Gene Cloning And Expression Profiling Of Eukaryotic Initiation Factor5a And Deoxyhypusine Synthase Of Eimeria Tenella

Eukaryotic initiation factor5A(eIF5A) is a key factor involved in protein synthesisprocess. The nascent eIF5A is an inert precursor in all of eukaryotic cells, and achieved itsacivity only after it is modified by hypusine in lysine with the structural contribution from thepolyamine spermidine. Hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine] is an unusual acimoacid naturally only occurring in eIF5A, and is formed in a novel posttranslationalmodification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusinehydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential forgrowth, proliferation, and survival of eukaryotic cells, if lack of hypusine-modified eIF5A incell owing to the inhibition of DHS and/or DOHH, the cell cycle will be aborted at G1stage.Recent studies have revealed eIF5A is homolog to prokaryotic elongation factor P (EF-P), andprovide new insights into the mechanism of eIF5A action at the elongateion step rather thaninitiation of translation. A series of researches have demonstrated that the modificationpathway of eIF5A by the DHS and/or DOHH is a potential drug-target in human and someparasitic protozoa, such as Plasmodium spp. and Trypanosoma spp. As we know, up to date,no any progress on the DHS/DOHH has been made in other important apicomplexan protozoa,like Toxoplasma gondii, Eimeri spp., as well as Piroplasma. For research on themodification reaction of eIF5A in Eimeria tenella (EteIF5A), analyse the structure andfunction of EteIF5A and EtDHS, and lay the foundation for assay the activity ofdeoxyhypusine synthase, we cloned and expressed EteIF5A and EtDHS, then analyse thedynamic profiling of EteIF5A,the results are as follows:1. In this study, we discovered the complete ORF of EteIF5A through bioinformaticspredicted, and then ORF potentially encoding for EteIF5A was firstly amplified by RT-PCRfrom Eimeria tenella (E. tenella) total RNA, using the gene specific primers based on thepredicted sequence. The full length cDNA of EteIF5A is486bp and encoding161animo acids. EteIF5A has significant similarity to other eIF5A, the similarity of animo acid sequencebetween EteIF5A and TgeIF5A is84%. EteIF5A was subcloned into the prokaryoticexpression vector pMAL-c2x, the soluble fusion protein of EteIF5A-MBP was achieved byEscherichia coli Transetta(DE3).2. In the other hand, the ORF of EtDHS was predicted through the same method andamplified by RT-PCR from Eimeria tenella total mRNA. The full length cDNA of EtDHS is1746bp, and encode581animo acids. EtDHS also has significant similarity to other DHS, thesimilarity of animo acid sequence between EtDHS and TgeIF5A is46.6%. When EtDHS wassubcloned into the prokaryotic expression vector pMAL-c2x, it will be terminate and can onlyexpress200anino acids in Escherichia coli expression system, and EtDHS can express whenwe designed the insert fragment begin with574bp and end with the stop codon.3. Five total RNA were extracted from5stages of E. tenella and subsequently weresynthesized the first strand cDNA. The β-actin of E. tenella was chosen as a control, and thetranscriptional levels of EteIF5A were detected by using of real-time PCR method. Thetranscriptional analysis results indicate that EteIF5A show different expression levels throughthe E. tenella life stages, and the highest expression of EteIF5A mRNA are in thesporozoites,while the lowest are in the sporulated oocysts.