Study On The Expression And The Host Cell Apoptosis Of Toxoplasma Gondii ROP16

Objective To clone and construct a recombinant expression plasmid of rhoptry protein(ROP)16 from tachyzoites of T. gondii in Escherichia coli, and to prepare the anti-ROP16 polyclonal antibody. To express ROP16 of Toxoplasma gondii in 293 T cells by constructing the eukaryotic expression vector, and explore the effect of 293 T cell apoptosis induced by ROP16.Methods:The primers were designed by DNAstar and ROP16 gene was amplificated by PCR with DNA of T. gondii tachyzoites as a template.The recombinant plasmids of ROP16/p TG19-T and ROP16/p ET32 a were constructed, and the positive recombinant plasmid was imported into E.coli Rosetta(DE3). The recombinant ROP16 was purified by KCl stain and gel slices, and the anti-ROP16 polyclonal antibody was prepared. T. gondii tachyzoite ROP16 expression was analyzed by Western blotting and IFA with anti-ROP16 polyclonal antibody.The eukaryotic expression recombinant plasmids p3XFLAG-Myc-CMV?-24-ROP16 was constructed and confirmed by PCR and double enzyme digestion. The positive plasmid was extracted with maxiprep kit and transfected into 293 T cells by Ronfect TM. Specimens were collected, quantitative and qualitative detections of expression of ROP16 in 293 T cells were tested.293 T cells were cultured and the control and transfected groups were set respectively. Apoptosis of both two groups were induced by 0.5 μg/ml actinomycin D for 24 h, specimens were collected after transfection for Annexin V-FITC/PI 、TUNEL,the rates of apoptosis were detected.The c DNA of samples were extracted and apoptosis-related factors like Bcl-2/Bax and Caspase3 were tested by RT-PCR.Results:2 121 bp ROP16 gene sequence was amplified from DNA in tachyzoites of T. gondii successfully. The recombinant plasmid ROP16/p ET32 a was constructed and recombinant ROP16 could express in E.coli Rosetta(DE3), and was primarily in the form of inclusion body. The specific band of 99.8 k Da was detected by SDS-PAGE, which is consistant with the theoretical value. The purified ROP16 was obtained by the KCl stain and gel slices. High potency polyclonal antibody was obtained. The specific band of 74 k Da was detected by Western blotting and the ROP16 was distributed in the cytoplasm of tachyzoites by IFA, it proved that anti-ROP16 polyclonal antibody was prepared successfully.The eukaryotic expression recombinant plasmids p3XFLAG-Myc-CMV?-24-ROP16 was constructed successfully, and specific bands were detected from specimens collected 3 time points after transfection by Western blotting, which is consistant with the theoretical value, and the expression is increasing.The apoptosis rates of 293 T cells in ROP16 transfected group were lower. Both rates had prominent difference(P<0.05).The value of Bcl-2 / Bax in ROP16 transfected group were higher than control group, while the value of Caspase3/actin were lower than that.Conclusions:The recombinant plasmid ROP16/p ET32 a was constructed and anti-ROP16 polyclonal antibody was prepared successfully, and the antibody has a high immune reaction activity by Western blotting and IFA.Toxoplasma gondii ROP16 was expressed in host cells successfully, and it inhibits apoptosis of host cells.