CDNA Library Construction Of PCR-selected Subtractive Hybridization Of Senescence-related Genes From Trichomonas Vaginalis And Cloning And Characterization Of Tv-Sir2-like Gene

Objective: (1) To construct PCR-selected subtractive hybridization cDNA libraries and screen senescence-related genes in T. vaginalis cells. (2) To clone and characterize the SIR2 homolog from a T.vaginalis cDNA expression library. Materials and methods: The mRNA was isolated from T.vaginalis cells which grew under conditions of normal caloric intaking and caloric restriction (CR), respectively, and the double-strand cDNA was synthesized. After two times subtractive hybridization followed by two polymerase chain reaction (PCR) amplification, the subtracted products were ligated with pGEM T-Easy vector. Thus, the forward and the reverse subtracted cDNA libraries were constructed. The cDNA clones were selected by blue-white screening and identified by enzyme restriction. Then the cDNA clones were sequenced and analyzed using NCBI tBlastx. A cDNA clone of SIR2 homolog was isolated from a T.vaginalis cDNA expression library, and named Tv-SIR2-like. The conserved domain of Tv-SIR2-like was further analyzed. Southern and Northern blots were performed. The recombinant plasmid pET-41 b/Tv-SIR2-like was constructed and the expression of the fusion protein was induced by IPTG in E.coli BL21. The fusion protein was analysed in SDS-PAGE. Result: 100clones were selected randomly and isolated from each subtracted cDNA library and sequenced. The results showed that all the clones contained cDNA fragments ranging from200 bp to700bp. From the subtracted cDNA library of normally cultured T.vaginalis cells, we obtained five unknown cDNA clones, which may be new genes, and three known genes, which were 40S ribosomal protein, N-acetylmuramoyl-L-alanine amidase and Fructose-1,6-bisphosph-ate aldolase. From the subtracted cDNA library of CR T.vaginalis cells, we obtained also five unknown cDNA clones, and three known gene, which were Ribulose, actin and Cysteine proteinase. Sequence analysis of Tv-Sir2p-like demonstrated that this protein shares a conserved core region with yeast Sir2p and its homologues, possessing all of the three characteristic motifs of Sir2p. These data suggest that this gene should be a homolog of SIR2, which may participate in the regulation of transcription silencing of T.vaginalis. The recombinant plasmid pET-41 /Tv-SIR2-like was constructed successfully. The Fusion protein of GST/Tv-SIR2-like was expressed in E.coli BL21 induced by IPTG. Conclusion: The differentially expressed cDNA libraries of T.vaginalis were constructed successfully. 16 positive cDNA clones were isolated and will be further analyzed. Sequence analysis showed that Tv-SIR2-like was a homolog of SIR2. It may regulate the life span, cell cycle of T.vaginalis. The recombinant plasmid pET-41/Tv-SIR2-like was constructed and the fusion protein was expressed.