The Prokaryotic Expression And Activity Analysis Of Caspase-1of Spodoptera Litura Sl-1Cells

Apoptosis is a process of programmed cell death. It is the center of the maintenance of cell and tissue homeostasis which participates in many physiological and pathological processes. Although apoptosis was first described over forty years ago, it still remains popular in many fields of research as its role in development, homeostasis, and host defense. The research of apoptotic pathway in mammals, nematodes and drosophila has made great progress, but still a crop of questions remained to solve. The apoptosis is highly conserved in evolution, though there are certain differences between the mammals and insects. Studies have shown that, Caspases played an important role in the process of apoptosis. When initiator Caspases received a apoptotic signal, the long N-terminus of the prodomain was cut to activate downstream effector Caspases. The intracellular proteins was cut by activated effector Caspases, which leads to apoptosis. The mechanism of Caspases activation between insects and mammals are different, and need further study. The Caspase-1of Spodoptera litura Sl-1cells is an effector Caspase similar to the caspase-3of mammalian cells. Its specific function and mechanism is still not clear. We intend to provide a reference for Lepidopterist Caspase-1function and lay the basis of the further study of Caspase-1’s mechanism by building S1-caspase-1prokaryotic expression cloning, expressing and purifying Sl-caspase-1protein to study its function preliminarily.In this study, S1-caspase-1prokaryotic expression plasmid was constructed, Sl-caspase-1fusion protein was expressed and purified,and its activity was determined. And polyclonal antibody of Sl-caspase-1was prepared and the location of the Sl-caspase-1in SL-1cells was analyzed.Firstly, specific primers were designed according to the known sequence of SL-Caspase-1gene. The open reading frame of SL-Caspase-1gene was amplified, and connected to PET-28a vector. The positive clones was picked up for prokaryotic expression which verified. Then the recombinant plasmid was transformed into E. coli BL21, induced by IPTG, finally protein crude extracts with fusion protein in was obtained. After Western-blot analyzed and verified the expression of fusion protein with His antibody, the purified fusion protein was obtained by affinity chromatography using a Ni column. Sl-caspase-1activity is determined by Ac-DEVD-AMC which is specific substrate of the Caspase-3. Purified fusion protein was separated by SDS-PAGE electrophoresis and Sl-caspase-1polyclonal antibody was obtained. Finally, we successfully observed the positioning of the Sl-caspase-1in Sl-1cells by using the polyclonal antibody to the immunfluorescence analysis.In this study Sl-caspase-1prokaryotic expression plasmid was constructed, preliminary study of Sl-caspase-1protein function, which laid the foundation for the further research on other proteins and the mechanism of SL-1cell apoptosis. Furthermore, it can provide a reference for the study on other Lepidoptera apoptosis. In addition, apoptosis plays an important role in the process of insects’ resistance to viral infection. Therefore, the study on the mechanism of SI-1cell apoptosis also laid a foundation for the researches on resistance mechanism of other agricultural pests.