Cloning And Expression Of Tv-LAG1 Gene And Screening Senescence-related Genes In The Protist Trichomonas Vaginalis

Objective: (1) To clone and characterize the LAGl homolog from a Trichomonas vaginalis cDNA expression library, to express the fusion protein of Tv-Laglp /GST, and to prepare the antibody against the fusion protein. (2) To screen senescence-related genes in T. vaginalis subtracted cDNA libraries. Materials and methods: (1) A cDNA clone of LAGl homolog was isolated from a T.vaginalis cDNA expression library, and named Tv-LAG1. The sequences of the cDNA clone and its deduced amino acid were further analyzed. The recombinant plasmid of pET-41/Tv-LAG1 was constructed, and the expression of the fusion protein was induced by isopropylthio-β -D-galactoside ( IPTG ) in E.coli BL21, the fusion protein was then purified with Ni-NTA affinity chromatography. SDS-PAGE analysis was performed to detect the expression of fusion protein. An antibody against the fusion protein was prepared by immunizing a rabbit and tested in Western blot. (2) Senescence-related genes in 15 cDNA clones isolated from T.vaginalis subtracted cDNA libraries were further analyzed by RT-PCR. Result: (l)The cDNA sequence of Tv-LAGl has a length of 910 basepairs ( including a poly A fragment at the 3'end) withan open reading frame of 834 bp. The deduced amino acid sequence from the open reading frame contains 277 residues corresponding to a putative molecular mass of 31984.82 and an estimated pi of 8.57. The comparison of the deduced amino acid sequence with the known sequences in data bases using BLASTP program demonstrated that Tv-LAGl is most homologous to LAG1 homolog 1 of Arabidopsis thaliana with 23 % identity(39 % similarity)over 258 amino acids and to human LAG1 homolog 2 (LASS2)with 28 % identity(39 % similarity) over 216 amino acids. It has less homology to yeast LAG1 with 21 % identity (39% similarity) over 263 amino acids. RPS-BLAST program revealed that the deduced amino acid sequence contains a Lagl motif and a TLC domain of six predicted transmembrane alpha helices conserved in Laglp ,Tram ,and Cln8 ( SMART accession number SM0724) ER membrance associated proteins. These data suggest that this gene should be a homolog of LAG1, which may participate in the regulation of cell cycle of T. vaginalis. The recombinant plasmid pET-41/Tv-LAGl was constructed successfully. The Fusion protein of Tv-LAGl / GST (about 38kDa) was expressed in E. coli BL21. The antiserum detects a clearly band in the fusion protein. (2)We obtained two known genes, which were 40S ribosomal protein and fructose-l,6-bisphosphate aldolase from the subtracted cDNA library of normally cultured (1% glucose) T. vaginalis cells. From the subtracted cDNA library of CR (0.05% glucose) T. vaginalis cells, we obtained one unknown gene (5UNA). Conclusion: (l)Sequence analysis showed that Tv- LAG1 was a homolog of LAGl. It may participate in the regulation of the cell cycle of T. vaginalis. The recombinant plasmid pET-41/Tv- LAGl wasconstructed and the fusion protein was expressed. (2)The genes identified from the T. vaginalis subtracted cDNA libraries will be further analyzed.