关键词: 　刚地弓形虫, 复合基因疫苗, SAG1-ROP5

Abstract: Objective  To observe the immune response induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii in mice.  Methods  The recombinant eukaryotic expression plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were constructed and identified by PCR, restriction enzyme digestion, and sequencing. The three recombinant plasmids were transfected into HeLa cells to express in vitro and identified by Western blotting analysis. Seventy Kunming mice were randomly divided into 5 groups with 14 each, i.e. pcSAG1 group, pcROP5 group, pcSAG1-ROP5 group, blank plasmid group and PBS control group. The mice were immunized intramuscularly with pcSAG1, pcROP5, pcSAG1-ROP5, pcDNA3.1, and PBS, respectively, every two weeks for three times. Sera were collected before each injection and 2 weeks after the last immunization. The titer of mice serum in pcSAG1-ROP5 group combined with recombinant protein SAG1, ROP5 and SAG1-ROP5 and the level of IgG against T. gondii in 5 groups were determined by ELISA. Three weeks after the last immunization, ten mice of each group were challenged with 103 tachyzoites of the virulent T. gondii RH strain to observe the survival time. One week later, the rest four mice in each group were sacrificed and the supernant of cultured splenocytes was collected for the detection of IFN-γ and IL-4.  Results  Western blotting showed that the recombinant plasmids pcSAG1, pcROP5 and pcSAG1?鄄ROP5 were expressed in HeLa cells with Mr 31 000, 57 000, and 88 000, respectively. The serum titer in pcSAG1-ROP5 group combined with SAG1, ROP5 and SAG1-ROP5 was 1 ∶ 320, 1 ∶ 160, and 1 ∶ 2 560, respectively. The IgG level kept rising in pcSAG1, pcROP5 and pcSAG1-ROP5 groups. Two weeks after the last immunization, the IgG level in pcSAG1-ROP5 group was higher than those in other groups（P<0.05）. After a lethal challenge of T. gondii RH strain, the survival time of the mice in pcSAG1-ROP5 group was （288±7） h, which was 48 h and 96 h longer than the groups of pcSAG1 and pcROP5, respectively （P<0.05）. Four weeks after the last immunization, IFN?-γ in splenocyte culture of pcSAG1-ROP5 group ［（908.52±6.31） pg/ml］ was higher than other groups （P<0.05）, with no significant difference in IL-4 （P>0.05）.  Conclusion  Compared with the single gene vaccines pcSAG1 and pcROP5, higher levels of IgG and IFN-γ and longer survival time are observed in mice immunized with pcSAG1-ROP5.

Key words: Toxoplasma gondii, Complex gene vaccine, SAG1-ROP5