果蝇凋亡蛋白Strica的原核表达、多克隆抗体的制备及在放线菌酮诱导下的激活状态检测

【目的】细胞凋亡是真核生物中保守的细胞程序化死亡，由蛋白酶caspase介导。Strica是果蝇Drosophila中一个特殊的caspase，至今未有其商品化抗体且生化功能未知。本研究旨在通过克隆、原核表达纯化果蝇Strica蛋白并制备其多克隆抗体来初步研究其功能。【方法】根据果蝇Strica的基因序列构建原核表达载体并转化至大肠杆菌Escherichia coli BL21(DE3)感受态细胞，表达Strica蛋白；将表达纯化后的Strica蛋白免疫家兔制备多克隆抗体；通过间接ELISA和Western blot分别测定抗体效价和灵敏度；利用前期构建的过表达载体pAc5-V5-Strica进行转染果蝇S2细胞，设计合成siRNA进行RNAi验证该抗体的特异性；利用该抗体通过免疫荧光实验检测Strica在果蝇S2细胞中的亚细胞定位；利用该抗体通过Western blot实验探究放线菌酮(10 μg/mL)处理后果蝇S2细胞中Strica蛋白的激活状态。【结果】获得了果蝇Strica重组蛋白；ELISA检测Strica抗体的效价大于1∶25 600；Western blot鉴定该抗体在1∶10 000的稀释度下能与重组蛋白反应；该抗体可以检测到S2细胞中过表达以及内源性Strica蛋白；Strica蛋白在S2细胞质中呈现散点分布；放线菌酮介导Strica蛋白的切割激活。【结论】成功制备了兔抗果蝇Strica多克隆抗体。Strica响应凋亡刺激物放线菌酮激活，推测Strica参与果蝇的细胞凋亡过程。本研究为进一步深入研究Strica的功能奠定了实验基础。

Prokaryotic expression,preparation of polyclonal antibody and detection of actinomycin-induced activation state of apoptotic protein Strica ofDrosophila

【Aim】 Apoptosis is a type of conserved programmed cell death in eukaryotes, and it is mediated by caspase protease. Strica is a special caspase in Drosophila. Its commercial antibody is not available and its biochemical function is unknown so far. The objective of this study is to clone, prokaryotically express and purify Drosophila Strica protein, and prepare its polyclonal antibody so as to preliminarily study its function. 【Methods】 Based on the gene sequence of Drosophila strica, a prokaryotic expression vector was constructed and transformed into Escherichia coli BL21 (DE3) competent cells to express Strica protein. Rabbits were immunized with the purified Strica protein to prepare polyclonal antibody. Antibody titer and sensitivity were determined by indirect ELISA and Western blot, respectively. pAc5-V5-Strica overexpression plasmid previously constructed was transfected Drosophila S2 cells, and siRNA was designed and synthesized to verify the specificity of the antibody through RNAi. Subcellular localization of Strica in Drosophila S2 cells was examined by immunofluorescence assay using the antibody, and the activation state of Strica protein in Drosophila S2 cells after actinomycin (10 μg/mL) treatment was explored by Western blot. 【Results】 The recombinant Drosophila Strica was obtained. The titer of Strica antibody detected by ELISA was more than 1∶25 600. Western blot showed that the antibody at a dilution of 1∶10 000 could react with the recombinant Strica. The antibody can detect the overexpressed and endogenous Strica protein in S2 cells. Strica protein is distributed scatteringly in the cytoplasm of S2 cells, and actinomycin induced the cleavage activation of Strica. 【Conclusion】 Rabbit polyclonal antibody against Drosophila Strica was successfully prepared. Strica can be activated in response to apoptosis stimulus actinomycin, suggesting that Strica is involved in the process of cell apoptosis of Drosophila. This study provides an experimental basis for further study of the function of Strica.