Cloning and expression of ubiquitin-conjugating enzyme E2 in Litopeaneus vannamei
Litopeaneus vannamei is one of the shrimps which have the highest farming production in the world. White Spot Syndrome Virus (WSSV) has been recognized as one of the major threats factors in shrimp aquaculture industry and has been causing severe damage. So the study for antiviral mechanism is extremely meaningful. Ubiquitin protea-some pathway (UPP) is an important cellular functions regulation system in eukaryote. Ubiquitin-conjugating enzyme E2 (UE2) is an integral part of the pathway. According to our preliminary work about transcriptome sequencing and digital gene expression sequencing of Litopeaneus vannamei, we found that UE2 gene was expressed significantly higher in hemocyte after WSSV injection. It hints that UE2 may take part in the process of virus infection of prawn. But the mechanism is not clear. In this study, the ORF sequence of ubiquitin-conjugating enzyme E2 (UE2) gene from Li-topeaneus vannamei was amplified by RT-PCR based on transcriptome sequencing. The open reading frame (ORF) was 447 bp, encoding 148 amino acid. The predicted molecular mass of UE2 protein was 16.84 KD, and the theoretical isoelectric point was 4.90.The homology and phylogenetic analysis revealed that the deduced amino acid sequence of UE2 exhibited high identity in different species and highest identity wih Fenneropenaeus chinensis.UE2 gene was ex-pressed in all the tissues examined by semi-quantitative RT-PCR. While the expressional profile was detected by real-time quantitative PCR, it showed that the UE2 gene was expressed higher in hepatopancreas and intestine than in the other tissues. Then the UE2 gene was cloned into the prokaryotic expression vector to yield an identified recombi-nant plasmid, which was then transformed into competent cells of BL21 (DE3) plysS after being confirmed by se-quencing. The recombinant protein approximately 17 kD was gained by inducing expression using IPTG. For antibody preparation, the protein was purified by means of affinity chromatography. These results have laid the foundation for further study of UE2 gene and UPP pathway in the process of virus infection of prawn.
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维普
