家蝇泛素编码区cDNA序列的克隆及在原核细胞中的表达

Cloning and prokaryotic expression of the cDNA sequence encoding ubiquitin from Musca domestica

泛素-蛋白酶体途径(ubiquitin-proteasome pathway)是具有高度选择性的蛋白质降解途径,该途径对细胞内蛋白的选择性降解起着重要作用。本研究根据GenBank已登录的真核生物泛素(ubiquitin)编码框的氨基酸序列,设计一对简并引物,RT-PCR克隆了家蝇Musca domestica泛素基因的编码区cDNA序列,并进行了测序。序列分析表明,该编码区的长度为228bp,编码76个氨基酸,命名为Mdubi,GenBank登录号为DQ115796。同源性比较发现,Mdubi氨基酸序列与其他真核生物泛素编码框同源性可达94%以上。RT-PCR检测表明,Mdubi在家蝇不同组织中均高效表达,且不受大肠杆菌Escherichia coli刺激的影响,是遍在性表达。为进一步研究Mdubi的结构和功能,将Mdubi克隆到原核表达载体pQE30上,构建重组质粒pQE30-UBI,转化大肠杆菌M15感受态细胞,在IPTG诱导下进行了高效表达,SDS-PAGE检测表明Mdubi在大肠杆菌中可表达相对分子质量为9.6kD的可溶性融合蛋白;Western blot分析表明表达产物能与Ni-NTA鏊合物特异性的结合,表明表达的Mdubi为N端带有6His标签的融合蛋白。利用Ni2+-NTA亲和柱一步纯化了Mdubi,以该融合蛋白免疫新西兰大白兔制备了抗Mdubi血清。本研究成功克隆了家蝇泛素的编码序列,并在原核细胞中得到了表达,为进一步研究泛素在家蝇体内的作用机制奠定了基础。

Ubiquitin-proteasome pathway is the most important and highly selective proteolytic pathway which plays an important role in degrading the intracellular proteins selectively. The cDNA sequence encoding ubiquitin from Musca domestica was cloned by RT-PCR and sequenced. Sequence analysis showed that the length of this ORF is 228 bp, encoding 76 amino acids, which was named Mdubi and registered in GenBank with accession no. DQl15796. Multiple sequence alignment indicated that Mdubi was very similar to the homologous proteins of other eukaryotic species and it shared more than 94 % amino acid sequence identity with ubiquitins from other eukaryotic species. The expression of Mdubi transcript was quantified by the semiquantitative RT-PCR, and the results demonstrated that the expression of Mdubi was ubiquitous and not influenced by stimulation of Escherichia coli. The Mdubi was inserted into expression vector pQE30 in vitro and transformed into E. coli M15. The M15 strain, containing Mdubi recombinant plasmid, expressed a 9.6 kD protein with 6His tag at N-terminus in agreement with the expected molecular weight after the induction with IPTG. The fusion protein was purified by Ni^2＋ -NTA column and used to raise antiserum. The successful cloning and expression of the coding sequence of M. domestica ubiquitin provided a basis for further study on its function.