Clonorchis sinensis tropomyosin: cloning and sequence of partial cDNA amplified by PCR

C. sinensis total RNA was containing large amount of 18S rRNA but little 28S rRNA. The size of the double-stranded cDNA synthesized from poly (A)+ mRNA was 0.4-4.2 kb long with tapering upto 9.5 kb. Degenerated oligonucleotides (as 2 sense and 3 antisense primers) were designed on the conserved regions of the known tropomyosin amino acid sequences. From one out of the PCR amplifications using total cDNA and matrix of primers, a specific gene product, 580 bp in size, was produced. Upon Southern hybridization of the PCR products with Schistosoma mansoni tropomyosin (SMTM) cDNA, only one signal appeared at the band of 580 bp product. This 580 bp product was considered to encode C. sinensis tropomyosin (CSTM) and cloned in pGEM-3Zf(-) for DNA sequencing. CSTM cDNA was 575 bp containing one open reading frame of 191 predicted amino acids, which revealed 86.3% homology with SMTM and 51.1% with Trichostrongylus colubriformis tropomyosin. CSTM cDNA obtained will serve as a probe in the studies of molecular cloning of CSTM.