RESUMEN
BACKGROUND: Schistosomiasis is a chronic parasitic disease that affects millions of people's health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). METHODS: In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure-activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. RESULTS: It was found that several new derivatives, including compounds 6a-6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a-6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. CONCLUSION: The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxadiazoles/farmacología , Schistosoma japonicum/efectos de los fármacos , Esquistosomiasis Japónica/tratamiento farmacológico , Animales , Antígenos Helmínticos/efectos de los fármacos , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/uso terapéutico , Estructura Molecular , Oxadiazoles/química , Oxadiazoles/uso terapéutico , Schistosoma japonicum/enzimología , Selenio/químicaRESUMEN
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Proteínas del Helminto/metabolismo , Schistosoma japonicum/enzimología , Animales , Secuencia de Bases , Clonación Molecular , Codón de Terminación , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Helmintos/genética , ADN de Helmintos/aislamiento & purificación , Femenino , Técnicas de Silenciamiento del Gen , Glutatión Peroxidasa/clasificación , Glutatión Peroxidasa/genética , Proteínas del Helminto/clasificación , Proteínas del Helminto/genética , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Óvulo/metabolismo , Estrés Oxidativo/fisiología , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Filogenia , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma japonicum/genética , Selenio/química , Selenocisteína/química , Caracoles/parasitologíaRESUMEN
OBJECTIVE: To clone and express the conservative region of gene encoding tyrosine kinase 4 of Schistosoma japonicum and identify the difference in gene expression between genders of S. japonicum. METHODS: The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) with primers designed according to SmTK4 encoding tyrosine kinase 4. The purified PCR product was ligated with pET28a and the recombinant protein was induced to express, and analyzed by SDS-PAGE, Western blotting and tools of bio-informatics. Subsquently, total RNA was respectively isolated from adult males, females and both worms of S. japonicum. The real-time PCR was performed with corresponding primers after reverse transcription to show the expression levels of the gene in both genders. RESULTS: A 582 bp in size of the DNA fragment was acquired by RT-PCR. Sequence analysis indicated that the fragment showed 91% in homology to that of SmTK4, and the deduced amino acid sequence showed to be 98% identical with that encoded by SmTK4. SDS-PAGE analysis revealed that the relative molecular weight (M(r)) of expressed protein rSjTK4 was approximately 26000. The bio-information analysis demonstrated that the protein had multiple sites of enzymatic activities. The relative number of copies of SjTK4 in male worms was 0.61 +/- 0.29, while 0.03 +/- 0.02 in female worms, showing that the mRNA level of TK4 in male worms was 18 times higher than that in females. CONCLUSION: The conservative region of gene encoding tyrosine kinase 4 of S. japonicum is successfully cloned and expressed. The mRNA level of TK4 in male worms is significantly higher than that in females.
Asunto(s)
Proteínas Tirosina Quinasas/genética , Schistosoma japonicum/enzimología , Secuencia de Aminoácidos , Animales , Western Blotting , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Femenino , Expresión Génica , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes , Schistosoma japonicum/genéticaRESUMEN
BACKGROUND: Schistosomiasis is a neglected tropical disease with high morbidity and mortality in the world. Currently, the treatment of this disease depends almost exclusively on praziquantel (PZQ); however, the emergence of drug resistance to PZQ in schistosomes makes the development of novel drugs an urgent task. Aldose reductase (AR), an important component that may be involved in the schistosome antioxidant defense system, is predicted as a potential drug target. METHODS: The tertiary structure of Schistosoma japonicum AR (SjAR) was obtained through X-ray diffraction method and then its potential inhibitors were identified from the Maybridge HitFinder library by virtual screening based on this structural model. The effects of these identified compounds on cultured adult worms were evaluated by observing mobility, morphological changes and mortality. To verify that SjAR was indeed the target of these identified compounds, their effects on recombinant SjAR (rSjAR) enzymatic activity were assessed. The cytotoxicity analysis was performed with three types of human cell lines using a Cell Counting Kit-8. RESULTS: We firstly resolved the SjAR structure and identified 10 potential inhibitors based on this structural model. Further in vitro experiments showed that one of the compounds, renamed as AR9, exhibited significant inhibition in the activity of cultured worms as well as inhibition of enzymatic activity of rSjAR protein. Cytotoxicity analysis revealed that AR9 had relatively low toxicity towards host cells. CONCLUSIONS: The work presented here bridges the gap between virtual screening and experimental validation, providing an effective and economical strategy for the development of new anti-parasitic drugs. Additionally, this study also found that AR9 may become a new potential lead compound for developing novel antischistosomal drugs against parasite AR.
Asunto(s)
Aldehído Reductasa/química , Antihelmínticos/aislamiento & purificación , Diseño de Fármacos , Inhibidores Enzimáticos/aislamiento & purificación , Proteínas del Helminto/química , Schistosoma japonicum/enzimología , Animales , Antihelmínticos/química , Antihelmínticos/farmacología , Bioensayo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Locomoción/efectos de los fármacos , Conformación Proteica , Schistosoma japonicum/anatomía & histología , Schistosoma japonicum/efectos de los fármacos , Schistosoma japonicum/fisiología , Análisis de SupervivenciaRESUMEN
BACKGROUND: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR. METHODOLOGY/PRINCIPAL FINDINGS: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells. CONCLUSIONS/SIGNIFICANCE: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/aislamiento & purificación , Antihelmínticos/farmacología , Simulación por Computador , Descubrimiento de Drogas , Schistosoma japonicum/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/genética , Animales , Muerte Celular/efectos de los fármacos , Clonación Molecular , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Genes de Helminto/genética , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Células Hep G2 , Humanos , Cinética , Schistosoma japonicum/citología , Schistosoma japonicum/efectos de los fármacos , Schistosoma japonicum/ultraestructura , Relación Estructura-Actividad , Análisis de Supervivencia , Factores de TiempoRESUMEN
The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His(6)-tagged glutathione S-transferase (His(6)-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His(6)-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A(340) upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His(6)-GSTase-CVIA-PE) or azide (His(6)-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His(6)-GSTase-CVIA-F).
Asunto(s)
Biocatálisis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Vidrio/química , Glutatión Transferasa/química , Animales , Activación Enzimática , Glutatión Transferasa/metabolismo , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/enzimología , Estereoisomerismo , Propiedades de SuperficieRESUMEN
Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692bp and had a 465bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37°C) revealed that its expression was enhanced 4.2-fold (p<0.001) at 30°C after 2h, however, the lower temperature (15°C) promoted CuZn SOD transiently (4.1-fold, p<0.001) and then the expression of CuZn SOD decreased to normal level (p>0.05). When exposed to H2O2 (0.1mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.
Asunto(s)
Rotíferos/enzimología , Rotíferos/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Carpas/genética , Catalasa/genética , Clonación Molecular , ADN Complementario , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Peróxido de Hidrógeno , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schistosoma japonicum/enzimología , Schistosoma japonicum/genética , Análisis de Secuencia de ADN , Superóxido Dismutasa/química , Temperatura , Vitamina ERESUMEN
Catecholamines, such as dopamine and noradrenaline, play important roles as neuromuscular transmitters and modulators in all parasitic helminthes, including Schistosoma japonicum. S. japonicum tyrosine hydroxylase (SjTH) was amplified by rapid amplification of cDNA ends polymerase chain reaction that shows strong homology to Schistosoma mansoni tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. The SjTH transcripts encoded the protein of 463 amino acids and a predicted size of 54 kDa. Purified recombinant SjTH as an N-terminal histidine fusion protein expressed in Escherichia coli showed catalytic activity that was confirmed with (3)H tyrosine uptake. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and similar sensitivity to be inhibited by high concentration of the substrate, tyrosine, as the mammalian enzyme. Also, purified SjTH showed characteristic inhibition by catecholamine products. The phosphorylated peptide from SjTH could interact with Sj14-3-3 signal protein. This evidence indicates that SjTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian hosts' enzyme, and its catalytic activity could be regulated by a phosphorylated or dephosphorylated form. This demonstration of SjTH further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.
Asunto(s)
Catecolaminas/metabolismo , Proteínas del Helminto/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Schistosoma japonicum/enzimología , Esquistosomiasis Japónica/parasitología , Tirosina 3-Monooxigenasa/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Datos de Secuencia Molecular , Fosforilación , Plásmidos , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma japonicum/genética , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Esquistosomiasis Japónica/metabolismo , Especificidad por Sustrato , Transformación Bacteriana , Tritio/análisis , Tirosina 3-Monooxigenasa/química , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/aislamiento & purificaciónRESUMEN
Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Glutatión Transferasa/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Evaluación Preclínica de Medicamentos/instrumentación , Resistencia a Medicamentos , Resistencia a Antineoplásicos , Glutatión/metabolismo , Isoenzimas/metabolismo , Luminiscencia , Ratones , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/efectos de los fármacos , Schistosoma japonicum/enzimología , Bibliotecas de Moléculas Pequeñas , Especificidad por SustratoRESUMEN
Tyrosinase (TYR) was thought to play a critical role during trematode egg production. In this study, we analyzed two genes (SjTYR1 and SjTYR2), derived from Schistosoma japonicum genome databases, which encode proteins with significant homologies to mammalian TYR. They exhibited the typical TYR topology, including two copper-binding domains and a highly conserved cysteine-rich domain. Semi-quantitative reverse transcription polymerase chain reaction showed that two SjTYR genes were mainly expressed in the female adult worm. A complementary DNA coding the putative common copper center domain of each SjTYR was cloned and inserted into a pET-28a-c(+) prokaryotic expression vector. After purification, the recombinant proteins expressed in Escherichia coli were used to produce their specific antibodies. The native active SjTYRs enzyme appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bonds. Both female and male worms possessed monophenol oxidase and diphenol oxidase activities of TYR. The relative enzymatic activities were 0.165 min(-1) mg(-1) and 0.0805 min(-1) mg(-1), which were inhibited by a copper-chelating agent (allyl thiourea) and correlated with disruption of female egg production. Our results revealed that SjTYRs might play a significant role during eggshell formation.
Asunto(s)
Proteínas del Helminto/biosíntesis , Proteínas del Helminto/genética , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Schistosoma japonicum/enzimología , Secuencia de Aminoácidos , Animales , Catecol Oxidasa/metabolismo , Clonación Molecular , Dimerización , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Expresión Génica , Perfilación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.
Asunto(s)
Schistosoma japonicum/enzimología , Schistosoma japonicum/genética , Succinato Deshidrogenasa/genética , Animales , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , ADN de Helmintos/genética , ADN de Helmintos/aislamiento & purificación , Femenino , Biblioteca de Genes , Proteínas del Helminto/genética , Humanos , Proteínas Hierro-Azufre/genética , Ratones , Ratones Endogámicos BALB C , PlásmidosRESUMEN
Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.