Molecular Cloning And Characterization Of Two Rab1 Protein In Trichomonas Vaginalis

Objective: (1) To clone and characterize two of the Rab1 homolog genes from Trichomonas vaginalis cDNA expression library, to express the TvRabl fusion proteins. And prepare the antibody against the fusion proteins.(2) To define the subcellular localization of the two Rabl proteins in Trichomonas vaginalis.Materials and Methods: (1) A cDNA expression library was constructed with T. vaginalis total RNA. Two cDNA clones, which showed high homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs, etc. The genomic DNA corresponding to the cDNA sequences were amplified using PCR techniques and sequenced. (2) The recomibinant plasmid of pQE80L/TvRabl and pQE80L/TvRab1-like was constructed, and the expression of the fusion proteins were induced by isopropylthio - β-D- galactoside (IPTG) in E. coli M15, the fusion proteins were then purifyied by Ni-NTA affinity chromatography. SDS-PAGE was performed to analyse the fusion proteins. The anti-sera were prepared by immunizing two guinea pigs with the fusion protein respectively, and then tested them in Western blot.(3) The subcellular localization was observed using fluoroimmunocyto-chemistry with corresponding antiserum.Results: (1) One of the cDNA sequence (TvRabla) has a length of 707 basepairs with an open reading frame of 606 bp. The deduced amino acid sequence from the open reading frame possesses 202 residues. Sequence analysis demonstrated that this cDNA clone was most homologous to the Rabla subfamily of different species. The other one (TvRab1-like) has a length of 717 bp with an open reading frame of 603 bp. The deduced peptide sequence contains 200 amino acid residues. Sequence comparison showed this cDNA clone had a significant similarity to Rabla GTPases of different species. Both the deduced amino acid sequences contain all the well known...