The protective immunity of a DNA vaccine encoding Schistosoma japonicum Chinese strain triose-phosphate isomerase in infected BALB/C mice.

The development of a DNA vaccine for schistosomiasis japonica and testing the protective efficacy after challenge in BALB/c mice were performed. Thirty-nine female BALB/c mice were divided into three groups. Each mouse of the control group was injected intramuscularly with 100 microg of pcDNA3.1 DNA. In the TPI group, each mouse was injected with 100 microg of pcDNA3.1-SjCTPI DNA. The TPI+IL-12 group was injected with 100 microg of pcDNA3.1-SjCTPI DNA and 100 microg of the mixture of pcDNA3.1-P35 and pcDNA3.1-P40 DNA. Each mouse was immunized three times at two-week intervals and challenged with 45 cercariae of Schistosoma japonicum Chinese strain four weeks post-immunization. Then the mice were sacrificed and perfused at 45 days after challenge; the recovered worms and hepatic eggs were counted. Cytotoxic T lymphocyte (CTL) activity mediated by SjCTPI was detected with the 51Cr release assay. ELISA was performed for the detection of anti-rTPI antibodies. Anti-rTPI antibody detection with ELISA after immunization showed ten serum samples from the control group were negative, five of ten serum samples from the TPI group were weakly positive, six of ten from the TPI+IL-12 group were also weakly positive. The CTL activity of the control group was 9.1%, while CTL activities of the TPI group and the TPI+IL-12 group were 27.6% and 54.4%, respectively. The worm and egg reduction rates of TPI group and the TPI+IL-12 group were 30.2%, 52.9%, 32.7%, and 47.0%, respectively in comparison with the control group. This study further proved the possibility of the SjCTPI DNA vaccine as a potential DNA vaccine for schistosomiasis.

Protective immunity induced with 23 kDa membrane protein dna vaccine of Schistosoma japonicum Chinese strain in infected C57BL/6 mice.

A 23 kDa membrane protein DNA vaccine for Schistosoma japonicum Chinese strain was developed and tested for its protective efficacy and immune responses in infected C57BL/6 mice. The cDNA encoding SjC23 amplified from pUC19-SjC23 were subcloned into an eukaryotic expression vector (pcDNA3.1). Forty-eight female C57BL/6 mice were divided into three groups. Each mouse of group A (control group) was immunized intramuscularly (i.m.) with 100 microg of pcDNA3.1; of group B (SjC23 group) was immunized (i.m.) with 100 microg of pcDNA3.1-SjC23; of group C (SjC23+IL-12) was immunized (i.m.) with a mixture of 100 microg of pcDNA3.1-SjC23, 100 microg of pcDNA3.1-p35 and 100 microg of pcDNA-p40. These were followed by two boosts of the same DNA once every two weeks. All mice were challenged with 45 cercariae of Schistosoma japonicum Chinese strain at week 8, and were killed and perfused at week 14. The numbers of recovered worms and hepatic eggs were counted. The expression of SjC23 and p35, p40 in muscle tissue was determined by immunohistochemical method. By culture of spleen cells, the production of IL-2, IL-4, IL-10 and IFN-gamma with the stimulation of specific antigen of the recombinant hydrophilic domain of SjC23 (rSjC23-HD) was determined after the last immunization (before challenge). Sera were collected from each group before immunization and two weeks before and after challenge. Anti-SjC23 antibodies were tested by Western blot. The results showed that SjC23 and p35, p40 of mouse IL-12 were expressed on the membrane and in the plasma of the muscle cells of immunized C57BL/6 mice. A rise of IL-2 and IFN-gamma in the SjC23 group and SjC23+IL-12 group was observed; No changes were found in IL-4 and IL-10. Detection of anti-SjC23 antibody with Western blot showed that after the third immunization (before challenge) all the serum samples from the control group were negative; 8 of 10 sera from the SjC23 group and 9 of 10 sera from the SjC23+IL-12 group were positive. The worm reduction rates in the SjC23 group and SjC23+IL-12 group were 26.9% and 35.4% respectively; the liver eggs reduction rates were 22.2% and 28.4%, respectively in comparison to the control group. This indicates that the pcDNA3.1-SjC23 DNA vaccine can induce partial protection against Schistosoma japonicum infection in C57BL/6 mice.

The protective immunity produced in infected C57BL/6 mice of a DNA vaccine encoding Schistosoma japonicum Chinese strain triose-phosphate isomerase.

The development of a SjCTPI DNA vaccine for Schistosoma japonicum and the detection of the immune responses to and the protective efficacy of immunization were performed and challenged in C57BL/6 mice. According to the gene sequence of SjCTPI and murine IL-12, three pairs of primers were designed. The full length cDNA encoding SjCTPI and P35, P40 amplified from pUC19-SjCTPI and murine IL-12 by PCR were subcloned into an eukaryotic expression vector (pcDNA3.1). Forty-five female C57BL/6 mice were divided into three groups; each mouse of the control group was injected with 100 pg of pcDNA3.1 by i.m. route; the TPI group was injected with 100 microg of pcDNA3. 1-SjCTPI; the TPI+IL- 12 group was injected with 100 microg of pcDNA3.1-SjCTPI and 100 pg of mixture of pcDNA3.1-P35 and pcDNA3.1-P40. Each mouse was immunized at weeks 1 and 5 and challenged with 45 cercariae of Schistosoma japonicum Chinese strain at week 9. The mice were killed and perfused 45 days after challenge; the numbers of recovered worms and hepatic eggs were counted. The expression of SjCTPI in muscle tissue was determined by an immunohistochemical method. Culture of spleen cells showed the production of IL-2, IL-4, IL-10 and IFN-gamma with the stimulation of specific antigen before and after challenge. Sera were collected from each group before immunization, before challenge and two weeks post challenge; ELISA and Western-blot tests were performed for detection of anti-rTPI antibodies. The antigen of SjCTPI was expressed in the membrane and plasma of the muscle cells of C57BL/6 mice. The obvious rising of IL-2 in TPI group and TPI+IL-12 group before and after challenge was seen. The anti-rTPI antibody detection with Western-blot showed that ten serum samples from the control group were negative; nine of ten serum samples from the TPI group were weakly positive, eight of ten from the TPI+IL-12 group were weakly positive. The worm and egg reduction rates of TPI group and TPI+IL- 12 group were 27.9% and 13.7%, 31.9% and 18.6% respectively in comparison with the pcDNA group. pcDNA3.1-TPI DNA vaccine could confer partial protection against a subsequent challenge of Schistosoma japonicum in C57BL/6 mice and might therefore be a potential DNA vaccine.

Clone of the gene of the dense granule antigen (GRA6) of the Toxoplasma gondii pig strain.

The gene of GRA6 of Toxoplasma gondii pig strain was cloned; and the value of recombinant GRA6 as a diagnostic antigen of toxoplasmosis in China was investigated. A pair of primers was synthesized according to the DNA sequence of GRA6 of T. gondii RH strain, and the DNA of pig strain tachyzoites was prepared. A single, specific DNA fragment was successfully amplified from the genomic DNA of tachyzoites of T. gondii. This gene fragment was cloned into the plasmid pUC19 to form the recombinant. The recombinant plasmid was purified and the foreign DNA fragment was sequenced. The result of DNA sequencing showed that the open reading frame of GRA6 is composed of 693 base pairs and encodes 230 amino acids and is 100% homologous with the sequence of GRA6 of T. gondii RH strain.