关键词: 中华蜜蜂, NPC1蛋白, 原核表达, 表达谱, RNA干扰, siRNA

Abstract: 【Aim】 Human Niemann-Pick disease type C (NPC) is an inherited disease due to mutations in the NPC1 gene. As one of the important receptors in host cells for virus entry mainly responsible for the transport of intracellular cholesterol, NPC1 protein has attracted much attention in recent years. This study aims to explore the relationship between NPC1 protein and Chinese sacbrood virus (CSBV) infection. 【Methods】 The AcNPC1 gene of Apis cerana cerana was amplified by PCR and then cloned into the prokaryotic expression vector pET-30a. The recombinant vector was transformed into Escherichia coli Rosetta^TM (DE3) strain. The recombinant protein was expressed by IPTG induction and purified by Ni-NTA column. The polyclonal antibody was prepared by immunizing mouse with the purified recombinant protein. siRNA targeting AcNPC1 was synthesized and fed to A. c. cerana larvae. The expression levels of AcNPC1 gene and VP1 gene of CSBV in the A. c. cerana larvae infected by CSBV at different time post RNAi were detected by RT-qPCR and analyzed. 【Results】 The recombinant expression vector pET-30a-AcNPC1 was successfully constructed. The soluble AcNPC1 protein of about 36 kD was effectively expressed in E. coli. The recombinant protein with high purity was acquired by affinity purification. The polyclonal antibody was successfully prepared from immunized mouse. Western blot analysis showed that AcNPC1 antibody could hybridize to two bands, which may be partially degraded during membrane protein extraction. The AcNPC1 mRNA levels in CSBV-infected larvae of A. c. cerana at 12, 24 and 48 h post infection were not significantly different from those in the normal larvae, while the AcNPC1 mRNA level in CSBV-infected larvae at 72 h post infection was significantly up-regulated as compared to that in the normal larvae. The RNAi test results revealed that all three interference fragments (siRNA1, siRNA2 and siRNA3) of AcNPC1 downregulated the expression of AcNPC1 in the normal larvae by 73.20%, 72.81%, and 54.78%, and in the CSBV-infected larvae by 43.90%, 59.85%, and 57.67%, respectively, at 24 h post RNAi. The VP1 gene of CSBV was down-regulated by 67.65% at 72 h post RNAi of AcNPC1. 【Conclusion】 Using prokaryotic expression system we effectively expressed AcNPC1 in vitro, and prepared its polyclonal antibody. The AcNPC1 expression in A. c. cerana larvae was interfered using the in vitro synthesized siRNA, suggesting that the expression level of target gene can be successfully downregulated by RNAi via feeding bees. However, the RNAi tests indicate that the decline of AcNPC1 expression may not directly affect the infection of CSBV. This study lays a foundation for further elucidation of the potential functions of AcNPC1.

Key words: Apis cerana cerana, Niemann-Pick type C1 protein, prokaryotic expression; expression profile, RNA interference, siRNA