果蝇spen蛋白的抗体制备、组织特异性表达及功能分析

Spen家族蛋白参与多种生物学过程,包括神经元细胞的命运、神经元突起延伸的调节、细胞周期调控等,并且是联系Notch信号途径和生长因子受体途径的关键分子。最近的研究表明spen基因在果蝇的眼睛、翅膀和腿组织中参与Wnt信号转导。但该基因在果蝇中的功能还有很多不明确之处。文章采用基因克隆、原核表达及亲和层析等方法制备并纯化了黑腹果蝇spen的C端6×His-spen融合蛋白,以纯化的融合蛋白免疫大鼠获得了抗spen的多克隆抗体。利用制备的抗体进行免疫染色结果显示spen蛋白定位于细胞核内,并且在大脑、脂肪体、血细胞、肠和唾液腺等组织中表达量较高。分析野生型和突变体果蝇血细胞的噬菌作用,发现spen蛋白低表达的突变体吞噬外来异物明显低于野生型,结果表明spen蛋白能够调节血细胞的吞噬功能。

Drosophila spen protein: generation of ployclonal antibodies, functional analysis and tissue-specific expression

The spen family of proteins participates in various biological processes. It is involved in neuronal cell fate, survival and axon guidance, and cell cycle regulation. Recent studies showed the Drosophila spen gene was required for Wnt-dependent signaling in the eye, wing and leg. However, the genetic role and biological function in Drosophila remain largely unclear. A Drosophila C-terminal fragment of spen was cloned and expresed in E. coli. The purified 6×His-spen protein was injected into SD rat to generate polyclonal antibodies. Subcellular localization and tissue-specific expression of spen protein were analysed by immunostaining and histoimmunochemistry. The results indicated that spen protein was localized in the nucleus and expressed at high levels in brain, fat body, hemocyte, gut and salivary gland. To assay the function of mutant hemocytes in vivo, wild-type and spen mutant larvae were infected with fluorescent microspheres. Wild-type hemocytes showed a strong fluorescence signal from the phagocytosed microspheres; however, spen mutant had a weak fluorescence signal, indicating that the mutant hemocytes were defective in the uptake of the microspheres.