CAJ | 학술논문

首先在凡纳滨对虾(Litopenaeus vannamei)转录组测序的基础上,应用RT-PCR方法获得了凡纳滨对虾泛素交联酶E2(UE2)基因的开放阅读框序列。该序列长为447 bp,编码148个氨基酸,理论相对分子量为16.84 kD,等电点为4.90。同源性比对和系统进化分析显示,不同物种的 UE2基因具有较高的同源性,其中凡纳滨对虾与中国明对虾(Fenneropenaeus chinensis)的同源性最高并聚为一支。半定量RT-PCR分析表明, UE2基因在所检测的组织中均有表达；实时定量PCR分析表明, UE2基因在肝胰腺和肠中的表达量最高,在心脏、鳃、胃和肌肉组织中的表达量略低,在血淋巴中的表达量最低。原核表达分析结果显示, PCR?T7/NT-Topo TA-UE2表达载体在1.0 mmol/L IPTG、37℃诱导3 h条件下可获得纯度较高的分子量约17 kD的蛋白。利用亲和层析的方法将蛋白进行纯化用以制备抗体。研究结果将为深入研究UE2基因在对虾白斑综合症病毒侵染过程中的作用机制奠定基础。
수선재범납빈대하(Litopenaeus vannamei)전록조측서적기출상,응용RT-PCR방법획득료범납빈대하범소교련매E2(UE2)기인적개방열독광서렬。해서렬장위447 bp,편마148개안기산,이론상대분자량위16.84 kD,등전점위4.90。동원성비대화계통진화분석현시,불동물충적 UE2기인구유교고적동원성,기중범납빈대하여중국명대하(Fenneropenaeus chinensis)적동원성최고병취위일지。반정량RT-PCR분석표명, UE2기인재소검측적조직중균유표체；실시정량PCR분석표명, UE2기인재간이선화장중적표체량최고,재심장、새、위화기육조직중적표체량략저,재혈림파중적표체량최저。원핵표체분석결과현시, PCR?T7/NT-Topo TA-UE2표체재체재1.0 mmol/L IPTG、37℃유도3 h조건하가획득순도교고적분자량약17 kD적단백。이용친화층석적방법장단백진행순화용이제비항체。연구결과장위심입연구UE2기인재대하백반종합증병독침염과정중적작용궤제전정기출。
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.