Expression Of P30of Toxoplasma Gondii Kunshan Strain In Lactococcus Lactis And Stundy On The Induction Of Immune Rasponses In Mice

Objective: To construct the plasmid L2-Ps-P30-T and express the P30gene ofToxoplasma gondii(T.gondii) Kunshan strain in Lactococcus lactis LM2345.To study the protective effects against Toxoplasma gondii after oral immunizationwith recombinant Lactococcus lactis expressing P30in mice.Methods. The fragments of pSK-P30digested by BamHI/XhoI were inserted intothe same site of psk-PsT, resulting plasmid pSK-Ps-P30-T. Then the plasmidpSK-Ps-P30-T were digested by restrictive enzyme PvuII and the expressionelement-Ps-P30-T were collected. The expression element-Ps-P30-T wereintroduced into L2-PTRK, a shuttle plasmid between E.coli and L Lactis, resultingplasmid L2pS-P30-T. After identification by restrictive enzyme and PCR, the plasmidL2pS-P30-T was transformed to Lactococcus lactis LM2345by electroporation.Then the recombinant L Lactis were cultured in GM17medium overnight andcollected using centrifuge at10000rpm for5min. Then the recombinant L Lactiswere lysed and the lysate were performed by SDS-PAGE and Wester blottingaccording to standard protocols. Sixty-nine BALB/c mice aged6-8weeks old wererandomly divided into three groups: vaccinated group, L. lactis control group, normalsaline control group. They were given5×109recombinant L. lactis cells (vaccinatedgroup), L. lactis cells (L. lactis control group) and normal saline (normal salinecontrol group), respectively. Serum samples of immunized mice (3mice) werecollected on day27,34and48, respectively. The mucosal secretions in the lumen ofthe intestine of immunized mice were collected on day48. IgG and SIgA weremeasured with ELISA. The spleen cell of immunized mice were collected on day48and cultured in DMEM for72h. The level of INF-γ、IL-4and IL-10in the superliquid were measured with ELISA.The mice were infected with T. gondii throughoral pathway on day48and sacrificed30days after the infection，and the tachyzoites in the livers and brains of mice were counted.Result. The results of restrictive enzyme digestion and PCR showed that the plasmidL2pS-P30-T was constructed correctly. Althought the expression of P30in L lactiswere not detected by SDS-PAGE, a protein band with a molecular weight of30kDa was detected with wester blotting and the protein can reacted with the antiserafrom a patient with acute toxoplasmosis. Compared with the two control group, thelevels of IgG in the serum and SIgA in the mucosal secretions of immunized miceshowed a significant increase. The survival proportion of immunized mice showed asignificant increase and the tachyzoite burdens of immunized mice showed significantdecrease compare to those in the control groups. Conclusion L2pS-P30-T had beenconstructed correctly and expression of P30was achieved in L Lactis. Oralimmunization with recombinant L.lactis expressing P30can significantly induce thehumoral immunity, mucosal immune responses and can increase the protectiveefficacy against T. gondii.