RElongation Factor 1-α、rβ-tubulin And RH1463201 Of Leishmania Donovani Induce The Th2 Immune Response In Balb/c Mice

Objective:To evaluate the rElongation factor 1-α、rβ-tubulin and rH1463201 of Leishmania donovani stimulating immune efficacy and inducing immune response type in the Balb/c mice.Methods: 1.PCR amplified the genomic DNA of EF 1-α、β-tubulin and H1463201 of Leishmania donovani(MHOM/CN/92/SC10H2),and the open reading frame were cloned into the pQE30 plasmid vector.The recombinant plasmids were transformed into E.coli M15 respectively,after inducted by IPTG,the recombinant proteins were purifed with Ni-column affinity chromatography.2.The Balb/c mice were immunized with the rElongation factor 1-α、rβ-tubulin and rH1463201,prepared the antiserum respectively.3.With Western Blot and ELISA methods,exploited the specificity,titer and IgG subtype of the mice antiserum.4.Prepared spleen cells of the immunized mice,after reinforce immunization with the antigenic protein in vitro,detected IL-4,IL-12 and IFN-γ level using ELISA method.Results: The recombinant plasmid pQE30-EF 1-α,pQE30-β-tubulin and pQE30-H1463201 could express target proteins in inclusion body form in E.coli M15.The antiserums against rEF 1-α,rβ-tubulin and rH1463201 could identify rEF 1-α,rβ-tubulin and rH1463201 specifically,and antiserums titers were 1:800,1:1600 and 1:1600 respectively.The mices,immunized with rEF 1-α and rH1463201,presented the higher levels of both IgG1 and IgG2 a,although the IgG1 production have been significantly higher than IgG2 a.Sera IgG1 and IgG2 a levels of rβ-tubulin-immunized mice was no difference(P>0.05).Addtionally,after booster immunization with rEF 1-α,rβ-tubulin and rH1463201 in vitro,IL-4 levels of the spleen cells were 12.05±2.135 pg/mL,109.34±9.279 pg/mL and 112.01±10.326 pg/mL,and higher than PBS control group(P<0.05),while IL-12 and IFN-γ were no difference than PBS control group.Conclusion: The EF 1-α,β-tubulin and H1463201 gene of Leishmania donovani were cloned and expressed successfully,and all the recombinant proteins induced the Th2 immune responses in Balb/c mice.