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

目的 通过cDNA文库筛选识别华支睾吸虫新基因 ,并将所发现的华支睾吸虫翻译控制肿瘤蛋白 (csTCTP)编码区克隆到原核表达质粒PGEX - 4T - 1和真核表达质粒 pcDNA3上 ,为进一步研究其功能奠定基础。 方法 将插入于 pBlue script质粒上的cDNA进行随机测序 ,在NCBI和ExPasy网站上进行序列分析 ,识别华支睾吸虫新基因 ,并应用Motifsan、Scan prosite等程序对其进行结构域分析。根据PGEX - 4T - 1和pcDNA3上的克隆位点及所发现的csTCTPcDNA编码序列设计引物 ,进行PCR扩增 ,扩增产物回收纯化后克隆到原核表达载体PGEX4T - 1和真核表达载体 pcDNA3上。构建的PGEX4T- 1-csTCTP、pcDNA3-csTCTP重组表达质粒经PCR、双酶切及测序证实。 结果 发现华支睾吸虫新基因———csTCTP ,完整阅读框含 5 10个碱基 ,编码 16 9个氨基酸 ,理论分子量为 19 4 5 96Kd。序列分析表明 ,csTCTP编码氨基酸序列与其它物种有较高的同源性 ,Motifscan及Scanprosite程序发现推导的氨基酸序列具有TCTP保守的完整功能域以及两个典型的TCTP完整标签。所构建的重组原核和真核表达质粒经PCR、双酶切及测序证实与目标基因相符。结论 发现华支睾吸虫翻译控制肿瘤蛋白基因 ,并成功构建原核和真核重组表达质粒。

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.