| Schistosomiasis is recognized as one of the major global public health problems. Chemotherapy is an important measure for control the disease and reducing the prevalence of schistosomiasis.At present,praziquantel(PZQ) is the only drug for the treatment of schistosomiasis caused by five main human schistosome species.In spite of several studies have been undertaken on the mechanism of PZQ action,the molecular target of PZQ has not yet been defined.We intended to investigate the molecular target of PZQ and provide some clues as to finding new antischistosome drug.1.Isolation,identification and analysis of differentially expressed proteins of Schistosoma japonicum proteomics between the treated group with PZQ and the untreatedAt first,we performed the isolation and identification of differentially expressed proteins of Schistosoma japonicum proteomics from each group using two-dimensional-nano-liquid chromatography coupled by tandem mass spectrometry (2D-nano-LC-MS/MS).The database was established for each group.There 437 proteins based on 1431 peptides were identified and 253 proteins were annotated with known function in the control group.In the other group 73 proteins based on 2117 peptides were identified and 73 proteins were marked with function.There were 16 proteins found in both the control and the treated group.The number of total protein category of the treated group was significantly less than that of the control,in which a series of molecules involved in cellular metabolism,transcriptional/translational regulation were expressed differentially between the control and the treated.The statistics analysis showed there were 12 significantly up-regulated proteins and 4 significantly down-regulated proteins in the treated compared with the control.The former were cytoskeleton and motor proteins,response stress and chaperones, intracellular signal molecules,on the other hand,the latter were molecules of metabolism and regulation.In addition,the tegument proteins obviously changed in level and category between the control and the treated group.The result indicated there was significant difference in protein level between the two groups.It is suggested that PZQ can increased or inhabited the expression of certain genes of Schistosoma japonicum.The expressed differentially proteins may be related to the mode of PZQ action and provide some potential target for PZQ,and also permit the identification of protein candidates for the development of new targets for drug against schistosomiasis.2.Screening the target-protein gene of PZQ antischistosoma using improved Drug-Western method from cDNA LibraryDrug-Western method is a novel and convenient method to isolate genes of drug-binding proteins directly from a cDNA expression library.The combination of drug and marker molecule acted as probe,then the probe was detected using secondary antibody labeled.The positive clones obtained was separated,sequenced and expressed so as to study the function and structure of the gene.Based on structure and characteristics of PZQ,we improved the Drug-Western method:PZQ labeled ~3H was regarded as probe directly to screen cDNA expression library of schistosoma. After probing,the radioactive signal was detected by X-ray.The positive clones were obtained and the corresponding gene was sequenced and aligned.The results showed 7 positive clones belonged to 5 genes:one of them encodes cytochrome c oxidase, another was Schistosoma japonicum special protein with unknown function (SJCHGC02820),and the other three were new genes.The drug-binding proteins were gained by screening the genes of drug-binding proteins directly from a cDNA expression library of schistosoma using improved Drug-Western method.The improved method provides a new approach for screening drug target of the specific structure drug.3.Biocharacteristics of schistosome voltage-gated calcium channel variantβsubunitThe result of PZQ proteomics analysis and the electrophysiological study suggested schistosome voltage-gated calcium channelβsubunit may be related to mode of PZQ.The expressed level was investigated in the PZQ-sensitive(adult worm) and PZQ-insensitive(schistosomula) stages.The sequence of theβsubunits were aligned in different species which are distinct in sensitivity to PZQ.The difference of the gene among species was analyzed using phylogenetic tree.We examined the transcript and protein levels of the gene by real time-PCR and Western blot analysis, and localized the encoding protein in the adult worm and schistosomula by indirect fluorescent-antibody assay.The phylogenetic analysis showed the variant and the conventionalβsubunits tended to separate,the former only was found in PZQ-sensitive parasites and the similarity was relatively low.Western blot analysis indicated the encoding protein could be detected in the soluble protein of adult worm rather than schistosomula.However,the real time-PCR revealed that the transcript levels did not express differentially in the adult worm and schistosomula.The immunolocalization showed the encoding protein located in the tegument,gut epithelial tissues,parenchyma and vitelline gland of adult worms,but the fluorescent signal was not detected in schistosomula.The findings provided the evidence for the known pharmacological effects of the drug.The results revealed the distribution of SjCavβvar may be target tissue that was firstly attacked by PZQ.Therefore, SjCavβvar played a role in drug action and may be the target for PZQ.Thus,the relatively low overall similarity to humanβ1-β4subunits(38.6%-41.4%) makes it a potential target for the development of new drugs.4.Functional analysis onβinteraction domain of schistosome voltage-gated calcium channel variantβsubunitTheβinteraction domain(BID) of schistosome voltage-gated calcium channel variantβsubunit is important to regulate sensitivity of voltage-gated calcium channel to drug.The recombinant BID protein(rSjccβkd) of schistosome voltage-gated calcium channel variantβsubunit was firstly expressed and refolded.The aims of study were(1) to detect the binding capacity of rSjccβkd with PZQ,(2) to observe effect of rSjccβkd to PZQ,(3) to investigate effect of antibody of rSjccβkd on aldult worms in vitro and(4) to examine the protective efficacy of rSjccβkd.The binding capacity of the rSjccβkd to PZQ was detected by radioactive counting(decay per minute,DPM).The worms were observed for contractility,motility,viability and the change in tugement was examined simultaneously after incubation with rSjccβkd protein and immune rabbit serum of rSjccβkd,respectively.The protective experiment of rSjccβkd was carried out in mice.The binding assay using ~3H-PZQ demonstrated specific binding of the PZQ to rSjccβkd.The DPM of refolded rSjccβkd binding to PZQ were almost 3 times higher than denatured rSjccβkd.The results implied that rSjccβkd have no effect on PZQ action on adult worms in vitro.When adult worms of Schistosoma japonicum were maintained in culture medium containing immune rabbit serum and exposed to PZQ,the number of died worms was 1/3 more than that only exposed to PZQ in the same observed period.The results revealed that rSjccβkd did not directly antagonize PZQ action,but the immune rabbit serum had synergetic effect to PZQ.The immunication test showed a certain extent protection with worm reduction rate of 24.7%and egg reduction rate of 16.7%.The radio-ligand receptor assays indicated that it was sample and effective method to screen drug target in vitro.
|
PDF Full Text Request