Orientia tsutsugamushi의 외막 단백질 발현 및 특성 연구

초록

Purpose : In order to better understand the outer membrane protein of O. tsutsgamushi, 22k, 47k, 56k, 110k Da protein were cloned and sequence analysed. In addition, 47k Da protein and truncated form of 56k Da protein were expressed as a recombinant protein and treated to L929 mouse fibroblast cell line. Methods : O. tsutsgamushi was propagated in mouse fibroblast L929 cells and genomic DNA of O. tsutsgamushi was extracted by Puregene DNA Extraction kit. DNA sequence of PCR products was analyzed by BLAST search with the NCBI. The gene encoding 47k Da and 56k Da proteins were cloned into pMAL expression vector and purified as maltose binding recombinant protein. After purification, truncated forms of 56k Da protein and 47k Da protein were pre-treated to L929 cell line before O. tsutsgamushi infection. For identifying an infection, an indirect immunofluorescent antibody(IFA) was carried out by multiwell slides of acetone-fixed O. tsutsgamushi. Results : In this study, outer membrane proteins of O. tsutsgamushi, 22k, 47k, 56k, 110k Da protein were cloned and analysed with NCBI database. The DNA sequence of the outer membrane proteins showed high homology. SDS-PAGE and western blotting of 47k Da protein and truncated forms of 56k Da protein were performed with MBP-antibody. Finally, we have compared inhibition rates of the O. tsutsgamushi infection by treatment of 47k Da protein and 56k Da protein in mouse L929 cell. Interestingly, infection rate of some truncated 56k Da protein with 47k Da protein treatment group was dramatically reduced. Conclusions : In the present study, we are elucidated the function of outer membrane proteins in O. tsutsgamushi and our data clearly show that outer membrane proteins as a target molecule for the vaccine candidate. Future work should focus on determination of the protective effect of the outer membrane proteins in an animal model.

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