摘要
Ubiquitin (UBI) plays a very important role in regulated non-lysosoma l ATP dependent protein degradation. In the present work, the coding sequence of Spodoptera litura UBI gene was isolated (GenBank Accession No. AF436066). Th e le ngth of this ORF is 228bp, encoding a protein with Mr of 8.56 kD and isoelectri c point of 6.56. Multiple sequence alignment indicated that S.litura UBI is very similar to the homologous proteins of other eukaryotic species and it has 84% id entity with S.litura nucleopolyhedrovirus (SpltMNPV) UBI at amino acid level . RT -PCR analysis showed that S.litura UBI gene is ubiquitously expressed in la rva t issues which are susceptible to SpltMNPV infection. By constructing E.coli e xpre ssion vector, S.litura UBI was highly expressed and the recombinant protein was purified using Ni-NTA resin column, and currently further study on the function of S.litura UBI in SpltMNPV infection is underway.
泛素介导的蛋白质降解途径对胞内蛋白的选择性降解起着重要作用。设计一对简并引物 ,从斜纹夜蛾(Spodopteralitura)细胞中克隆了泛素基因的编码区 ,GenBank登录号AF4 36 0 6 6。序列分析表明 ,该编码区的长度为 2 2 8bp ,编码由 76个氨基酸组成的、分子质量为 8 5 6kD的蛋白 ,其等电点为 6 5 6。同源性比较发现 ,斜纹夜蛾泛素基因不仅与其它真核生物的泛素基因在氨基酸水平上具有 96 %以上的相似性 ,而且与斜纹夜蛾核多角体病毒 (SpltMNPV)泛素基因的同源性为 84 %。RT PCR分析发现 ,泛素基因在所检测的斜纹夜蛾幼虫多种组织 ,尤其是脂肪体中均有表达。采用构建的原核表达载体pQEUB ,在大肠杆菌M15中诱导并高效表达出了带有His tag的重组融合蛋白 ,薄层扫描分析得知靶蛋白约占总蛋白的 37%。利用Ni NTA亲和层析胶纯化得到重组融合蛋白 ,经SDS PAGE鉴定为单一区带 ,为进一步研究S .litura泛素在SpltMNPV感染中的作用打下了基础。
基金
国家重大基础研究项目 (973 ) (No .G2 0 0 0 0 1 62 0 9)
国家自然科学基金项目 (No.3 0 1 70 0 40 )资助~~
