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Silkworm BmeIF3s5 Cloning, expression and subcellular localization

Author: ZuoWei
Tutor: ZhangYaoZhou
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Silkworm Eukaryotic translation initiation factor 3 subunit 5 Western blot Subcellular localization
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 16
Quote: 0
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Abstract


Eukaryotic translation initiation factor (eIFs) in protein synthesis plays a key role in the 13 factors have been identified in elF3 ( of 8 or more subunits ) is one of the largest molecular weight and the translation initiation process plays a central role . eIF3s5 which is a subunit of eIF3 and eIF3 mediates most of the functionality is achieved. Our approach by RT-PCR amplified from home to silkworm chrysalis eIF3s5 complete ORF gene sequence , and named BmeIF3s5 (Bombyx mori eIF3s5), GenBank accession number DQ443287, the gene sequences of length 834 bp, encoding 278 amino acid residues , predicted molecular weight of 29.60kD. Homology comparison showed that the gene encoding the amino acid sequence in invertebrates with high conservatism. The BmeIF3s5 gene fragment was cloned into the expression vector pET-28a () on the corresponding restriction sites , the recombinant plasmid pET-28a ()-BmeIF3s5, by PCR, restriction enzyme digestion and sequencing analysis confirmed the reorganization is correct. The recombinant plasmid transformed E. coli Rosetta, engineered bacteria induced by IPTG after cell lysis for SDS-PAGE analysis , compared with the negative control positions around the 33.28 kD with a specific protein bands . Recombinant protein in the form of inclusion bodies , the sonicated cells , separated and washed inclusion bodies , dissolving , 12000rpm resultant centrifugal supernatant by affinity chromatography purified fusion protein . After further purified by FPLC reverse phase column chromatography , and then analyzed by mass spectrometry showed that the fusion protein has a molecular weight 34.03 kD, with the theoretical value 33.28 kD ( fusion tag 3.81 kD, BmeIF3s5 29.60 kD) match. The purified fusion protein to immunize rabbits polyclonal antibodies , ELISA detection of the antibody serum titer can reach 1:25600 or more. Analysis by Western blotting experiments BmeIF3s5 proteins in silkworm different times and different tissues , the results show that BmeIF3s5 detected in various developmental stages of the silkworm and various organizations have different levels of expression , including silk gland , eggs , moths expression higher levels . By subcellular localization of the protein found primarily in the cytoplasm . This study further explored BmeIF3s5 proteins in silkworm growth and development of the biological function of the process laid the foundation.

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