Study On Protective Immune Response Induced By Complex Gene Vaccine Encoding Toxoplasma Gondii ROP18 And ROP12 In Mice

Toxoplasma gondii(T. gondii) is an obligate intracellular protozoan parasite that can infect a wide variety of vertebrates, including birds and mammals which are on land or in the sea. Toxoplasma gondii can cause retinopathy, encephalitis and other diseases. It also can cause pregnant women miscarriage, stillbirth, malformation and congenital infection.Rhoptry proteins which play an important role in the formation and modification process of parasitophorous vacuoles can affect the growth and virulence of Toxoplasma gondii living in host cells in some way.In this study, the eukaryotic expression vector pVAX1-ROP18-ROP12 was constructed by molecular biology method and transfected into human cervical carcinoma cells(HeLa cells). The expression of the recombinant plasmid in HeLa cells was identified by RT-PCR. Then the BALB/c mice were immunized with the recombinant plasmid pVAX1-ROP18-ROP12 by intramuscular injection. The protective immunity in mice was assessed by immune-related indicators.First, primers were designed according to the published TgROP18 and TgROP12 gene. Using Trizol Reagent, the total RNA of T. gondii tachyzoites was extracted. The TgROP18 gene fragments and the TgROP12 gene fragments were amplified by reverse transcription PCR(RT-PCR).After digestion and ligation, the eukaryotic expression vector pVAX1-ROP18-ROP12 was constructed. We identified it using PCR,restriction enzyme digestion and sequence analysis. The results show that the amplification products of TgROP18 and TgROP12 were respectively 1662 bp and 711 bp. The PCR products of the recombinant plasmid pVAX1-ROP18-ROP12 was 2 373 bp in size. The restriction digestion results of the recombinant plasmid pVAX1-ROP18-ROP12 were correct.The sequencing result of TgROP18-TgROP12 complex gene was 100%consistent with the reported TgROP18 gene sequence and that was 99%consistent with TgROP12 gene sequence. It demonstrated that eukaryotic expression plasmid pVAX1-ROP18-ROP12 was successfully constructed.Then, we transfected it into HeLa cells. After 48 hours, the total RNA of HeLa cells in each group was extracted. Using the RT-PCR analysis, we identified the expression of the recombinant plasmid pVAX1-ROP18-ROP12 in HeLa cells. Then we largely extracted the plasmid pVAX1-ROP18-ROP12.Finally, we immunized the mice with recombinant plasmid pVAX1-ROP18-ROP12 by intramuscular injection. Meanwhile, we established single-antigenic plasmid groups(pVAX1-ROP18 plasmid group and pVAX1-ROP12 plasmid group), plasmid pVAX1 group, PBS group and control group. All groups were immunized 4 times for 2-week intervals and the dosage of last immunization was doubled. Before each injection and two weeks after the last injection, the blood of mice in each group was collected and the sera was separated. Then the serum antibodies was determined. Two weeks after the last immunization, six mice in each group were picked out and their spleens were removed aseptically. The splenocytes was made and seeded in cell culture plates. The splenocytes culture supernatant was collected in different times and the cytokines were measured. Meanwhile, the remaining mice in each group were infected with T. gondii tachyzoites to observe and record the survival time.The results showed that with the increase of imunization's times, the sera antibodies and cytokines in the experimental group were gradually increased. After the last immunization, the sera antibodies and cytokines in the experimental group significantly increased, which has a statistical difference compared with other groups(P<0.05). After challenge infection with Toxoplasma gondii tachyzoites, the survival time of mice in the experimental group was significantly longer, compared with other groups(P<0.05). It showed that the plasmid pVAX1-ROP18-ROP12 can induce a stronger immune response, which will provide information for the further study of ROP18-ROP12 complex gene vaccine.