| Objectives: Toxoplasma gondii is a protozoan parasite worldwide. It is a important opportunistic protozoan, parasitiged in the human body and nucleated cells. A total of more than 200 species of animals and human beings can be infected. It is estimated that the infection rate in the world is about 30%. A person with compromised immunity or a new-born baby is often the victim of Toxoplasma gondii, resulting in severe effects. So the parasite directly affects the health of mankind and the development of society and is harmful to food safety and animal breeding. However, we have no effctive drugs to prevent Toxoplasma gondii infection nowdays. So development of safe and effective vaccine to prevent the disease is particularly important. Researches shows that toxoplasma rhoptry proteins are secreted from rhoptry and plays an important role in attacking the host cell and poisoning it. And It is considered to has great potential for vaccine development. In this research, recombinant plasmid p VAX1-ROP10 was constructed by molecular biological methods and BALB/C mice were immunized by intramuscular injection. The results obtained provides an theory foundation for the further study of ROP10 gene DNA vaccine.Methods: Firstly, the total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The primes were designed based on published genes of Genbank(Accession No. DQ124368) and the ROP10 gene was amplified by RT- PCR and was inserted into the eukaryotic expression vector p VAX1. The recombinant plasmid p VAX1-ROP10 was verified by colony PCR, enzyme digestion analysis and sequencing analysis. Then the recombinant plasmid was transformed into competent Escherichia coli XL1-Blue. The positive clones were screened, cultured expandedly and the p VAX1-ROP10 recombinant plasmid was extracted and transfected into He La cells by Lipofector cationic liposome. The total RNA of He La cells was extracted and the β-actin gene and ROP10 gene were amplified by RT-PCR. The RT-PCR production was verified by electrophoresis to test whether the ROP10 gene was expressed in the He La cells. Eight-week-old BALB/C mice were immunized by quadriceps injection three times with 2 weeks intervals. The serums were taken from the mice before each injection and two weeks after the last injection separatedly. By indirect ELISA, the levels of Ig G antibody and cytokines(IFN-γ, IL-2, IL-4, IL-10) in serum of each group were measured. At the same time, the survival time of mice in each group was observed after infection with toxoplasma gondii.Results: The results showed that the RT-PCR product of ROP10 gene was 1761 bp and the recombinant plasmid p VAX1-ROP10 was constructed and the reresults of colony PCR and double enzyme digestion by Eco R I and Not I and sequencing analysis were identified correctly. The p VAX1-ROP10 recombinant plasmid was extracted and transfected into He La cells. The RT-PCR products of β-actin gene and ROP10 gene were verified by 0.8% agarose gel electrophoresis. Brights bands were observed respectively at the positions of 613 bp and 1761 bp which were consistent with the expected sizes. This proved the ROP10 gene was able to express in eukaryotic cells in vitro. After immunization, the level of serum Ig G antibody was significantly increased in experiment group along with the increase of the immunization frequency, but were not increased significantly in the empty plasmid group, the PBS control group and the blank control group. After the last injection, the serum Ig G taken from experiment group increased significantly compared with the control groups(P<0.05). The average serum concentration of IFN-γ, IL-2, IL-14, IL-10 in the experiment group were 465.31±12.21, 168.52±11.22, 150.35±11.34, 166.00±12.34 separately and that in empty plasmid group were 50.32±6.73, 48.63±6.02, 52.00±6.83, 53.67±5.94 and in PBS control group were47.02±4.68, 42.34±5.24, 47.30±5.23, 50.63±5.94, and in blank control group were 48.39±5.23, 46.32±5.25, 49.23±7.84, 50.00±6.82. The results showed that the level of cytokines in the experiment group increased significantly compared to the empty plasmid group, the PBS control group and the blank control group(P<0.05). Challenge experiments showed that the mean survival time of mice in the experiment group was significantly longer than the other groups(P<0.05).Conclusion: The eukaryotic expression plasmid p VAX1-ROP10 was constructed successfully and expressed in the He La cells. Immunized with recombinant plasmid p VAX1-ROP10, the immunity response in mice can be enhanced, which provides an experimental basis for further study of ROP10 DNA vaccine. |
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