利用免疫荧光标记技术分析TVDV在介体蚜虫消化道内的分布

Analysis of the Distribution of TVDV in the Alimentary Canal of Myzus persicae by Immunofluorescence

【目的】制备TVDV CP蛋白抗血清,明确TVDV侵入蚜虫消化道的部位以及TVDV在蚜虫体内的分布。【方法】首先构建原核表达载体p DEST17-TVDV-CP,所得载体转化到Rocceta菌株中诱导表达TVDV CP蛋白,收集目的蛋白免疫小鼠制备TVDV CP特异性抗血清。再利用制备的抗血清,通过免疫荧光技术标记TVDV,对TVDV在介体蚜虫中的分布解剖观察。【结果】TVDV CP蛋白在Rocceta菌株中,IPTG溶度为1、0.5、0.2、0.1 mmol/L,温度为28℃都能成功诱导表达;所得抗血清对TVDV侵染蚜虫总蛋白进行western blot检测,检测到大小约24 ku特异性条带;对免疫荧光标记的蚜虫消化道进行共聚焦观察,在蚜虫中肠上皮细胞检测到标记的TVDV荧光信号。【结论】制备了TVDV CP特异性抗血清,明确了TVDV分布于介体蚜虫消化道的中肠,由介体蚜虫以循回方式传播。

[Purpose]The objective of this study was to generate the specific antiserum of TVDV CP and to study the distribution of TVDV in the alimentary canal of Myzus persicae.[Method]The prokaryotic expression vector of p DEST17-TVDV-CP was constructed and transformed into Escherichia coli Rocceta to express the coat protein（CP） of TVDV. The specific antiserum of TVDV CP was obtained through immunizing mice with the purified CP proteins. Then, the TVDV CP antiserum was used to immunolabel TVDV in the transmission vector of M. persicae.[Result]The TVDV CP could be induced at 28 ℃ by 1, 0.5, 0.2 or 0.1 mmol/L IPTG in Rocceta; an about 24 ku protein could be detected using the specific antiserum through analyzing the total protein of TVDV-infected aphids by Western-blot; TVDV were detected in the midgut epithelial cells of M. persicae by immunofluorescence.[Conclusion]The specific antiserum of TVDV CP was generated; TVDV distributed in the midgut epithelial cells of M. persicae, and were transmitted by M. persicae in a circulative manner.