Abstract: 【Aim】 Activating enhancer binding protein 4 (AP-4), which is involved in the Wnt/β-catenin signaling pathway, is an important transcription factor that has attracted extensive attention in recent years. This study aims to express HaAP-4 gene of Helicoverpa armigera by using prokaryotic expression system, to prepare the polyclonal antibody, to determine the spatio-temporal expression profiles of HaAP-4 and to investigate the possible role of HaAP-4 in regulating the expression of sterol carrier protein-2 gene (HaSCP-2) of H. armigera. 【Methods】 The HaAP-4 gene of H. armigera was amplified by PCR and cloned into prokaryotic expression vector pET-28a. The recombinant vector was transformed into  Escherichia coli BL21 strain. The recombinant protein was expressed by IPTG induction and purified by Ni-NTA column. The polyclonal antibody was prepared by immunizing New Zealand rabbits with the purified recombinant protein. The expression levels of HaAP-4 in different developmental stages (egg, larva, prepupa, pupa and adult) and different tissues (midgut, fat body, head and epidermis) of the 5th instar larva and prepupa of H. armigera were determined by RT-qPCR. HaAP-4 siRNA was synthesized and transfected into Ha cells of H. armigera. The mRNA levels of HaAP-4 and HaSCP-2 in Ha cells after RNAi with HaAP-4 siRNA were detected by RT-qPCR. 【Results】 The recombinant expression vector pET-28a-HaAP-4 was successfully constructed. The soluble HaAP-4 protein of about 55 kD was effectively expressed in E. coli. The highly purified recombinant protein was obtained by purification, and the polyclonal antibody was successfully prepared using the purified recombinant protein. Western blotting results showed that the antibody had high specificity, and ELISA results revealed that the antibody had high titer. Developmental stage-specific expression profiles revealed that HaAP-4 was expressed in all developmental stages, with high expression levels in the egg, 5th larval and prepupal stages. Tissue expression profiles revealed that HaAP-4 was expressed in various tissues of the 5th instar larva and prepupa, showing high expression levels in the midgut and head, but low expression levels in the epidermis and fat body. The RNAi experiments revealed that the knockdown of HaAP-4 expression had a significant impact on the transcription of HaSCP-2, resulting in a decrease of the mRNA expression level of HaSCP-2 by about 55%. 【Conclusion】 The HaAP-4 can be effectively expressed in vitro by using prokaryotic pET-28a expression system, and the polyclonal antibody against HaAP-4 can be obtained. The results of RNAi experiments suggest that HaAP-4,which is highly expressed in the midgut, may up-regulate the expression of HaSCP-2 and play an important role in the lipid physiological metabolism of H. armigera. This study lays a foundation for further elucidating the potential function of HaAP-4 in H. armigera.

Key words: Helicoverpa armigera, activating enhancer binding protein, prokaryotic expression, polyclonal antibody, expression profile, RNA interference