Objective To evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii ( T.gondii ) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene. Methods Truncated...Objective To evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii ( T.gondii ) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene. Methods Truncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction (PCR) and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 μg plasmid DNA entrapped in liposome. Four weeks after the final booster injection,blood samples were collected and subjected to enzyme-linked immuno sorbent assay (ELISA) to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice. Results Green fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T.gondii tachyzoites and primarily interferon-γ and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1. Conclusions Immunization with a liposome-encapsulated DNA construct encoding the T.gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses.展开更多
文摘Objective To evaluate the immune responses induced by experimental DNA construct encoding Toxoplasma gondii ( T.gondii ) surface antigen1 (SAG1) and rhoptry protein 1 (ROP1) in mice as a hybrid gene. Methods Truncated SAG1 and ROP1 DNA fragments were amplified using polymerase chain reaction (PCR) and inserted into pEGFP-N3 vector to construct recombinant plasmid pSAG1-ROP1. NIH3T3 mammalian cells were transiently transfected with the DNA construct. Female BALB/c mice were given three intramuscular injections of 10 μg plasmid DNA entrapped in liposome. Four weeks after the final booster injection,blood samples were collected and subjected to enzyme-linked immuno sorbent assay (ELISA) to investigate humoral and cell-mediated immune responses. Reversal transcript-polymerase chain reaction (RT-PCR) was used to evaluate the transcription of inoculated DNA-liposome complex in the injected site. Dot-blot hybridization was employed in order to detect whether or not the injected DNA was incorporated into the genomic DNA of the immunized mice. Results Green fluorescence was observed in pSAG1-ROP1-transfected cells. Western blot analysis showed antibody recognition of the expressed SAG1-ROP1 was between 58 kDa and 75 kDa. No expression was observed in blank control plasmid-transfected cells. The sera of immunized mice exhibited antibodies to T.gondii tachyzoites and primarily interferon-γ and interlukin-2. RT-PCR showed that the duration of transcribed inoculated liposome entrapped DNA in the injected muscular tissue was at least ten days post the first injection. Dot-blot hybridization revealed that the presence of foreign DNA in the splenocytes and peripheral blood leukocytes was transient and that no foreign DNA had inserted into the genomic DNA of mice immunized with pSAG1-ROP1. Conclusions Immunization with a liposome-encapsulated DNA construct encoding the T.gondii SAG1 and ROP1 can induce humoral and cell-mediated immune responses.
