家蚕转胶蛋白(BmTGN)的原核表达及定位分析

ransgelin is a ubiquitous protein among smooth muscle tissues of normal adult vertebrates. Transgelin expression is one of the first markers of smooth muscle differentiation during embryogenesis. And most researches of transgelin are focused on rather vertebrate than invertebrate to date. We obtained a cDNA sequence of transgelin in Bombyx mori from NCBI library and named BmTGN (Bombyx mori transgelin). Concurrently bioinformatic methods were applied to analyze the obtained sequence .The ORF of BmTGN gene was 567 base pairs that could encode 188 amino acids. Using pET-28a(+) as the expression vector, and constructed the procaryotic expression plasmid pET-28a(+)-BmTGN. The recombinant plasmid was transformed into E.coli Rosetta. The analysis of SDS-PAGE showed that the recombinant protein was expressed in about 25 kD location. And the result showed that the molecular weight of this fusion protein was 24.589 kD, characterized by mass-spectrum after being further purified on 10 RI FPLC. It was consistent with thecoretical value 24.45 kD (His-tag 3.56 kD, BmTGN 20.89 kD). By immunization of New Zealand rabbit with purified recombinant protein high titer poly-clonalantibodies were prepared (1:6400). We compared the quantity of BmTGN mRNA in different stages and different tissues of silkworm by real-time PCR. BmTGN expression was also analyzed by Western blotting. The results showed that BmTGN existed in all development stages and organizations tested at different levels of expression, especially higher in moth stage and midgut. The experiment with poly-antiboby indicated that BmTGN was mainly distributed in cytoplasm through the method of subcellular localization.