Abstract
Storage protein 2 (SP2) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis. Endothelial cell (EC) apoptosis is an important contributing factor in the development of atherosclerosis; therefore, study of the antiapoptotic activity of SP2 on ECs provides information related to the treatment of atherosclerosis and other cardiovascular diseases. In this study, the sp2 gene was cloned and expressed in Escherichia coli to produce a 6xHis-tagged fusion protein, which was then used to generate a polyclonal antibody. Western blot results revealed that SP2 levels were higher in the pupal stage and hemolymph of fifth-instar larvae but low in the egg and adult stages. Subcellular localization results showed that SP2 is located mainly on the cell membrane. In addition, a Bac-to-Bac system was used to construct a recombinant baculovirus for SP2 expression. The purified SP2 was then added to a culture medium for human umbilical vein ECs (HUVECs), which were exposed to staurosporine. A cell viability assay demonstrated that SP2 could significantly enhance the viability of HUVEC. Furthermore, both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on staurosporine-induced HUVEC apoptosis.
Highlights
The vascular endothelium provides a cellular interface between the circulating blood and the vascular smooth muscle of the blood vessel walls
Cellfectin II Reagent and a Ni-NTA Purification System were sourced from Invitrogen (Carlsbad, USA); a Cell Death Detection ELISA kit was sourced from Roche Diagnostics (Castle Hill, Australia); the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was purchased from Invitrogen (Carlsbad, USA); staurosporine was sourced from the Beyotime Company (Shanghai, China)
Sequence analysis revealed that the open reading frame (ORF) of sp2 was 2112 bp in length and encoded a protein of 703 amino acids, with a predicted molecular mass of 83.45 kD and a theoretical isoelectric point of 5.7
Summary
The vascular endothelium provides a cellular interface between the circulating blood and the vascular smooth muscle of the blood vessel walls. Additional studies have shown that hemolymph from the silkworm (Bombyx mori) can inhibit insect and mammalian cell apoptosis that is induced by viruses and several chemical inducers, such as staurosporine, camptothecin, and actinomycin D [6,7,8]. As one such antiapoptotic component in silkworm hemolymph, the 30 K protein has been studied widely [6, 7]. SP2 is presumed to be used as a store of the amino acids required for the development of adult tissues [11]
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