Construction Of The Multi-stage DNA Vaccine Against Cysticercosis And Evaluation Of The Immunological Effect

Objective:(1) To construction includes solium and cysticercosis oncosphere ofspecific multi-phase composite DNA vaccine.(2) To evaluate the immunologicaleffect of the composite DNA vaccine.Methods:(1) Total RNAs were extracted from. The sequence of porcineTSOL18andcC1genes were cloned by RT-PCR, and by connecting with a flexible polypeptidelinker (Gly4Ser)3,the recombinant gene, TSOL18+linker+cC1(TSOL18/L/cC1), wasconstructed. The TSOL18/L/cC1gene was inserted into pMD18-T vector, then thegenes cloned were sequenced. Plasmid pMD18-TSOL18/L/cC1DNA was extractedand incubated with enzymes Xho I and Nhe I, then TSOL18-cC1genes was purifiedand connected with pVAX1eukaryotic expression vector, which incubated withenzymes Xho I and Nhe I also. pVAX1-TSOL18, pVAX1-cC1was built by the sameways. The DNA of plasmid pVAX1-TSOL18/L/cC1was constructed and transfectedto the293Tcells according to different ratio, at the same time groups of tranfectedwith DNA of plasmid pVAX1and untranfected were set up, then the cells werecollected after transfection for72h. The transcription of TSOL18/L/cC1mRNA in thecells were identified by RT-PCR and the expression of TSOL18/L/cC1fusion proteinwas detected by Western-blot.(2) Use pVAX1-TSOL18, pVAX1-cC1,pVAX1-TSOL18/L/cC1to immune BALB/c mice by muscle means, then test theimmunization cytokines such as cellular immunity indicators by flow cytometry, andsubclass specific antibodies and the dynamic changes of humoral immune targetswere detected by direct ELISA.Results:(1) Construction of recombinant plasmid by enzyme digestionpVAX1-TSOL18/L/cC1electrophoresis and sequencing results with the expecteddesign of the gene sequence. mRNA expressed was showed in other groups thanpVAX1empty vector transfected and untransfected group of the transfectedeukaryotic cells by RT-PCR. Through Western-blotting analysis showed that in293T cells after eukaryotic transfection to express the target protein. Afterimmunohistochemical staining positive brown granules was showed in therecombinant plasmid in muscle fibers of muscle tissue of the injection site. In addition,there were more brown granules in pVAX1-TSOL18/L/cC1group thanpVAX1-TSOL18and no obvious brown particles in skeletal muscle organizations ofempty vector group and control group.(2) pVAX1-TSOL18/L/cC1the serum IgGantibodies were significantly higher than that of pVAX1-TSOL18, pVAX1-cC1groupand control group and pVAX1(P <0.05). pVAX1-TSOL18/L/cC1group,pVAX1-TSOL18, pVAX1-cC1group of IFN-γ secreting CD4~+and CD8~+Tcells ratiowere significantly higher than the PBS group and pVAX1group (P<0.05),pVAX1-TSOL18/L/cC1group was significantly higher than pVAX1-TSOL18,pVAX1-cC1group (P<0.05); pVAX1-TSOL18/L/cC1group, pVAX1-TSOL18,pVAX1-cC1group secreted IL-4in CD4~+T cells was significantly higher than thePBS group and pVAX1group (P<0.05), while the pVAX1-TSOL18/L/cC1group,pVAX1-TSOL18, pVAX1-cC1group and blank PBS group and the pVAX1group,there were no significant differences (P>0.05); pVAX1-TSOL18, pVAX1-cC1groupsecreted IL-4in CD8~+T cells was significantly higher than the PBS group and pVAX1group (P <0.05), while the pVAX1-TSOL18/L/cC1group and PBS group andpVAX1group no significant difference between groups (P>0.05).Conclusions:1) Successfully constructed the specific complex multi-gene vaccinecontained oncosphere solium and cysticercosis and initially revealed their expressionin vivo and vitro.2) pVAX1-TSOL18/L/cC1multi-phase composite cysticercosisDNA vaccine have more better immune protection than pVAX1-TSOL18,pVAX1-cC1single-gene vaccines.