昆虫学报 ›› 2020, Vol. 63 ›› Issue (11): 1314-1324.doi: 10.16380/j.kcxb.2020.11.004

• 研究论文 • 上一篇    下一篇

中华蜜蜂NPC1蛋白AcNPC1的原核表达、多克隆抗体制备及功能鉴定

刘永梅, 史红霞, 李颜, 马振刚, 王林玲, 许金山, 周泽扬, 党晓群*   

  1. (重庆师范大学, 重庆市动物生物学重点实验室, 重庆市媒介昆虫重点实验室, 重庆 401331)
  • 出版日期:2020-11-20 发布日期:2020-12-08

Prokaryotic expression, polyclonal antibody preparation and functional characterization of the Niemann-Pick type C1 protein AcNPC1 of Apis cerana cerana (Hymenoptera: Apidae)

LIU Yong-Mei, SHI Hong-Xia, LI Yan, MA Zhen-Gang, WANG Lin-Ling, XU Jin-Shan, ZHOU Ze-Yang, DANG Xiao-Qun*    

  1. (Chongqing Key Laboratory of Animal Biology, Chongqing Key Laboratory of Vector insect, Chongqing Normal University, Chongqing 401331, China)
  • Online:2020-11-20 Published:2020-12-08

摘要:  【目的】人类 C 型尼曼匹克病(Niemann-Pick disease type C, NPC)主要是由NPC1基因突变引起的一类遗传疾病。NPC1蛋白主要负责胞内胆固醇运输,同时也作为病毒侵染宿主细胞的重要受体之一近年来备受关注。本研究旨在探究NPC1蛋白与中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus, CSBV)感染的关系。【方法】通过PCR扩增中华蜜蜂Apis cerana cerana AcNPC1基因;将其克隆至pET-30a原核表达载体,转化至大肠杆菌Escherichia coli RosettaTM(DE3)进行IPTG诱导表达,采用镍柱纯化重组蛋白,免疫小鼠制备多克隆抗体。设计并合成AcNPC1 siRNA,添食中华蜜蜂幼虫;运用RT-qPCR检测和分析中华蜜蜂CSBV添毒幼虫中AcNPC1的表达量,以及RNAi干扰后不同时间AcNPC1基因和CSBV病毒衣壳蛋白VP1基因在感染CSBV的中华蜜蜂幼虫中的表达水平。【结果】成功构建重组表达质粒pET-30a-AcNPC1,在大肠杆菌中获得高效表达的重组蛋白,目的蛋白大小约为36 kD,经亲和纯化获得了纯度较高的重组蛋白,免疫小鼠成功制备了多克隆抗体,免疫印迹分析发现AcNPC1抗体能杂交到两条带,有可能是在提取膜蛋白过程中该蛋白发生了部分降解。中华蜜蜂CSBV添毒幼虫中AcNPC1表达与正常幼虫中的相比,在12, 24和48 h并无显著变化,在72 h出现显著上调。RNAi实验结果表明,中华蜜蜂正常幼虫添食24 h后AcNPC1的3个干涉片段(siRNA1, siRNA2 and siRNA3)均对AcNPC1具有显著性下调作用,AcNPC1表达分别下调了73.20%, 72.81%和54.78%;给CSBV添毒幼虫分别添食3个干涉片段,AcNPC1表达分别下调了43.90%,59.85%和57.67%。CSBV VP1基因在干扰AcNPC1基因72 h后表达下调了67.65%。【结论】利用原核表达系统能有效地在体外表达中华蜜蜂AcNPC1,并制备了其多克隆抗体。利用体外合成的siRNA对中华蜜蜂幼虫中AcNPC1的表达进行干扰,表明在蜜蜂中通过饲喂的方式进行RNAi可以成功下调靶标基因表达水平。不过,RNAi实验结果提示AcNPC1表达下调后可能并不直接影响CSBV的感染。本研究为进一步阐明AcNPC1的潜在功能打下了基础。

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 RosettaTM (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