[Toxoplasma gondii ROP38 promotes the maturation of dendritic cells mediated by TLR4]

Objective To investigate the effect of rhoptry protein 38 (ROP38) from Toxoplasma gondii (T. gondii) on the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4) induction in vitro. Methods The total RNA from T. gondii RH strain was extracted by guanidine thiocyanate method, and then cDNA was synthesized with reverse transcription reaction. After ROP38 gene was amplified by PCR, the recombinant pGEX-4T-ROP38 was constructed and expressed under IPTG induction. The recombinant ROP38 protein was detected by SDS-PAGE and Western blot analysis. The secondary structure and antigenicity of the ROP38 were predicted through DNAStar8.0 and ProtScale. In vitro, DCs were isolated and cultured for 6 days, then reacted with ROP38 antigen for 2 hours. The CD11c was detected by flow cytometry, and the data were analyzed with ANOVA by SPSS 23.0 software. Results The amplified gene was about 516 bp as expected. The sequence analysis showed that its homology was 99% compared with the reported sequence (XM_002366710.2) from GenBank. It was found that the relative molecular mass (M_r) of recombinant ROP38 was 45 kD. The prediction of structure indicated that there were 5 α-helices, 5 β-sheets, 5 hydrophilic regions and 8 potential epitopes in ROP38 protein. In vitro, the expression of CD11c on DCs was significantly up-regulated after stimulated with ROP38, and the expression level of CD11c in ROP38 infection group was significantly higher than that of the other groups. Conclusion ROP38 promotes the maturation of DCs mediated by TLR4.