| Objective: (1) To clone and characterize the Silent information regulator2 (SIR2) homolog from the parasitic protist Trichomonas vaginalis cDNA expression library, to express the fusion protein of TvSIR2 and TvSIR2-like/GST(isolated previously in our laboratory). To prepare the anti-serum against the fusion proteins and localize them in the protist. (2) To clone and characterize the β -GAL homolog from the Trichomonas vaginalis cDNA expression library, to express the fusion protein of Tv β -GAL. Materials and methods: (1) A cDNA clone of SIR2 homolog was isolated from a T.vaginalis cDNA expression library, which was named TvSIR2. The sequence of the cDNA clone and its deduced anino acid was further analyzed. The recombinant plasmids of pET-41a/TvSIR2 and pET-41a/TvSIR2-like were constructed, and the expression of the fusion proteins were induced by isopropylthio - β -D-galactoside (IPTG) in E.coli BL21, the fusion proteins were then purified with Ni-NTA affinity chromatography. SDS-PAGE analysis was performed to detect the expression of fusion protein. Two antibodies against the fusion protein were prepared by immunizing two pigs and tested in Western blotting. Immunofluorescence was made to localize the two proteins in the protist. (2) A cDNA clone of β -galactosidase homolog was isolated from a T.vaginalis cDNA expression library, which was named Tv β -GAL. The sequence of the cDNA clone and its deduced anino acid was further analyzed. The recombinant plasmid of pQE-80L/Tv β -GAL was constructed, and the expression of the fusion protein was induced by IPTG in E.coli SG13009, SDS-PAGE analysis was performed to detect the expression of fusion protein. Result: (1) The cDNA sequence of TvSIR2 has a length of 1034 basepairs(including a poly A fragment at the 3'end)...
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