Abstract
The bumblebee (Bombus lantschouensis Vogt) is an important pollinator of wild plants. Sphingomyelin phosphodiesterase (SMPD) is a hydrolase that plays a major role in sphingolipid metabolism reactions. We report the preparation and characterization of a polyclonal antibody for bumblebee SMPD. We then use the polyclonal antiserum to detect the SMPD protein at different development stages and in different tissues. Our results showed that a 1228bp fragment homologous with the B. terrestris SMPD gene was successfully amplified. The molecular weight of the fusion protein was about 70 kDa by SDS-PAGE. An effective polyclonal antibody against SMPD was also obtained from mice and found to have a higher specificity for bumblebee SMPD. Western blotting detection showed that SMPD was expressed at a high level in queen ovaries, although expression was lower in the midgut and venom gland. SMPD expression decreased from the egg stage until the pdd stage. We interpret our results as showing that the development of an effective polyclonal antiserum for the SMPD protein of a bumblebee, which provides a tool for exploring the function of the SMPD gene. In addition, the work has confirmed that SMPD should be considered as an important enzyme during bumblebee egg and larval stages.
Highlights
Sphingomyeline phosphodiesterase (SMase) is a hydrolytic enzyme that is involved in metabolic reactions of sphingolipids
We described the clone and expression of the recombinant protein with a partial fragment of Sphingomyeline phosphodiesterase gene (SMPD) gene from B. lantschouensis
The DNA sequence and homogenous analyses of this product has clearly elucidated that it is a segment of SMPD gene
Summary
Sphingomyeline phosphodiesterase (SMase) is a hydrolytic enzyme that is involved in metabolic reactions of sphingolipids. Sphingomyeline phosphodiesterase gene (SMPD) was first cloned from Bacillus cereus (Yamada et al, 1988). For the optimal activity pH value, the acid SMase (aSMases) was reported to be 4.5~5 (Brady et al, 1966; Jenkins et al, 2009), neutral SMase (nSMases) near pH 7 (Sabourdy et al, 2008), and the alkaline SMase is about pH 9 (Kraut, 2011). The acid SMase was the most explored in the past few years (Brady et al, 1966; Jenkins et al, 2009; Bartelsen et al, 1998)
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