Development Of Recombinant BCG Of Toxoplasma Gondii Rhomboid Gene And Its Immune Effect Of Canine

Toxoplasma gondii, an obligate intracellular protozoan parasite, is foundworldwide. It infects a wide range of warm-blooded animals and can cause severe zoonotic parasites to the production of human health and animal husbandry.There is no ideal drugs and vaccines in the prevention and treatment of toxoplasmosis, the development of safe, effective inexpensive vaccines become toxoplasmosis prevention focus. Currently candidate antigen genes of Toxoplasma genetically engineered vaccine are mainly SAG1, SAG2, P28, P23, MIC antigens,invasion-promoting factor (PEF),54ku membrane protein antigens, and densegranules. Rhombiod has become gondii vaccine candidate genes and targets ofdrug research hotspot. IL-2as immune regulatory network, can play an important role in synergistic or antagonistic cytokines.Currently, the research about canine interleukin-2(IL-2) recombinant BCGpreventing toxoplasmosis have not been reported, in this study was to constructcanine interleukin-2-Rhomboid recombinant BCG, further immune dogs to detect clinical immune function.Cloning and bioinformatics analysis of canine interleukin-2gene Extract theRNA of dog spleen, and design primers according to canine interleukin-2genesequence in NCBI Genebank. Amplify dog interleukin-2gene open reading frame byreverse transcription PCR and connect it to the pMD18-T vector. Sequencing resultsshowed465bp PCR product show a100%homology with the original.The expression and identification of recombinant plasmid pMV2(3)61-IL2-Rho in BCG The gene of interleukin-2and Toxoplasma gondii Rhombiod werecloned into E. coli-Mycobacterium shuttle expression vector pMV261and integratingexpression vector pMV361together. Recombinant plasmid pMV261-IL2-Rho andpMV361-IL2-Rho were electrotranformated into BCG respectively. Positive colonies were selected with Kan+culture media and subculture. After45℃heat-inducedexpression of the target protein, expression products were analyzed by SDS-PAGEand Western-blotting assays. The results showed that the shuttle expression vectorpMV261-IL2-Rho and integration expression vector pMV361-IL2-Rho aresuccessfully constructed. Rhomboid gene can be stably expressed in Mycobacteriumtuberculosis and have good immunity immunogenicity. The results make importantfoundation for further Toxoplasma gondii rBCG development.Protective immunity of rBCG pMV2(3)61-Rho and pMV2(3)61-IL2-Rhoagainst Toxoplasma gondii challenge The rBCG pMV261-IL2-Rho, pMV361-IL2-Rho, pMV261-Rho, and pMV361-Rho respectively immunize dogs. The levels ofhumoral and cellular immunity were detected two weeks later after the thirdimmunization. The results showed that the experimental group immunized antibodytiters increased with the increase of times, and recombinant BCG pMV261-IL2-Rhoand pMV361-IL2-Rho experimental group are significantly different from the controlgroup. Th1-type and Th2-type cytokines were detected with the increase in thenumber of immune each test, and IL-2, IFN-γ levels rise. However, the PBS and BCGcontrol group did not change significantly. Temperature tests showed small changes inexperimental group of dogs’ body temperature compared with the control group.Detection of parasites concentration in the blood show recombinant BCG has a certainprotective effect against T. Gondii attacks, and Interleukin-2enhances the protectiveeffect of rBCG.