KR101161920B1 - A composition and method for screening anti-malarial drug by using the active form of recombinant calpain in Plasmodium falciparum - Google Patents

Abstract

The present invention provides a composition for screening an antimalarial drug including calpain, which is a cysteine protease of tropical malaria protozoa, and a method of screening using the same as a target material. In detail, the present invention clones the calpaine gene of the tropical malaria protozoa, completes the active form of recombinant calpain through a bacterial protein expression system, identifies its enzymatic properties and functions, and By developing a method for screening malaria medicaments, it can be widely applied to diagnosis of malaria patients, prevention or treatment of malaria.

Calpine, Cysteine Protease, Tropical Fever Malaria, Antimalarial Agents

Description

A composition and method for screening anti-malarial drug by using the active form of recombinant calpain in Plasmodium falciparum}

The present invention relates to a method for screening an antimalarial drug comprising synthesizing an active recombinant protein of calpain, a cysteine protease of tropical malaria protozoa, and using it as a target.

1, Snow, R. W. et al., (2005) Nature, 434, pp214217

Guerrant, R. L. et al., (2001) Essentials of tropical infections diseases, pp 341-355, Churchill Livingstone press

3 Banerjee, R., et al., (2002) Proc. Natl. Acad. Sci. USA 99, pp 990 995

4 Sijwali, P.S. et al., (2001) Biochem. J. 360, pp481489

[Reference 5] Rosenthal, R.J. (2004) Int. J. Parasitology. 34, pp1489-1499

6 Eggleson, K.K., et al., (1999) J. Biol. Chem. 274, pp3241132417

7, Kim, Y. M., et al., (2004) J. Biochem. 193, pp 189-195

8, Francis, SE et al., Annu. Rev. Microbiol. 51, pp97-123

Document 9 Laemmli, U.K. (1970) Nature, 277, pp 680-685

10 Debiasi, R. L., et al., (1999) J. Virol. 73, pp695-701

[11] Lambros, C., et al., (1979) J. Parasitol., 65, pp418420

12, Arthur, J.S., et al., (1995) FEBS Lett. P368, pp397400

The present invention relates to a composition and method for the screening of antimalarial drugs using calpain, a cysteine protease of tropical fever malaria protozoa.

Malaria is the five major diseases designated by the World Health Organization (WHO) and is a fatal disease with a death rate of 500,000 worldwide in one year (Snow, RW et al., (2005) Nature, 434, pp214217). Malaria is caused by protozoan parasites of the genus Plasmodium. Four species that infect humans include Plasmodium falciparum (tropical malaria), Plasmodium bibox ( P. malaria), Plasmodium malaria ( P. malariae ) and Plasmodium obale ( P. ovale ) Among them, Plasmodium falciparum mainly causes acute malaria and often fatal malaria (Guerrant, RL et al., (2001) Essentials of tropical infections diseases, pp341-355, Churchill Livingstone press), Associated morbidity is significant, and most of the world's population is at risk for this disease. Malaria is a public health problem in areas where 40% of the world's population lives, and the disease has serious social and economic consequences for these communities. The disease has recently been revived by the abandonment or collapse of regulatory measures and the increased resistance of the vector to insecticides and falciparum malaria for chemotherapy. Therefore, it is urgent to develop effective prevention and treatment for malaria.

The most important start in the development of new antimalarial drugs is to find new therapeutic targets that are key to the growth of malaria parasites and thereby develop drug screening systems. In order to develop a therapeutic method and a therapeutic agent for suppressing malaria recently, the integrated analysis of genome sequence, DNA polymorphisms, mRNA and protein expression is carried out through the analysis of the infectious agents, the host mosquito and host human, human and malaria. Research is underway to understand and identify the interrelationships and actions of protozoa. Based on this, efforts are being made to reduce malaria mortality and mortality by identifying and verifying drug targets for new malaria therapeutics and developing malaria vaccines. It is becoming. The Plasmodium sequencing projects have reported 25-30 Mb of P. falciparum genome, from which 14 chromosomes and about 5,000 genes have been identified. Analysis of P. falciparum genomic data has attracted attention of various proteases as targets for antimalarial drugs, and the specific design of inhibitors for these enzymes has been made, further targeting them. Antimalarial drugs are under development.

     In clinical manifestations caused by malaria protozoa, most of the sporezoite delivered by mosquitoes breaks down in the liver, creating a large amount of merozoite, which repeatedly invades human red blood cells. It appears by destroying red blood cells. Malaria protozoa can obtain amino acids, which are necessary for breaking down the red blood cell membrane, invading other red blood cells and growing in red blood cells, through biosynthesis, host plasma, and hemoglobin degradation of the host, but most of them are obtained by breaking down hemoglobin of red blood cells. Protozoal proteases are involved. Malaria protozoa transfer more than 80% of hemoglobin from red blood cells to acidic food vacuole and break it down into heme and globin, and break down globin into amino acids for protein synthesis and energy metabolism. Hemoglobin breakdown mechanisms in malaria protozoa include three types of proteases (4 types of aspartic proteases: plasmepsin I, II, IV, and HAP), and 3 types of cysteine proteases. (cysteine proteases: falcipain-1, -2, and -3), ③ a metalloprotease (falcilysin) has been reported to be involved (Banerjee, R., et al., (2002) Proc. Natl.Acad.Sci. USA 99, pp990995; Sijwali, PS et al., (2001) Biochem. J. 360, pp481489; Rosenthal, RJ (2004) Int. J. Parasitology. 34, pp1489-1499; Eggleson, KK , et al., (1999) J. Biol. Chem. 274, pp3241132417).

The development of antimalarial drugs targeting three types of proteases is underway. To date, crystallization of plasmepsin II has been successful, and recombinant plasmin I / II and falcipain-2 have been successfully developed. As a result, the characteristic design of inhibitors for these enzymes is being made. The present inventors have succeeded in the expression and purification of recombinant proteins from the Plascinsin II and IV genes of malaria protozoans. As a result of previous studies to determine their function, the present inventors confirmed that hemoglobin was effectively degraded under acidic conditions (Kim, YM). , et al., (2004) J. Biochem. 193, pp 189-195). In addition, as a result of treatment of various kinds of protease inhibitors with these recombinant plasmons, the enzyme activity was completely inhibited by pespstatin A, an aspartic protease inhibitor, and calpine, a kind of cysteine protease. It was confirmed that the activity of plaxinin was also inhibited by the inhibitor ALLN. In palcifein-targeted studies centered on Roseenthal (University of California, USA), falcifein inhibitors such as peptidyl fluoromethyl ketone, vinyl sulfone, and aldehydes were tested in vitro. It showed antimalarial effects in vitro and in vivo (Rosenthal, RJ (2004) Int. J. Parasitology. 34, pp1489-1499), and Goldberg (University of Washington, USA) Demonstrated the antimalarial effect of cysteine protease inhibitors of E64 in addition to pepstatin A, a representative inhibitor of plascine (Francis, SE et al., Annu. Rev. Microbiol. 51, pp97-123). However, the study of the antimalarial effects of these two researchers using plascin and falcipine inhibitors did not completely control the mechanism of these proteases. For example, inhibition of falcifein did not inhibit the enzymatic activity of the upper regulatory step, plascine, and the inhibitor pepstatin A did not inhibit the mechanism of action that precedes plascine activation.

There is a disadvantage in that it takes about one week for the culture and the determination of efficacy, and there is a difficulty that the skilled person should measure visually to determine the efficacy. We tried to construct a high-efficiency system for in vitro screening for drug efficacy on the worm spp. That is difficult and time consuming to determine.

     Malaria has been shown to be once killed by special drugs and insecticides such as DDT, but the prevention and treatment is currently very difficult due to the emergence of drug-resistant malaria prostheses and insecticide-resistant mosquitoes. Sulfonamide-based medicaments are used as toxoplasma worm treatments, but to date the drug is not effective. Although there are no selective drugs for the mosquitoes, antibiotics, which are microbial drugs, have been mainly used. However, it has recently been found that the genetic structure of the microorganisms and the moss. Therefore, there is a need for new drugs and prophylactic agents that take this into consideration.

Calpine is a type of cysteine protease known to be involved in cell signal transduction, cell motility, apoptosis, and cell cycle regulation in humans and mammals. Although the function and enzymatic activity of calpein have not been clearly reported in malaria protozoa, it is assumed that it is essential for growth of malaria protozoa.

In our previous studies, it was confirmed that malaria calpain inhibitors such as ALLN and ALLM inhibit the mechanism of plasmepsin processing. From this, the study of malaria calpain would be important as a higher-level regulating mechanism that could overcome the limitations of the study of plascine and falcifein and encompass plascin and falcifein for more complete blockade of hemoglobin degradation mechanism. It was judged. Therefore, the present inventors confirmed the regulation mechanism at the upper level encompassing proteases such as plaxinin and falcifein as shown in FIG. 1, and targeted calpine for the development of more effective antimalarial drugs. I chose to.

To date, research has been carried out to identify the function of specific genes in malaria protozoa. In particular, enzymatic properties have been identified through expression of active recombinant proteins of proteolytic enzymes that are important for survival of malaria protozoa. However, there is no report on the expression of active recombinant protein against calpain, one of the cysteine proteases of malaria protozoa. The present inventors attempted to propose a new drug target for the development of antimalarial drugs by elucidating the enzymatic properties and roles of calpine by synthesizing and purifying recombinant active calpine using an expression vector manufacturing technique. Accordingly, the present inventors completed the present invention as a result of studying an in vitro search method capable of screening new antimalarial drugs from various compounds or natural products using calpine protein having enzymatic activity.

In accordance with the above object, the present invention provides a composition for screening an antimalarial drug comprising a calpain gene of P. falciparum .

The calpein gene as defined herein is a nucleotide sequence having at least 90% homology to the nucleotide sequence set forth in SEQ ID NO: 3, preferably, a base sequence having at least 95% homology thereof to SEQ ID NO: 3, more preferably , SEQ ID NO: 3 or a nucleotide sequence having a homology of 98% or more thereof.

The present invention provides a method for screening an antimalarial drug, characterized by using the composition as a target material.

In addition, contacting the composition and the test substance; Identifying reactions between them; And a final step of determining whether the composition exhibits the activity of inhibiting the expression of a gene comprising the antimalarial drug.

The present invention provides a composition for screening an antimalarial agent comprising a protein expressed from the calpein gene of a tropical fever malaria parasite.

A protein expressed from a calpein gene as defined herein is an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90% homology thereof, preferably an amino acid sequence having at least 95% homology thereof, or more than More preferably, it comprises an amino acid sequence having at least 98% homology with SEQ ID NO.

The present invention provides a method for screening an antimalarial drug, wherein the composition is used as a target.

The screening method as defined herein comprises the steps of contacting the composition with a test substance; Identifying reactions between them; And a final step of determining whether the composition exhibits the activity of inhibiting the function of the protein included therein.

In addition, the test substance may include a well-known antimalarial agent or an antimalarial activity to be tested, such as Altimicin, for example, a natural substance or a compound through a chemical library. It features.

Hereinafter, the method for obtaining the active recombinant calfane protein of the present invention will be described in detail.

A first step of cloning the gene of calpein of tropical fever malaria parasites; A second step of cloning a gene of domain IIa including an enzyme active site from the gene to express an active recombinant calfane protein; A third step of transforming the recombinant vector comprising the protein into E. coli; A fourth step of purifying the recombinant calfane protein from the transformed E. coli through affinity chromatography; It is characterized in that it comprises a fifth step of obtaining the active recombinant calpine protein of the present invention by performing a refolding (refolding) process of the protein through two-step dialysis (two-step dialysis) .

The present invention also provides a composition for screening an antimalarial agent comprising a calpain gene of the above-mentioned P. falciparum or a protein expressed from the calpein gene.

As described above, the active recombinant calfane of tropical fever malaria synthesized and purified according to the present invention has the activity of cysteine protease and is involved in the plasmepsin IV, a protease involved in hemoglobin degradation of malaria. It has been shown to regulate the activation process. Calcine of the malaria protozoa can be a good drug target for antimalarial drugs, and can develop antimalarial screening methods using active recombinant calfane as a target.

Hereinafter, the present invention will be described in detail by the following reference examples, examples and experimental examples. However, this is only to illustrate the present invention, but the content of the present invention is not limited thereto.

Reference example. Experimental material

Pepstatin A and trans-epoxysuccinyl-L-rusilamido-4-guanidino-butal (trans-epoxysuccinl-L-Leucylamido-4-guanidino-butane; E-64) (Mannheim, Germany), phenylmethane-sulfonyl fluoride (PMSF), N-acetyl-L-leusil-L-norrucinin (N-accetyl-L-leucyl-L-norleucinal ALLN), gelatin and other chemical samples were purchased from Sigma (St. Louis, MO, USA). Expression vectors pET21b (+), Ni-NTA resin and E. coli strain BL21 were purchased from Novagen (Madison, WI, USA). Gel extraction kits and DNA miniprep kits were purchased from Qiagen (Valencia, CA, USA). All restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA, USA).

Example 1. Cloning and Purification of Recombinant Calpine Protein

1-1. Vector cloning

From the genomic DNA (ATCC, # MRA-151G) isolated from P. falciparum 3D7 strain (Forward Primer CGGGATCCC GGAATGGGTAAAAGCAAAGAACGTAAAGGT; Reverse Primer CCGCTCGAGCGG CTTTGTGTCCTCTCTACAA) A gene of about 1.26 kb, which contains a calpine domain IIa catalytic region, was obtained. The italics indicated on each primer are the sites where restriction enzymes act. The forward primer has Bam HI and the reverse primer has Xho I. In order to obtain an active recombinant protein from a 1.26 kb calpein gene, DNA ligase was obtained after treatment of restriction enzymes Bam HI and Xho I to pET21b (+), which is amplified by PCR, and a protein expression vector. (T4 ligase) was used to link the Pf-calpein gene and vector to each other to complete a new construct for recombinant calfane protein expression.

primer SEQ ID NO: Base sequence Domain IIa (f) One 5'-CGGGATCCCGGAATGGGTAAAAGCAAAGAACGTAAAGGT-3 ' Domain IIa (r) 2 5'-CCGCTCGAGCGGCTTTGTGTCCTCTACAAATTCAACACTGTT-3 ' Recombinant Calpain
protein gene sequence 3 5'-ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCCGGAATGGGTAAAAGCAAAGAACGTAAAGGTTACTTAAAATGTAATACAAGCAAATATGATAAAATTAAAAATAATAATTATATTAATGATGTGTATAATAATAATGCAAGTGTTAAAGATATACAAAAATTTAAACAAAAGGATAAAGAGACTGATAAATTGAATGATAAATGGAATGACAAATCGTATGACAAATCGTATGATAAATGTAATGACAAATGTAATGACAAATGTAATGACAAATATAATGACAAATCGTATGACAAATCGTATGACAAATCTAATTATAAACAAAAAAGTAAAACAAACGAATCGAAGAATATCAAATTAGAATGGGAGAAATGCTTATTAGATACTTGTGATCATAAATATATAGTAAATGAGGATTGTATTAAAAAGAATTACATGTGTTGTAAAAAATGTGATAAGAAGAGATGTATATTTCATTACGTAAATGGTTTTAAATTATATGGATGGTTGAATTATAAATGGACAGATCCCGATGGGTTCTTAGAATTAAGTATTAGACAAAAAAGAAAATTTGATTCTTGGAAAAGATTATCAGAATTATATGAAAATCCAATTGTTATGTCACCATATTTATATAATAATTGTATAAGACAAGGTTTTGTTGCGGATTGTTCTTTTCTGTCTTCATTAACAGTATTAATAGAATATGAGAAAAAACATAAGATACCTGTTATTTCTAGTATTATAACACCTTGTAGTTGGAATTATTTATGTATAAATAAAACGTGGCCTATTTTTAATCCTTGTGGTATGTATATATGTAGACTTCATTGTAATGGTATACAAAGAAAGATAATAATAGATGATTATGTACCGGTTAAGAACAATAATTCATTGCTTGTGGCTTATTCAAATAATCAAAAAGAATTATGGGTTACCTTATTAGAAAAAGCTTTTGTTAAATTGATGGGAGGATCATATTCTA TGTTTGGTTCTAATCCTGGTTCTGATTTGTATTATTTGACAGGTTGGATACCAGTAACCATATCTTTGAAATCTAAGATTAGTAGTTCTCCCCCTTCTTTTGACAAACATAGTACGCATTCTTTAATAAGGGATGAGCGTATAGATGAATTGGTATATGAAACTAACGACCTAATGATAAATAAGAAGAGAGAGAGAGAGAGA Recombinant Calpain
protein amino acid sequence 4 N'-MASMTGGQQMGRDPGMGKSKERKGYLKCNTSKYDKIKNNNYINDVYNNNASVKDIQKFKQKDKETDKLNDKWNDKSYDKSYDKCNDKCNDKCNDKYNDKSYDKSYDKSNYKQKSKTNESKNIKLEWEKCLLDTCDHKYIVNEDCIKKNYMCCKKCDKKRCIFHYVNGFKLYGWLNYKWTDPDGFLELSIRQKRKFDSWKRLSELYENPIVMSPYLYNNCIRQGFVADCSFLSSLTVLIEYEKKHKIPVISSIITPCSWNYLCINKTWPIFNPCGMYICRLHCNGIQRKIIIDDYVPVKNNNSLLVAYSNNQKELWVTLLEKAFVKLMGGSYSMFGSNPGSDLYYLTGWIPVTISLKSKISSSPPSFDKHSTHSLIRDERIDELVYETNEVMINK KHGYVNSVEFVEDTKPLEHHHHHH Stop-C '

1-2. Transforming Escherichia coli

After transfection the Pf -calpain-pET21b (+) architecture (construct) obtained in Example 1-1 in BL21 E. coli (E.coli) with a CaCl ₂ treatment, containing the ampicillin (ampicillin) LB Agar The plate was cultured to obtain a transgenic colony containing Pf -calpain-pET21b (+). 500 ml of LB medium containing 50 μg / ml empicillin was transformed with transformed Escherichia coli (KCTC 11498BP, Genebank of Biotechnology) while stirring (250 rpm) at 37 ° C until the absorbance value (OD ₆₀₀ ) reached 0.6. After incubation, IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration of 1 mM) was added to induce protein expression while incubating for 4 hours. The cultured cells were harvested by centrifugation at 4000 × g, 4 ° C. for 20 minutes after incubation.

1-3. Recombinant Calpine Protein Purification

IPTG-derived cells were stirred for 60 minutes at room temperature in 5 ml of 6 M Gu-HCl, 0.1 M sodium phosphate buffer, 0.01 Tris-Cl, pH 8.0 per gram of wet weight. Cell lysate was centrifuged at 12,000 × g, 4 ° C. for 30 minutes. Transfer the supernatant to a new tube, attach 1 ml of 50% Ni-NTA (Novagen, BugBusterNi-NTA HisBind Purification Kit, Cat # 70751-3) slurries, and gently soften the solution for 60 minutes at room temperature. After stirring, the residue was purified by affinity chromatography. Protein-bound resin was loaded on a column (Novagen, BugBusterNi-NTA HisBind Purification Kit, Cat # 70751-3), 4 ml of 8 M urea, 0.1 M sodium phosphate buffer, Wash twice with 0.01 M Tris-Cl, pH 6.3. The bound protein was then followed by 8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-Cl, pH 5.9 and 8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris-Cl, pH 4.5 After elution, each fraction was collected to confirm purified rPf-calpinein protein through protein analysis by SDS-PAGE and Western blot. The purified rPf-calpinein protein fraction was refolded through two-step dialysis in two consecutive steps for 6 hours at 0.01 M Tris-HCl, pH 7.5. The sample was used as an example (hereinafter referred to as 'rPf-calpain').

As a result, as shown in Figure 2A, it was confirmed that IPTG-induced cells express the expected protein of the rPf-calpinein including His-Tag, the main protein of about 50 kDa.

Experimental Example 1. Expression and Purification of Recombinant Calpine Protein

The rPf-calpinein obtained in the above example was quantified by Bradford protein assay (Bio-Rad), and by SDS-PAGE and Western blot method (Laemmli, UK (1970) Nature, 277, pp680) -685) was analyzed as follows.

Protein samples were added to 12% gels according to a Mini Protein III electrophoresis apparatus (Bio-Rad, Hercules, Calif., USA), followed by Coomassie brilliant R-250 blue. ). To confirm the presence of rPf-calpain following separation, the unstained polyacrylamide gel was transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Bioscience), 5%. After blocking with skim milk, polyclonal anti-His antibody (1: 5000 dilution), anti-Pf-calpine (5%) on 5% skim milk Pf-CapnA) antibody (1: 2000 dilution), anti-plasmepsin II antibody (1: 5000 dilution; MR4 # MRA-66) or anti-plasmepsin IV antibody (1: 1000; MR4 # MRA-814A) was incubated at room temperature for 2 hours and then incubated with goat anti-mouse covalently-HRP or goat anti-rabbit covalently-HRP antibody (1: 5000 dilution) for 1 hour.

As a result of the experiments, as shown in FIGS. 2B and 2C, rPf-calfane reacts with anti-Pf-calpein antibody and anti-His-Tag antibody, and as shown in FIG. 2D, It was confirmed that the calfane protein reacted was about 46 kDa.

Experimental Example 2. Fluorogenic-substrate assay

2-1. Optimal pH Determination of Calpine Activity

      In order to measure the activity of the rPf-calpinein obtained in Example 1, a fluorescence-based assay was performed by Debiasi, RL, et al., (1999) J. Virol. 73, pp695-701. According to the experiment was performed as follows.

rPf-calpain was reacted with an amino acid substrate, Suc-LLVY-AMC 50, at 200 ° C. in 100 mM Tris-HCl, pH 7.5 for 1 hour at 37 ° C. Fluorescence values of aminomethylcoumarin were measured at intervals of 5 minutes immediately after substrate addition in an excitation 360 & emission 460 nm using a fluorometer (Infinite F200, Tecan, Mannedorf, Switzerland). To determine the optimal pH conditions for the highest activity of recombinant calfane, calphine activity was measured in 100 mM sodium acetate buffer from pH 4.5 to pH 7.5 and in 100 mM Tris-HCl buffer from pH 6.5 to pH 9.5. Each activity was measured according to pH conditions step by step.

As a result, as shown in Figure 3A, after rPf-calcane and the amino acid substrate was reacted for 1 hour at 37 ℃, the activity was measured, it was confirmed that the highest at pH 7.5.

2-2. Influence of Recombinant Calpine Activity Inhibitors

In order to measure the activity of rPf-calpine, N-acetyl-L-leucil-L-norleucine (N-accetyl-L-leucyl-L-norleucinal; ALLN) 50, a calphine specific inhibitor, is generally used. Trans-epoxysuccinl-L-Leucylamido-4-guanidino-butane (E-64), a cysteine protease inhibitor, trans-epoxysuccinyl-L-leusilamido-4-guanidino-butane 50, PMSF 100, a serine protease inhibitor, and Pepstatin A 100, an aspartic protease inhibitor, were added at 37 ° C. for 30 minutes before adding Suc-LLVY-AMC. Pretreatment with rPf-calpine was measured in the same manner as in Experimental Example 2-1.

As shown in FIG. 3B, the activity of rPf-calpinein was about 70% or more in the experimental group treated with 50 ALLN and about 60% compared to the control group in the 50 E-64 treated group. On the other hand, the experimental group treated with 100 PMSF and 100 pepstatin A showed no change.

Experimental Example 3. Determination of proteolytic activity by gelatin zymography

     In order to measure the proteolytic activity of the rPf-calpinein obtained in Example 1, the experiment was carried out using gelatin SDS-PAGE (Gelatin SDS-PAGE) as follows.

SDS-PAGE was performed using a 10% polyacrylamide gel containing 0.1% gelatin as a copolymerized substrate under non-reducing conditions. rPf-calfane was mixed with 2-mercaptoethanol (2-mercaptoethanol) with a limiting Tris-glycine SDS sample buffer (2x) and left at room temperature for 10 minutes. Gels were run on 1 × Tris-glycine SDS running buffer at 125 V, followed by protein degradation followed by gel removal, gentle stirring at room temperature for 1 hour on 2.5% Triton X-100 solution to remove SDS, Incubated for 12 hours on Zymogram developing buffer and 2.5% Triton X-100 containing 50 mM Tris-HCl, 0.2 M NaCl, 5 mM CaCl2. The protease used in Experimental Example 2-2 was diluted in a Zymogram development buffer and used at a final concentration of 50. The gel was stained for 1 hour in 0.5% Coomassie blue R-250. Then desalted in 40% methanol and 10% acetic acid.

As a result, as shown in Figure 4, the clear band (Control) of about 50 kDa having a proteolytic activity against the indigo blue background, the rPf-calcane obtained in Example 1 has a proteolytic activity Could have guessed. In addition, in the result similar to Experimental Example 2, it was shown to be inhibited by protease inhibitors 50 μM ALLN and 50 μM E-64, while not inhibited by 50 μM PMSF or 50 μM pepstatin A. I could confirm it. Therefore, it was confirmed that the protease activity of rPf-calpinein was inhibited by specific calpine inhibitor and cysteine protease inhibitor.

Experimental Example 4. Morphological Observation of Maturity Forms in Blood

Parasitemia of malaria parasitemia in the blood maturation of P. falciparum 3D7 strain (ATCC, # MRA-151G) according to the treatment of calpine inhibitor ALLN ) And morphological changes were observed as follows.

The method reported by Lambros et al. (Lambros, C., et al., (1979) J. Parasitol., 65, pp418-420) was applied with a slight change. Sorbital was used to homogenize the maturation stages, rings, trophozoite and schizont in the blood of tropical malaria. After centrifugation of 4 ml of cultured tropical fever malaria at a rate of 600 g, the sap was obtained and the supernatant was discarded. Thereafter, 4 ml of 5% sorbital was added to the sap solution and left at room temperature for 10 minutes. After centrifugation at 600 g, the sediment was washed three times with malaria medium. The parasitemia (blood malaria parasitic rate) was calculated under a 1000-fold magnification microscope using the finally obtained salivary solution. Treatment of ALLN at maturation stages in homogenized tropic malaria blood was carried out using a 6-well culture dish (NUNC, A / S) to incubate the ring stage of homogenized tropic malaria with different concentrations of ALLN (1 uM, 10 uM and 100 uM) for 48 hours. That is, 5% hematocrit and 2% parasitemia are based on 0 hours. Parasitemia is calculated periodically every 6 hours for 48 hours.

As a result, as shown in Figure 5, when treated with 100NM ALLN for 48 hours, the parasitemia was 0%, whereas the malaria medium added only 6.15% parasite disease showed. Overall, the experimental group treated with ALLN showed a direct relationship with the treatment concentration and treatment time of ALLN.

In addition, the maturation stage of ALLN-treated tropical fever malaria was observed using optical and confocal microscopy. The optical microscope was used to calculate parasitemia under 1000-fold magnification and to analyze morphological changes of tropical malaria. For analysis of confocal microscopy, tropical malaria is lightly plated on a slide glass and fixed at 4 ° C. for 24 hours using 1% paraformaldehyde. Thereafter, the tropical malaria fixed with phosphate-buffered saline was washed and treated with 0.1% Triton-X 100 for 30 minutes at room temperature and washed once with phosphate buffer. Tropical malaria was treated with 1% bovine serum albumin at room temperature for 1 hour, washed with phosphate buffer solution, and then treated with anti-calpinein antibody (1: 100 dilution) at room temperature for 4 hours. Afterwards, Cy-3 conjugated goat-anti-rabbit antiserum was treated at room temperature for 1 hour and washed with phosphate buffer. For nucleation, 1 ug / ml of 4,6-diamidino-2-phenylindole (DAPI) is treated with tropical malaria and washed once with phosphate buffer. To observe using a confocal microscope, the slide glass was mounted using a mounting solution and tropical heat malaria was observed by LSM 510 Meta confocal microscope (Carl Zeiss). As shown in FIG. 6, when the 10 uM ALLN was treated for 48 hours, the parasitic disease showed 0.69%, but the maturation stages in the blood seen on the optical microscope (FIG. 6B) and confocal microscopy (FIG. 6A) were determined by ring ( It was confirmed that only ring) step was observed. Therefore, it could be assumed that the treatment of ALLN can be suppressed from the ring stage.

Experimental Example 5. Determination of Plasepsin II and IV Activity by SDS-PAGE

Plasepsin (hereinafter referred to as PM) II and PM IV are known to be activated by autocatalytic processing under acidic conditions (pH 4.5) and by the calpain inhibitor ALLN. Activation is known to be inhibited (Arthur, JS, et al., (1995) FEBS Lett. P368, pp 397400). Self-catalysis of PM occurs under acidic conditions by the conversion of the pro-domain from the PM precursor to the active, mature form.

Recombinant PM II and IV (Kim, YM, et al., (2004) J. Biochem. 193, pp 189-195) were placed in 100 mM acetate buffer pH 4.5 and 100 mM Tris-HCl buffer, respectively. Each was added at pH 7.5 and treated at 37 ° C. for 1 hour. To determine the effect of rPf-calpinein obtained in Example 1 on the action of PM II and IV (see FIG. 1), 0.1 M Tris-HCl, 5 μg of rPf-calpinein was mixed with 5 μg of each PM under an optimal condition of pH 7.5, and then incubated at 37 ° C. for 2 hours, followed by SDS-PAGE analysis in the same manner as in Experimental Example 1.

As a result, as shown in Figure 7, both PM II and IV were converted into a mature form (lane 2), while it was confirmed that the self-catalysis did not occur in the pH 7.5 conditions (lane 3). Also, when rPf-calpine was mixed, PM IV was converted to mature form without prodomain by lane r, whereas PM II was inactivated by rPf-calpine. It could be confirmed that it did not happen (lane 4). Therefore, it was confirmed that the PM IV precursor (P; precursor) was converted into a mature form (M;) by rPf-calpine, and it was confirmed that rPf-calpinein was involved in PM action.

[Microbial Deposit]

[Deposit Name] Biotechnology Research Institute Genetic Bank (KCTC)

[Accession Number] KCTC 11498BP

[Consignment Date] 2009. 04. 07

[National Development Research Project Supporting This Invention]

[Task unique number] RT105-03-02

[Ministry of Knowledge] Ministry of Knowledge Economy

[Name of Research Project] Local Technology Innovation Project

[Research Project Name] Acquired Common Infectious Disease R & D Project

[Organization] Wonkwang University

[Research Period] May 1, 2005 ~ April 30, 2010

1 is a diagram showing the role of calpein in the process of the active conversion of malaria plasminin (plasminin) itself,

2 is a diagram showing the result of expression and purification of recombinant protein from the calfamine gene of tropical fever malaria parasites ((A) Coomassie blue stained electrophoresis result M; Molecular Marker, 1; Grind, 2; Ni-NTA purified fraction; (B) anti- Pf- Calpain antibody; (C) analysis of expressed recombinant calfane protein by Western blotting using anti-poly-HisTag antibody (D) anti- Analysis of Caffeine Proteins from Cell Crushes of Tropical Malaria Protozoan by Western Blotting Using Pf- Calpain Antibody and (C) Anti-poly-HisTag Antibody)

Figure 3 is measured by the fluorescence-substrate assay using a recombinant calfamine activity (Suc-LLVY-AMC) (A) activity change of the recombinant calfamine protein according to the hydrogen ion concentration (B) of the protease Is a diagram showing the change in activity of recombinant calfane protein by inhibitors,

      4 is a diagram showing the measurement of proteolytic activity and inhibition pattern of recombinant calfane protein,

      Figure 5 is a diagram showing the degree of change of malaria parasites caused by calpaine activity inhibitor ALLN,

      6 is a view showing optical and confocal microscopic observation of malaria protozoa treated with ALLN, a calpine activity inhibitor,

      Figure 7 is a diagram showing the interaction analysis of recombinant calpein with Plascinsin II and IV (P; precursor form of plascin, M; mature form of Plascin. Lane 1: no culture; Lane 2: 100 mM sodium Acetate buffer, pH 4.5; Lane 3: 100 mM Tris-HCl buffer, pH 7.5; Lane 4: 5 mg plascine plus 5 mg recombinant calfane, 100 mM Tris-HCl buffer, pH 7.5, 2 hr, 37 ℃).

<110> Industry-Academic Cooperation Foundation Wonkwang University <120> A composition and method for screening anti-malarial drug by          using the active form of recombinant calpain in Plasmodium          falciparum <130> DIF / 2009-03-0014 / SJ <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 39 <212> DNA <213> Plasmodium falciparum <400> 1 cgggatcccg gaatgggtaa aagcaaagaa cgtaaaggt 39 <210> 2 <211> 42 <212> DNA <213> Plasmodium falciparum <400> 2 ccgctcgagc ggctttgtgt cctctacaaa ttcaacactg tt 42 <210> 3 <211> 1257 <212> DNA <213> Plasmodium falciparum <400> 3 atggctagca tgactggtgg acagcaaatg ggtcgggatc ccggaatggg taaaagcaaa 60 gaacgtaaag gttacttaaa atgtaataca agcaaatatg ataaaattaa aaataataat 120 tatattaatg atgtgtataa taataatgca agtgttaaag atatacaaaa atttaaacaa 180 aaggataaag agactgataa attgaatgat aaatggaatg acaaatcgta tgacaaatcg 240 tatgataaat gtaatgacaa atgtaatgac aaatgtaatg acaaatataa tgacaaatcg 300 tatgacaaat cgtatgacaa atctaattat aaacaaaaaa gtaaaacaaa cgaatcgaag 360 aatatcaaat tagaatggga gaaatgctta ttagatactt gtgatcataa atatatagta 420 aatgaggatt gtattaaaaa gaattacatg tgttgtaaaa aatgtgataa gaagagatgt 480 atatttcatt acgtaaatgg ttttaaatta tatggatggt tgaattataa atggacagat 540 cccgatgggt tcttagaatt aagtattaga caaaaaagaa aatttgattc ttggaaaaga 600 ttatcagaat tatatgaaaa tccaattgtt atgtcaccat atttatataa taattgtata 660 agacaaggtt ttgttgcgga ttgttctttt ctgtcttcat taacagtatt aatagaatat 720 gagaaaaaac ataagatacc tgttatttct agtattataa caccttgtag ttggaattat 780 ttatgtataa ataaaacgtg gcctattttt aatccttgtg gtatgtatat atgtagactt 840 cattgtaatg gtatacaaag aaagataata atagatgatt atgtaccggt taagaacaat 900 aattcattgc ttgtggctta ttcaaataat caaaaagaat tatgggttac cttattagaa 960 aaagcttttg ttaaattgat gggaggatca tattctatgt ttggttctaa tcctggttct 1020 gatttgtatt atttgacagg ttggatacca gtaaccatat ctttgaaatc taagattagt 1080 agttctcccc cttcttttga caaacatagt acgcattctt taataaggga tgagcgtata 1140 gatgaattgg tatatgaaac taacgaggta atgataaata agaaacatgg atatgttaac 1200 agtgttgaat ttgtagagga cacaaagccg ctcgagcacc accaccacca ccactga 1257 <210> 4 <211> 418 <212> PRT <213> Plasmodium falciparum <400> 4 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Pro Gly Met   1 5 10 15 Gly Lys Ser Lys Glu Arg Lys Gly Tyr Leu Lys Cys Asn Thr Ser Lys              20 25 30 Tyr Asp Lys Ile Lys Asn Asn Asn Tyr Ile Asn Asp Val Tyr Asn Asn          35 40 45 Asn Ala Ser Val Lys Asp Ile Gln Lys Phe Lys Gln Lys Asp Lys Glu      50 55 60 Thr Asp Lys Leu Asn Asp Lys Trp Asn Asp Lys Ser Tyr Asp Lys Ser  65 70 75 80 Tyr Asp Lys Cys Asn Asp Lys Cys Asn Asp Lys Cys Asn Asp Lys Tyr                  85 90 95 Asn Asp Lys Ser Tyr Asp Lys Ser Tyr Asp Lys Ser Asn Tyr Lys Gln             100 105 110 Lys Ser Lys Thr Asn Glu Ser Lys Asn Ile Lys Leu Glu Trp Glu Lys         115 120 125 Cys Leu Leu Asp Thr Cys Asp His Lys Tyr Ile Val Asn Glu Asp Cys     130 135 140 Ile Lys Lys Asn Tyr Met Cys Cys Lys Lys Cys Asp Lys Lys Arg Cys 145 150 155 160 Ile Phe His Tyr Val Asn Gly Phe Lys Leu Tyr Gly Trp Leu Asn Tyr                 165 170 175 Lys Trp Thr Asp Pro Asp Gly Phe Leu Glu Leu Ser Ile Arg Gln Lys             180 185 190 Arg Lys Phe Asp Ser Trp Lys Arg Leu Ser Glu Leu Tyr Glu Asn Pro         195 200 205 Ile Val Met Ser Pro Tyr Leu Tyr Asn Asn Cys Ile Arg Gln Gly Phe     210 215 220 Val Ala Asp Cys Ser Phe Leu Ser Ser Leu Thr Val Leu Ile Glu Tyr 225 230 235 240 Glu Lys Lys His Lys Ile Pro Val Ile Ser Ser Ile Ile Thr Pro Cys                 245 250 255 Ser Trp Asn Tyr Leu Cys Ile Asn Lys Thr Trp Pro Ile Phe Asn Pro             260 265 270 Cys Gly Met Tyr Ile Cys Arg Leu His Cys Asn Gly Ile Gln Arg Lys         275 280 285 Ile Ile Ile Asp Asp Tyr Val Pro Val Lys Asn Asn Asn Ser Leu Leu     290 295 300 Val Ala Tyr Ser Asn Asn Gln Lys Glu Leu Trp Val Thr Leu Leu Glu 305 310 315 320 Lys Ala Phe Val Lys Leu Met Gly Gly Ser Tyr Ser Met Phe Gly Ser                 325 330 335 Asn Pro Gly Ser Asp Leu Tyr Tyr Leu Thr Gly Trp Ile Pro Val Thr             340 345 350 Ile Ser Leu Lys Ser Lys Ile Ser Ser Ser Pro Pro Ser Phe Asp Lys         355 360 365 His Ser Thr His Ser Leu Ile Arg Asp Glu Arg Ile Asp Glu Leu Val     370 375 380 Tyr Glu Thr Asn Glu Val Met Ile Asn Lys Lys His Gly Tyr Val Asn 385 390 395 400 Ser Val Glu Phe Val Glu Asp Thr Lys Pro Leu Glu His His His His                 405 410 415 His His          

Claims (9)

A composition for screening an antimalarial drug comprising a calpain gene having the nucleotide sequence set forth in SEQ ID NO: 3 of P. falciparum. delete A composition for the screening of an antimalarial drug comprising an active recombinant calfane protein having the amino acid sequence of SEQ ID NO: 4 expressed from the calpein gene of the tropical fever malaria parasite according to claim 1. delete Contacting the composition of claim 1 with the test substance; Identifying reactions between them; And a final step of determining whether the composition exhibits the activity of inhibiting the expression of a gene comprising the antimalarial agent. delete Contacting the composition of claim 3 with the test substance; Identifying reactions between them; And a final step of determining whether the composition exhibits the activity of inhibiting the function of the protein comprising the antimalarial agent. delete delete

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