Lu Xiao-zhao, Peng Hong-juan, Chen Xiao-guang
Department of Parasitology, First Military Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2003 Apr;23(4):296-9.
To subclone the novel gene screened from the Schistosoma japonicum cercariae cDNA library through expressed sequence tag (EST) strategy and analyze its functions.
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 (eIF2 alpha) 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 eIF2 alpha from 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.
A novel cDNA sequence coding for a eIF2 alpha was found from the cDNA library of Schistosoma japonicum cercariae, which was highly homologous to the known Schistosoma mansoni eIF2 alpha mRNA, with the homology of 87% at the nucleotide level and 79% at the amino acid level.
The novel gene found by EST strategy may encode a eIF2 alpha which is highly homologous to Schistosoma mansoni eukaryotic eIF2 alpha mRNA.
通过表达序列标签（EST）策略从日本血吸虫尾蚴cDNA文库中筛选新基因并分析其功能。
对插入pTriplEx2载体的cDNA片段进行测序，并用BLASTn程序检索结果。发现该cDNA与曼氏血吸虫真核翻译起始因子2α亚基（eIF2α）mRNA高度同源。根据已知的EST序列，设计与pTriplEx2质粒中5'端测序引物匹配的3'端引物，用于从cDNA文库中扩增eIF2α的全长开放阅读框（ORF）序列。经过适当纯化后，将PCR产物连接到pGEM-T载体上，对重组T载体进行测序以获得全长ORF，使用NCBI blast程序检索该序列进行同源物鉴定。使用NCBI BLAST网站上的BLAST 2 Sequence程序在氨基酸和核苷酸水平上对高度同源的序列进行比较。还使用在线可用软件检索基序和保守结构域。
从日本血吸虫尾蚴cDNA文库中发现了一个编码eIF2α的新cDNA序列，它与已知的曼氏血吸虫eIF2α mRNA高度同源，核苷酸水平同源性为87%，氨基酸水平同源性为79%。
通过EST策略发现的新基因可能编码一种与曼氏血吸虫真核eIF2α mRNA高度同源的eIF2α。
