Acta Entomologica Sinica ›› 2022, Vol. 65 ›› Issue (11): 1437-1443.doi: 10.16380/j.kcxb.2022.11.004

• RESEARCH PAPERS • Previous Articles     Next Articles

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

MENG Kun1, HU Xu-Hao1, SHI Liu-Liu2,*   

  1. (1. Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan, Hubei 442000, China; 2. School of Basic Medicine, Hubei University of Medicine, Shiyan, Hubei 442000, China)
  • Online:2022-11-20 Published:2022-12-02

Abstract: 【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.

Key words: Drosophila, apoptosis, caspase, Strica, prokaryotic expression, polyclonal antibody