摘要
目的对用表达序列标签(EST)策略从日本血吸虫(Schistosomajaponicum)尾蚴cDNA文库中筛选出的新基因进行全长cDNA克隆及功能预测。方法将插入于pTriplEx2 质粒上的cDNA进行测序,测序结果经BLASTn程序搜索,发现该插入cDNA序列与曼氏血吸虫真核生物翻译起始因子2α亚基(eukaryotic translation initiation factor 2 alpha subunit, eIF2α) 基因高度同源。根据该EST序列设计与pTriplEx2 质粒上的5'端测序引物相匹配的PCR下游引物,从该cDNA文库中扩出包含eIF2α全长ORF的cDNA片段。将PCR产物纯化后克隆到pGEM-T载体上,并对此克隆进行测序得到其全长cDNA序列。用NCBI 站点的BLASTx及BLASTn程序对所获得的新基因序列进行同源性搜索;用blast two sequence程序对同源性高的基因进行核苷酸及氨基酸水平的同源性比较。同时用网上分析软件进行基序和保守区域的搜索。结果发现一个与曼氏血吸虫eIF2α亚基mRNA高度同源的日本血吸虫新基因,核苷酸与氨基酸水平的同一性分别为87%和79%,编码由327个氨基酸组成的蛋白序列。结论用EST策略筛选到一个日本血吸虫新基因,编码eIF2α亚基,全长编码序列与曼氏血吸虫eIF2α亚基mRNA高度同源。
Objective To subclone the novel gene screened from the Schistosoma japonicum cercariae cDNA library through expressed sequence tag (EST) strategy and analyze its functions. Method The cDNA fragment inserted in pTriplEx2 vector was sequenced and the result retrieved with BLASTn program. It was found that this cDNA was highly homologous to Schistosoma mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2α) mRNA. According to the known EST sequence, the 3'-terminal primer that matched the sequencing primer for the 5'-terminal in pTriplEx2 plasmid was designed and used to amplify the full-length open reading frame (ORF) sequence of the eIF2αfrom the cDNA Library. After proper purification, the PCR product was linked to pGEM-T vector and the recombinant T-vector was sequenced to obtain the full length ORF, which was retrieved for homologue identification using NCBI blast program. The sequences that were highly homologous underwent comparison at the levels of amino acids and nucleotides using BLAST 2 Sequence program on NCBI BLAST site. The motif and conserved domain were also retrieved with the software available online. Result A novel cDNA sequence coding for a eIF2αwas found from the cDNA library of Schistosoma japonicum cercariae, which was highly homologous to the known Schistosoma mansoni eIF2αmRNA, with the homology of 87% at the nucleotide level and 79% at the amino acid level. Conclusion The novel gene found by EST strategy may encode a eIF2αwhich is highly homologous to Schistosoma mansoni eukaryotic eIF2αmRNA.
出处
《第一军医大学学报》
CSCD
北大核心
2003年第4期296-299,共4页
Journal of First Military Medical University
基金
联合国发展计划署/世界银行/世界卫生组织热带病研究和培训特别规划署基金(A00690
A00191)~~
关键词
日本血吸虫
表达序列标签
基因克隆
真核生物翻译起始因子2α
Schistosoma japonicum
Schistosoma mansoni
expression seqnence tag
gene clone
eukaryotic initiation factor 2
