›› 2008, Vol. 51 ›› Issue (5): 473-479.

• RESEARCH PAPERS • Previous Articles     Next Articles

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

JIN Feng-Liang   

  • Online:2010-07-27 Published:2008-05-20

Abstract: Ubiquitin proteasome pathway is the most important and hi ghly 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 a nalysis 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. DQ115796.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 semi-quantitative 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 transfo rmed 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 Ni2+-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.

Key words: Musca domestica, ubiquitin, cloning, prokaryotic expression, antiserum