Related Experiment Videos
Cloning and function analysis of full-length cDNA sequence of Schistosoma japonicum eukaryotic translation initiation
Xiao-zhao Lu1, Hong-juan Peng, Xiao-guang Chen
1Department of Parasitology, First Military Medical University, Guangzhou 510515, China.
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 (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.
Result:
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.
Conclusion:
The novel gene found by EST strategy may encode a eIF2 alpha which is highly homologous to Schistosoma mansoni eukaryotic eIF2 alpha mRNA.