Endemic status and control of animal schistosomiasis in China / 中国血吸虫病防治杂志

This paper describes the damages caused by animal schistosomiasis and the role of animals in the transmission of schistosomiasis in China and reviews the progress of animal schistosomiasis control and the endemic status of animal schistosomiasis at various stages. Although the endemic situation of animal schistosomiasis has been effectively controlled, there are still multiple factors that affect the transmission of schistosomiasis, and there are still risks leading to reemergence or rebounding of schistosomiasis in local areas. Strengthening of schistosomiasis surveillance as well as the prevention and control effects in key areas, consolidation of schistosomiasis control achievements, resolving of key technical problems in the elimination of animal schistosomiasis and development of precise technical measures and strategies are needed to accelerate the progress towards the elimination of schistosomiasis in China.

Cloning, expression and characterization of a gene encoding signal transduction protein Wnt4 of Schistosoma japonicum / 生物工程学报

Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4 (GenBank accession No. DQ643829). Sequence analyses showed that SjWnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4. The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential express. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) cells, and the production of recombinant Sjwnt4 protein fused to a GST tag was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.

Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization / 生物工程学报

In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

Studies on phage displayed mimotopes of a protective monoclonal antibody (SSj14) against Schistosoma japonicum / 生物工程学报

To obtain peptides mimicking epitope of a protective McAb SSjl4 specific to Schistosoma japonicum and investigate their immuno-protection effects. A phage random 12 peptide library was screened using purified McAb SSj14, 33 clones were picked up for specificity identification by ELISA. The epitope of each positive clones were detected by the sequencing analysis technique. The antigenicity of three positive clones (P1, P2 and P11) and their mixture cock-tail were further confirmed by Western-blotting, and their protective efficiency were evaluated by mice vaccination experiment. IL-12 level between the vaccinated mice and control mice were compared. 30 positive phage clones were obtained, which represented 11 different epitopes respectively, there were a similar sequence "H-N/Q-X-S-P/F-X-X-L-A-T" among all of the epitopes. Western-blotting showed that all of the three tested clones were recognized by McAb SSj14. Significant adult worm reduction (13.84% to approximately 52.83%), liver tissue egg reduction (34.17% to approximately 65.47%) as well as fecal egg reduction (28.89% to approximately 73.78%) were observed in mice vaccinated with phages of P1, P2, P11 and mixture of three clones when compared with those of the blank control group, among them, the mice vaccinated with the mixture of phage clones got higher protection than any of the mice injected with only one kind of clone phages. At the same time, the IL-12 level in serum of vaccinated mice was found higher than those of the blank control one, this suggest that IL-12 may correlate with the protective efficiency induced by the clone phages. The study provides a new way for developing an effective vaccine against S. japonicum.

Protective effect induced by vaccination with a partial cDNA expression library of Schistosoma japonicum in mice / 生物工程学报

The cDNA fragments of interest were amplified using Sj lambda ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p-CMV-GH; A small number of DNA fragments inserted in the recombinants was identified by restriction cleavage, EST sequencing and bioinformatical analysis; mice were injected intramuscularly with the expression library (L-CMV-SjR) or sublibraries(L-CMV-SjR1, L-CMV-SjR2 and L-CMV-SjR3), immunized mice were challenged with Schistosoma japonicum cercariae on day 35, the levels of IgG antibodies in sera from the immunized mice were detected by ELISA. The results demonstrated that a partial cDNA expression library of S.j, with approximately 10(5) transformants, was constructed, most of the recombinants contained the insert DNA fragments of interest, and these fragments had the features of protein-coding sequences for Schistosome. There were no significant differences for the levels of IgG antibodies in sera from all of the immunized groups. Mice immunized with L-CMV-SjR, L-CMV-SjR1 and L-CMV-SjR2 developed significant protective effect against Sj infection compared to control mice injected with the empty plasmid, the rate of worm reduction was about 30%.

Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain) / 生物工程学报

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain). Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain). The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum. This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction. SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.