Identification Of Translin Protein In Bombyx Mori

As translocation protein translin is an evolutionarily conserved 27 kD protein that binds to specific DNA and RNA sequences and has diverse cellular roles,such as the chromosome recombination, translocation and cell division. In its tertiary structure there is a conserved leucine zipper area which is necessary for the formation of polymer.Translin can bind mRNA 3'UTR of mRNA and then regulate mRNA transport and translation. Translin protein is also involved in telomere length regulation, with a number of tumor pathogenesis.We cloned an 708 bp ORF fragment from Bombyx mori pupae which was compared with other known translin genes throngh BLAST in NCBI, then we named it BmTLP(for translin-like protein from Bombyx mori).This gene encoded 235 amino acid residues with the predicted molecular weight of 27.259 kD and isoelectric point of 5.87. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The recombinant protein was purified by Ni²⁺ affinity chromatography. We purify the protein in addition by FPLC reversed-phase column chromatography for further purifying and MS analysis confirmed that the protein expression is correct. The purified fusion protein was used to immunize a male New Zealand rabbit.The titer of the polyclonal antibodies reaches 1:25600 measured by ELISA. Western blotting and real time RT-PCR were performed in different stages and in different issues of the fifth instar larva of Bombyx mori.The analysis suggested that the protein was more expressed in head; bowels; silk gland; epidermis; testicle and stomata,but we didn't detect the expression in ovaries; fat body and tuba Malpighii. The protein transcribe and express more in pupal period but egg stage is lowest. After carrying out subcellular localization about BmTLP, we found that it mainly existed in cytoplasm and the location is difference with the cell cycle.