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Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.

A 1949 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA with 3 pair of primers that were designed according to published SmGCP gene encoding gynecophoral canal protein of Schistosoma mansoni and SjGCP1 gene encoding the conservative region of gynecophoral canal protein of Schistosoma japonicum. Sequence analysis indicated that this fragment, named SjGCP, with 85% identity to SmGCP, contained a complete open reading frame (ORF) of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain). The amino acid sequence shared 83.7% identity with gynecophoral canal protein of Schistosoma mansoni. This fragment was cloned into the expression vector pET28c(+) and subsequently expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 80 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.

A 1 131 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with primers designed according to published SmAct2 encoding Schistosoma mansoni actin. Sequence analysis indicated that this fragment, with 92% homology to SmAct2, was a complete open reading fragment (ORF) of actin gene of Schistosoma japonicum (Chinese strain). This gene was cloned into the expression vector pET28a( ) and subsequently expressed in Escerichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 47 kD. Western blotting showed that the recombinant protein had good reactivity with the rabbit serum immunized with Sj worm antigen, indicating that this gene encode actin of Schistosoma japonicum(Chinese strain).

A 600 bp DNA fragment was amplified by PCR, from an adult Schistosoma japonicum cDNA library. Sequence analysis revealed that this fragment containedthe S. japonicum Chinese Mainland strain fatty acid binding protein (Sj-14FABPc) gene. This gene was then cloned into the expression vector pGEX-2T, and subsequently expressed in Escherichia coli. The recombinant GST-fusion protein could be purified by glutathione agarose affinity chromatography. Its molecular weight was about 41 kD. The yield of expression was around 25 mg/L E. coli culture. The immunological test suggested that the recombinant protein had good antigenicity, and could be developed into a new vaccine molecule of S. japonicum.

Triose-phosphate isomerase is an important candidate for schistosoma antigens. An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA. Sequence analysis revealed that this fragment contained S. japonicum (Chinese strain) triose-phosphate isomerase gene. Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E. coli. The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography. Its molecular weight was determined to be 54 kD. The yield of expression was around 30 mg/L E. coli culture. Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S. japonicum multi-valent recombinant vaccine.

The gene of the 28 kD glutathione S-transferase (GST) from the Chinese strain of Schistosoma japonicum had been expressed in the silkworm (Bombyx mori) cells and larvae by Bombyx mori nuclear polyhedrosis virus (BmNPV) vector which had been modified. The GST gene was inserted into the right position of the BmNPV genome as identified by Southern hybridization. The product was a 28 kD protein, had GST activity and antigenicity. The yield in BmN cells (1x10(6) cells/ml) was 0.77 mg and more than 5 mg in a silkworm larvae. All the results showed that it is possible to develop the GST expressed in the silkworm larvae to a new kind of genetic engineered schistosomiasis vaccine.