| Toxoplasma gondii is an obligate intracellular protozoan parasite thatis able to infect any warm-blooded animal. It is estimated that1/3populations are infected or once infected with Toxoplasma gondii. Becausethis parasite can lead to retinal scarring, brain damage and miscarriage inpregnant women it has already been a major protozoan in etiology ofhuman disease. Toxoplasma rhoptry proteins secreted by the secretoryorganelles rhoptry plays an important role in the parasite invasion of thehost cells, and is closely related to the process of formation andmodification of parasitophorous vacuoles (PV). In this experiment aeukaryotic expression recombinant plasmid pcDNA3.1(+) containingROP11gene from toxoplasma gondii RH strain (pcDNA3.1(+)-ROP11)was constructed. The recombinant plasmid was transfered into HeLa cellsand the expressions of the plasmid were detected by indirectimmunofluorescence assay. Then BALB/C mice were immunizedintramuscularly with the recombinant plasmid pcDNA3.1(+)-ROP11andthe protective immunity effects were evaluated by detecting serum IgG、IL-2、IL-4、IL-10and IFN-γ levels and the survival time of mice afterinfection with toxoplasma gondii were recorded so as to provide theoreticaland material basis for further Toxoplasma vaccine research.Firstly, total RNA was extracted from RH strain tachyzoites ofToxoplasma gondii. According to the open reading frame of full-lengthsequence (Accession No. DQ077905) of the ROP11gene primers weredesigned and amplified by reverse transcription PCR. The fragment ofROP11gene was then cloned into the eukaryotic expression vector pcDNA3.1(+) and a recombinant plasmid pcDNA3.1(+)-ROP11wasconstructed. The products of PCR digested with EcoR I and Not I wereconnected to the pcDNA3.1(+) eukaryotic expression vector digested alsowith EcoR I and Not I and the recombinant plasmids were identified bycolony PCR, EcoR I and Not I double enzyme digestion and sequencing.The results showed that the product of RT-PCR was1548bp and the resultsof colony PCR and double enzyme digestion are correct. Compared to thesequence of ROP11in GenBank, eight nucleotides were discovered to havevaried and five amino acid changed and at the fifty-six amino acid site onemore asparagine was found inserted. These results showed that theeukaryotic expression plasmid pcDNA3.1(+)-ROP11had beensuccessfully built.After the expression vector plasmid constructed successfully, therecombinant plasmid was transformed into competent Escherichia coliXL-Blue. The positive clones were screened, cultured expandedly and thepcDNA3.1(+)-ROP11recombinant plasmid was extracted and transfectedinto HeLa cells by Lipofector cationic liposome. By Indirectimmunofluorescence assay yellow-green fluorescence was observed in thecytoplasma of recombinant plasmid transfected HeLa cells, but none in thecontrol group, which proved that eukaryotic expression plasmid was able toexpress the target protein in eukaryotic cells.A large number of plasmids were extracted by the alkaline lysismethod and the mice were immunized. Forty8-week-old BALB/C micewere randomly divided into four groups, i.e. ten in each: the experimentgroup, the empty plasmid group, the PBS control group and the blankcontrol group. The mice were immunized by quadriceps injection threetimes with2weeks intervals. The experiment group were injected with100μg(1μg/μl) of recombinant plasmid pcDNA3.1(+)-ROP11; the emptyplasmid group with100ug (1μg/μl) of pcDNA3.1(+) plasmid; the PBSgroup with100ul of PBS buffer and the blank control group with nothing. In the last injection the dosage of injectants were doubled in all mice. Theserums were taken from the mice before each injection and two weeks afterthe last injection separatedly and antibodies and cytokines（IFN-γ、IL-2、IL-4、IL-10）were detected. To observe the protective immunity effects themice in all groups were challenged intraperitoneally (IP) with1×103tachyzoites of the toxoplasma gondii RH strain two weeks after the lastinjection. After immunization, the serum IgG antibody of experiment groupwas significantly increased along with the increase of the immunizationfrequency, but were not increased significantly in the empty plasmid group,the PBS control group and the blank control group. The serum IgG fromexperiment group taken after the last injection had also had a statisticallysignificant (P<0.05) compared with that fom the empty plasmid group, thePBS control group and the blank control group. In the experiment group,the average level of IFN-γ was364.51pg/ml and had a statisticallysignificant increase (P<0.05) compared to the empty plasmid group（49.55pg/ml）, the PBS control group（46.24pg/ml）and the blank control group（47.52pg/ml）; the average level of IL-2was151.54pg/ml and had astatistically significant increase (P<0.05) compared to the empty plasmidgroup（49.40pg/ml）, the PBS control group（43.34pg/ml）and the blankcontrol group（43.23pg/ml）; the average level of IL-4was131.45pg/mland had a statistically significant increase (P<0.05) compared to the emptyplasmid group（50.28pg/ml）, the PBS control group（53.17pg/ml）and theblank control group（49.14pg/ml）; the average level of IL-10was159.82pg/ml and had a statistically significant increase(P<0.05) compared to theempty plasmid group（56.58pg/ml）, the PBS control group（52.18pg/ml）and the blank control group（49.29pg/ml）. The results showed that all IgGand cytokines in the experiment group were increased significantly (P<0.05) compared to the empty plasmid group, the PBS control group and theblank control group. Anti–infection immunity challenge experimentsshowed that the mean survival time of mice in the experiment group was 236.3h and was significant longer than that of the empty plasmid group97.8h, the PBS control group96.3h and the blank control group96.2h(P<0.05). The results showed that immunizition of mice withpcDNA3.1(+)-ROP11recombinant plasmid could enhance humoralimmunity and cellular immune functions, which provided an experimentalfoundation for further research in biological functions of ROP11gene andin anti T. gongii nucleic acid vaccine. |
PDF Full Text Request