| Toxoplasma gondii is an obligate intracellular parasite belonging to the protozoan phylum Sporozoa. Roughly one fourth of the human beings carry live Toxoplasma cysts in their brains with no overt effects. In immunocompromised patients T. gondii may cause severe degeneration in the central nervous system and congenital transmission of tachyzoites could also result in grave consequences for infected fetuses and newborns. But healthy individuals are rarely affected by T. gondii infection since the immune system could recognize and eliminate the parasites of tachyzoites.Recently, the development of vaccines against toxoplasmosis has progressed considerably due to the application of the technology of molecular biology. Research showed that the inactivated vaccine was insufficient to induce proper immune responses. A vaccine based on the live attenuated S48strain was developed for veterinary uses. However, it is not suitable for human use for the possibility of reverting to a pathogenic strain. With expensive preparation, weak antigenicity and very complicated production process, polypeptide vaccine of genetic engineering has not been widely used. Under the present scenario, development of effective vaccine are very crucial to protect susceptible population and to promote aristogenesis.There are a variety of studies suggesting that apical complex in particular rhoptries is a major players in T. gondii invasion and host cell interaction. The TgROP18, a member of the rhoptry proteins, has been implicated as an important virulence factors.In this study, we constructed a plasmid expressing rhoptry protein18(ROP18) of T. gondii, and evaluated its anti-infections immune response in mice. First of all, extract total DNA from T.gondii tachyzoites of RH strain. TgROP18gene was amplified by PCR and cloned into pET30a(+) vector.The constructed pET30a(+)-TgROP18was transformed into E.coli BL21(DE3) first and selected through the E.coli BL21(DE3) for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by coomassie brilliant blue staining. Western blotting assay with mice anti-T.gondii serum was used to analyze its antigenicity. The results showsd that the TgROP18gene was successfully cloned by PCR and the length was about1665bp and the recombinant plasmid pET30a(+)-TgROP18was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately59.80kD and could be recognized by mice anti-T.gondii serum. The conclusion is that the soluble His-TgROP18protein maintains its immunoreactivity.Next, we assessed for the protective immunity induced by Toxoplasma gondii Rhoptry Protein18against Toxoplasmosis in Mice.A total of60mice were equally divided into4groups. The mice in each group were injected with His-TgROP18-FAC(A), His-TgROP18(B), FAC(C), PBS(D) respectively, and mice were immunized using the same protocol on days0,20and34. One week after the final inoculation, the mice in all groups were challenged intraperitoneally (IP) with1×103tachyzoites of the virulent T. gondii RH strain was used for challenging infection, and blood was collected from the mouse caudal vein to immunization. Sera were separated and stored at-20℃until used for cellular immune response analysis. The results of mean concentration were as follows:A groups:IL-454.17ng·L-1, IL-10106.88ng·L-1, IL-175.02ng·L-1, INF-γ166.96ng·L-1; B groups:IL-452.56ng·L-1, IL-10103.34ng·L-1, IL-174.99ng·L-1, INF-γ142.42ng·L-1; C groups IL-473.06ng·L-1, IL-10144.40ng·L-1, IL-1713.34ng·L-1, INF-γ73.64ng·L-1; D groups: IL-468.29ng·L-1, IL-10145.29ng·L-1, IL-1714.97ng·L-1, INF-γ76.50 ng·L-1.Significantly high levels of IFN-γ were observed in spleen cell cultures from mice immunized with His-TgROP18-FCA and His-TgROP18compared with mice immunized with FAC and PBS. On the other hand, low levels of IL-4、IL-17and IL-10showed a slightly but significantly reduced response from the serum of mice immunized with His-TgROP18-FCA and His-TgROP18, compared with those of mice with FAC and PBS (P<0.05).These results demonstrate that TgROP18has been induced significant humoral and cellular Th1immune responses. After lethal challenge, the mice immunized with the TgROP18showed a significantly increased survival time of22～25days compared with control mice which died within6～7days. Our data demonstrate:1. The TgROP18gene was successfully cloned by PCR;2. The recombinant plasmid pET30a(+)-TgROP18was successfully constructed;3. ROP18triggered a strong humoral and cellular response against T. gondii, and the antigen was a promising vaccine candidate against toxoplasmosis, and worthing further development. |
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