Objective To analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.

Nineteen stocks of Trypanosoma cruzi originating from several endemic countries for Chagas‘ disease in Central and South America were subjected to two-dimensional protein electrophoresis analysis. The presence or absence of a total of492polypeptide spots among19gel profiles was determined. The stocks were classified into three major distinctive groups derived from (I) Central America and the northern part of South America; (IIa) Central America and the northern part of South America; and (IIb) central and southern parts of South America, which showed perfect concordance with the previously reported classification based on isozyme and DNA sequence analyses. Late log phase of each epimastigote was inoculated to human cell lines WI-38and Hs224.T originating from the lung and muscle, respectively, and the number of trypomastigotes released was counted. The number of trypomastigotes from T. cruzi in group I released from the two cell lines was significantly higher than that in group III (p&It;0.05). The findings suggested that the phenetic distance appearing within the T. cruzi may, to some extent, be associated with the intracellular growth of T. cruzi, one of the characteristic features of growth found in the species.

OBJECTIVETo explore the diagnostic efficiency of circulating antigen using the TM5.28 mAB-biotin-avidin system for the detection of schistosomiasis japonica.

METHODSA mAb-biotin-avidin system was set up using a TM5.28 mAB which was prepared against a gut associated antigen of Schistosoma japonicum. Detection was performed on the sera from 50 acute schistosomiasis patients, 224 chronic patients, 49 advanced patients and 46 schistosomiasis patients who were followed up at 6 months and 12 months post treatment. In addition, 19 cases of clonorchiasis, 31 cases of paragonimiasis, 23 cases of hepatitis B and 100 healthy individuals were also included.

RESULTSThe system showed sensitivity of 83.1% and specificity of 94.0% when applied to detect chronic schistosomiasis and healthy persons respectively, while 94.0% to acute schistosomiasis. The Youden's index of the system was 0.771. The rate of cross-reaction to paragonimiasis, clonorchiasis and hepatitis B was 12.9%, 15.8% and 13.0% respectively. The rates of negative turning were 43.9% and 62.1% respectively in chronic schistosomiasis at the 6 month and 12 month intervals after treatment. Geometric mean of the OD values also decreased from 0.172 before treatment to 0.081 at 6 months and 0.068 at 12 months after treatment with a reduction rate of 60.30%. The detection rate in the heavy infected population reached a maximum of 90.0%. This was similar in moderate and light infected populations, i.e., 83.9% and 82.1%, respectively.

CONCLUSIONThe TM5.28 mAb-biotin-avidin system showed a relatively high efficiency in the diagnosis of schistosomiasis and a high negative turning rate after treatment. It is, therefore, a valuable tool for the estimation of prevalence in endemic populations, as well as individual diagnosis and for assessing the effect of chemotherapy.

Animals ; Antibodies, Helminth ; immunology ; Antibodies, Monoclonal ; immunology ; Avidin ; immunology ; Biotin ; immunology ; Cell Fusion ; Humans ; Mice ; Mice, Inbred BALB C ; Schistosomiasis japonica ; diagnosis ; immunology ; Serologic Tests

OBJECTIVETo identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.

METHODSThe egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.

RESULTSEighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.

CONCLUSIONThe cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.

Animals ; Antigens, Helminth ; genetics ; Base Sequence ; China ; DNA, Complementary ; analysis ; Ovum ; Schistosoma japonicum ; genetics ; immunology

In malaria endemic areas, people naturally acquire an age-related immunity to malaria. Part of this immunity involves anti-malarial specific antibodies. Acquisition of these malaria-specific antibodies depends not only on exposure to malaria parasites but also on the human genetic predisposition. CTLA-4 is a costimulatory molecule that delivers an inhibitory signal to suppress T-cell as well as B-cell responses. We investigated associations between malaria-specific antibody levels and CTLA-4 polymorphisms in 189 subjects living in a hyper-endemic area of Papua New Guinea (PNG), where both P. falciparum and P. vivax are prevalent. We determined P. falciparum⁄ P. vivax specific IgG⁄IgE levels (Pf-IgG, Pv-IgG, Pf-IgE, Pv-IgE) and polymorphisms in the CTLA-4 gene at position -1661 promoter region (A⁄G), the +49 exon 1 non-synonymous mutation (A⁄G), and the +6230 3‘-UTR (A⁄G). All quantified antibody levels were significantly higher in subjects > 5 years (n = 150) than in subjects ≤ 5 years of age (n = 39). In children ≤ 5 years old, significant associations were detected between CTLA-4 +49 (GG⁄AG vs. AA) and Pv-IgG (median 18.7 vs. 13.7 Μg⁄ml, P = 0.017) and Pv-IgE (266.6 vs. 146.5 pg⁄ml, P = 0.046). No significant difference was observed in subjects > 5 years old. These results suggest that the CTLA-4+49 polymorphism influenced Pv-IgG and Pv-IgE levels among children less than five years old in the studied population, which may regulate the age- and species-specific clinical outcomes of malaria infection.

Immunoepidemiological studies from endemic areas have revealed age-dependent resistance correlation with increased level of IgE and decreased level of IgG4 antibodies in responses to schistosomes’ soluble worm antigen. However, there have been limited studies on analyses of major antigens that provoke IgE and IgG4 immune response during chronic stage of schistosomiasis. In this study, for the first time, immunoproteomics approach has been applied to identify S. japonicum worm antigens in liquid fractions that are recognized by IgE and IgG4 antibody using plasma from chronically infected population. ProteomeLabPF 2D fractionated 1-D and 2-D fractions of SWA antigens were screened using pooled high IgE/IgG4 reactive plasma samples by dot-blot technique. In 1-D fractions, IgE isotype was detected by fewer antigenic fractions (43.2%). The most recognized isotype was IgG3 (79.5%) followed by IgG1 (75.0%) and IgG4 (61.4%). Liquid chromatography MS/MS protein sequencing of reactive 2-D fractions revealed 18 proteins that were identified, characterized and gene ontology categories determined. 2-D fractions containing proteins such as zinc finger, RanBP2-type, domain-containing protein were strongly recognized by IgE and moderately by IgG4 whereas fractions containing proteins such as ubiquitin-conjugating enzyme and cytosolic II 5'-nucleotidase strongly recognizing by IgG subclasses (IgG1, IgG3 and IgG4) but not IgE. By this study, a simple and reproducible proteomic method has been established to identify major immunoreactive S. japonicum antigens. It is anticipated that this will stimulate further research on the immunogenicity and protective potential of proteins identified as well as discovery of novel compounds that have therapeutic importance.