Expression And Functional Analysis Of Transgelin From Silkworm Pupa(Bombyx Mori)

Transgelin 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. 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 protein library and named BmTGN (Bombyx mori transgelin). Concurrently bioinformatic methods were applied to analyze the obtained sequence and its induced amino acid sequence, including open reading frame (ORF), homologous sequences and so on. The ORF of BmTGN gene was 567 base pairs that could encode 188 amino acids with a predicted molecular weight of 20.89 kD. Additionally, the sequence of the amino acids encoded by this gene was more conservative in invertebrates through the homology comparision.Using pET-28a(+) as the expression vector, and constructed the procaryotic expression plasmid pET-28a(+)-BmTGN which was identified by PCR, double digestion and sequencing. The recombinant plasmid was transformed into E.coli Rosetta. After inducing with IPTG, the analysis of SDS-PAGE showed that the recombinant protein was expressed in about 25 kD location.The recombinant protein was soluble and was purified with gel filtration chromatography and metal-chelating affinity chromatography. The molecular weight of this fusion protein was about 25 kD. 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. It laid a good foundation for further researching the biological function of BmTGN gene.