein sequence of the parasitic virus according to the present invention are useful for the treatment of diseases caused by the paragonimus by molecular biologic methods In addition, the recombinant protein is an important antigen protein in the production of a diagnostic kit for pulmonary metapneumonia.

Claims (7)

A DNA sequence encoding the cysteine protease of the parasitic worm virus consisting of the nucleotide sequence shown in FIG. 5. A cysteine protease consisting of the amino acid sequence shown in FIG. 5 and having a molecular weight of about 28 kDa. Plasmid pETPwCP containing a gene encoding the cervical protease of the parasitic worm. An E. coli strain BL21 containing the vector pETPwCP according to claim 3. A synthetic probe comprising the following base sequence: 5'-GAGTGGTATTTATTCAGAGCCAAaC-3 '(PwCPF3) 5'-GATTKGGCAATTCTACCGCATTGCC-3 '(PwCPR1) 6. The probe of claim 5, wherein the probe is for use in the diagnosis of infectious disease of a parasitic organism in a human or animal. Partial DNA sequence shown in Figure 4B.