华支睾吸虫翻译控制肿瘤蛋白(TCTP)基因的鉴定及其编码区序列克隆

Identification and sequence cloning of a novel gene encoding translationally controlled tumor protein(TCTP) of Clonorchiasis sinensis

Objective To identify the novel genes of Clonorchiasis Sinensis by screening the cDNA library and clone the screened gene——Translationally controlled tumor protein(TCTP) to the prokaryotic expression vectors and eukaryotyic expression vectors.Methods The homologue of the novel sequences with a high identity was compared on amino acid and nucleotide level with blast programme on NCBI BLAST site.The motifs of the protein coded by the novel gene were searched with MotifScan and Scanprosite in a proteins sequence on ExPASy site.Bisides,A pair of specific oligonucleotide primers via ECOL1,Xhol1 restriction sites respectly were designed and synthesed according to the coding region of csTCTP gene found by screening library ,The coding region of csTCTP gene was amplified by PCR and then cloned into the prokaryotic expression vectors PGEX-4T-1 and eukaryotyic Expression vectors pcDNA3 via ECOL1 and Xhol1 restriction sites,and transformed into E.coli JM109 respectively.The positive recombinant PGEX-4T-1-csTCTP-and pcDNA3-csTCTP were screened and identified by endonuclease digestion,PCR and sequence.Results A novel cDNA sequence coding csTCTP was found from the cDNA library of Clonorchiasis Sinensis,which was highly homologous to the known TCTP family.Translation of nucleotides sequence revealed putative open reading frame of 169 amino acids with a molecular mass of 19.4596 Kda and PI of 5.06.The identity of its amino acids sequence compared with other species.A search of amino acid sequence of csTCTP with the pattern data base of Prosite revealed that SmTCTP contained 100% identical TCTP1 signature and TCTP2 sequences.The recombinant plasmids PGEX-4T-1-csTCTP-and pcDNA3-csTCTP were constructed.Conclusion A novel gene coding TCTP homologe of Clonorchiasis Sinensis was found and cloned and its Prokaryotic expression vectors and eukaryotyic expression vectors were constructed successfully.