Study On Immunization With Multiepitope Antigenic Gene Of Toxoplasma Gondii And Application Of The Recombinant Antigen In Toxoplasmosis Immunodiagnosis

Partâ… Study on vaccination with multiepitope antigenic geneof Toxoplasma gondii1. Preparation of vaccineRecombinant plasmid pcDNA3-MAG was constructed to express multi-epitopeantigenic gene of T.gondii in eukaryotic expression system. Plasmid DNA ofpcDNA3-MAG, pcDNA3-SAG1 and pcDNA3 were purified in large scale to prepareDNA vaccine, the values of OD₂₆₀/OD₂₈₀ were 1.809, 1.912 and 1.816 respectively.To enhance the immune effect of DNA vaccine, based on previous experience,lipofectin was used as an adjuvant of DNA vaccine.In addition, rcombinant antigens of MAG(rMAG),rSAGland thioredoxin (Trx,expressed by vector plasmids pET32a), were prepared to be used as protein vaccinesto boost the DNA vaccines respectively. 2.Immunization of miceThe above vaccines were divided into two series according to their composition,DNA immunization and DNA vaccine prime/protein vaccine boosting immunization.There are total 7 groups BALB/C mice in the study, named from A to G.3. Immune response and protectionIgG antibodies, IFN-γand IL-4 in the mice were detected by ELISA before andpost immunization 1,3,5 weeks. All mice were challenged with highly virulenttachyzoites of RH and survival time of immunized mice was compared. The resultswere analysed by SPSS 12.0.As the results showed that DNA immunization of MAG could elicit strongercellular and humoral immunity(P＜0.001) than that of both SAG1 gene and vectorplasmid, which results in longer survival time of the mice after being challenged withhigh virulent strain of T.gondii,RH (P＜0.001) and even one of the mice(8.33%)survived for more than 90 days after the deathal challege.With the aim to enhance the effectivity of vaccine, DNA vaccine of MAG wasboosted with rMAG, which results in so strong immunol responses that longer livetime of the mice was obtained(P＜0.001) and 4 mice 10 the group(33.3%) survived formore than 90 days after the deathal challege.At the same time, in another parallelgroup which was immunized by DNA of SAG1 and boosting with rSAG1, 2 micesurvived after the challenge.To evaluate if the epitopes encoded by the MAG were expressed and presentedand if they elicited specific immune respose in the mice, three recombinant antigens,rSAG1, rGRA2 and rROP2 were prepared.The results of Western-blot show that an-tiserum raised against both DNA-mediated immunization of MAG and DNA prime/protein boost immunization of MAG. could be well recognized with the aboverecombinant antigens respectively,which indicats that the epitopes encoded by the MAG were well expressed and presented in the mice and consequently elicitedspecific humoral immune respose.Thus, vaccine based on the DNA of MAG could elicit both cellular and humoralimmune response after immunizing to mice and could afford part of immunologicalprotection against T.gondii infection.PARTâ…¡Application of the recombinant antigen of MAGin immunodiagnosis of toxoplasmosis1. Preparation and identification of sera of mice infected with T.gondiiMice were infected with T.gondii B36 strain and their sera were collected. Withthe results of direct detecting cyst or tachyzoite with microscope, sera were judgedpositive or negative. 17 mice were ensured to be infected by T.gondii and sera fromthem were considered positive as chronic infection of T.gondii.2. Soluble Expression and characterizing of the rMAG in E.coliRecombinant plasmids pET32a-MAG was constructed to express multi-epitopeantigenic gene of T.gondii in E.coli. Large scale rMAG was expressed by inducingwith IPTG and was purified by both immobilized metal affinity chromatography(Ni-NTA resin) and electroelution from SDS-polyacrylamide gels. The purifiedrMAG could be well recognized by mouse antisera and rabbit antisera in respondingto the infection of B36 strain and RH strain respectively.3. Application of rMAG in serological diagnosis of toxoplsmosisCoating with the purifed rMAG with the concentration of 3μg/ml, ELISA kitwas constructed to detect antibody in sera from both mouse and rabbit. A number of 117 known sera from 17 mice infected by B36 strain were detectedby rMAG-ELISA kit. The results show that the kits' sensitivity and specificity are88.88% (104/117×100%) and 91.67% respectively, the total consistency is 89.36%.A number of 24 rabbit sera, in which 18 were derived from rabbits infected withRH T.gondii and the other 6 from normal rabbits, were detcted by rMAG-ELISA kit.94.4%(17/18×100%) of the positive sera were detected by the kit, the totalconsistency is 91.5%((17+5)/24×100%).The results of this part show that rMAG could be used as good coating antigento construct ELISA kit for both acute and chronic infection of T.gondii.