Study On Coloning The Rhoptry Protein 1 Gene Related To Penetrating Factor From Toxoplasma Gondii And Its DNA Immunity In Mice

OBJECTIVE Amplifing the genes of coding rhoptry protein 1 (ROP1) from the genomic DNA groups of 4 different isolates of Toxoplasma gondii, RH, ZS1, ZS2 and GT1; Constructing the prokaryotic and eukaryotic expression recombinants containing ROPlgene and expressing ROP1 protein in vitro. DNA vaccinating BALB/c mice with the eukaryotic recombinant plasmid, to find out the heredity stable of the gene; research the immunogenicity of the expression proteins; observe the expression protein in the muscle tissue from the immunized mice and the humoral and cellular immune respond elicited by the DNA immunization, the effects coadministration with IFN- γ gene, and the protective from the infection of T.gondii; explore the new way for DNA vaccine of T. gondii; provide the scientific basis for the immunity prevention from T. gondii infection.RESEARCH CONTENTS Including ROP1 gene cloning and DNA based immunization. Methods ① Amplifying, cloning and expression ROP1 gene from T.gondii The gene fragment cording ROP1 from the genomic DNA of T.gondii RH,ZS1,ZS2, and GT1 isolates were amplified by means of polymerase chain reaction (PCR). The gene fragments from RH and ZS2 isolats were inserted into cloning vector, pUC18. By using restrictive enzymes and PCR, the recombinants were identified. Subcloning the prokaryotic expression recombinant, pBV220-ROPl, the recombinant protein was expressed in E.coli, DH5 α ; induced by temperature and analyzed by SDS-PAGE and Western-blot. Subcloning the eukaryotic expression recombinant, pcDNA3-ROPl, the recombinant was introduced into mammalian cells, HEPG-2, using lipsome-mediated transfection. The transfected cell clones were selected by G418 culturing and the cells and their cultured fluid were tested by SDS-PAGE and Western-blot. Another recombinant, pcDNA3-IFN was also...