›› 2017, Vol. 60 ›› Issue (3): 274-285.doi: 10.16380/j.kcxb.2017.03.005

• 研究论文 • 上一篇    下一篇

褐飞虱吞蛋白的基因克隆、多克隆抗体制备及表达定位

俞叶微#, 许益鹏#, 赵晨星, 韩善捷, 安鹏, 俞晓平*   

  1. (中国计量大学生命科学学院, 浙江省生物计量与检验检疫重点实验室, 杭州 310018)
  • 出版日期:2017-03-20 发布日期:2017-03-20

Gene cloning, polyclonal antibody preparation and expression localization of the endophilins from Nilaparvata lugens (Hemiptera: Delphacidae)

YU Ye-Wei#, XU Yi-Peng#, ZHAO Chen-Xing, HAN Shan-Jie, AN Peng, YU Xiao-Ping*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2017-03-20 Published:2017-03-20

摘要: 【目的】探析吞蛋白(endophilin)在褐飞虱Nilaparvata lugens生长繁殖过程中的作用。【方法】克隆褐飞虱吞蛋白基因,并进行生物信息学分析;在大肠杆菌Escherichia coli Rosetta中诱导表达褐飞虱吞蛋白基因,融合蛋白经Ni柱亲和层析法纯化后,免疫新西兰兔子,获得相应的多克隆抗体;将所得的抗体用于检测褐飞虱卵巢中吞蛋白的表达和定位。【结果】克隆得到褐飞虱两个吞蛋白基因endophilin A (Endo A)和endophilin B (Endo B),GenBank登录号分别为KY126096和KY126095,分别编码387个和352个氨基酸,都含有吞蛋白典型的BAR结构域和SH3结构域,但它们在结构上存在差异。ELISA和Western blot检测结果表明,制备的兔抗血清效价达1∶1 000 000,并表现出较好特异性。将获得的抗体应用于免疫荧光实验表明, Endo AEndo B蛋白在褐飞虱卵巢中普遍表达,在卵巢滤泡细胞的细胞间隙、细胞膜、细胞质中广泛分布,而且与脂类物质的分布模式类似,与正在侵入褐飞虱卵巢的类酵母共生菌共定位。【结论】获得了褐飞虱吞蛋白基因Endo AEndo B序列,并明确了其生物信息学特征,成功制备了Endo A和Endo B多克隆抗体,分析了Endo A和Endo B在褐飞虱卵巢中的表达情况,认为其可能与褐飞虱卵巢发育和成熟以及类酵母共生菌入侵褐飞虱卵巢有关。这些结果为进一步研究Endo A和Endo B在褐飞虱中的生物学功能奠定了基础。

关键词: 褐飞虱, 吞蛋白, 基因克隆, 多克隆抗体, 蛋白定位, 卵巢

Abstract: 【Aim】 To explore the function of endophilin in the growth and reproduction of the brown planthopper, Nilaparvata lugens. 【Methods】 The endophilin genes of N. lugens were cloned and analyzed by bioinformatics. Then, recombinant prokaryotic expression vectors were constructed and transferred into Escherichia coli Rosetta to induce the expression of fusion proteins. After purification by Ni affinity chromatography, the fusion proteins were used to immunize the New Zealand rabbits to obtain polyclonal antibodies against endophilins. The obtained antibodies were used to detect the expression and localization of endophilins in N. lugens ovary. 【Results】 Two endophilin genes, endophilin A (Endo A) and endophilin B (Endo B), were cloned from N. lugens, with the GenBank accession numbers of KY126096 and KY126095, respectively. Endo A and Endo B encode 387 and 352 amino acids, respectively. Both encoded proteins contain the typical BAR domain and SH3 domain, but show different structure. The titer of the obtained antibodies was estimated as high as 1∶1 000 000 dilution ratio through ELISA, and the antibodies had good specificity as shown by Western blot. Using immunofluorescence, the antibodies were used to detect the expression of Endo A and Endo B in N. lugens ovary. The results indicated that Endo A and Endo B were universally expressed in N. lugens ovary. In ovary, Endo A and Endo B were widely distributed in the extracellular space, cytomembrane and cytoplasm of follicle cells of N. lugens ovary, and this distribution pattern was similar to that of lipids. Meanwhile, Endo A and Endo B were colocalized with yeast-like symbionts invading into N. lugens ovary. 【Conclusion】 The nucleotide sequences and the biological characteristics of Endo A and Endo B were clarified. With the obtained polyclonal antibodies of Endo A and Endo B, the expression of Endo A and Endo B in N. lugens ovary was profiled, and the results suggest that Endo A and Endo B might be associated with the development and maturity of N. lugens ovary as well as the invasion of the yeast-like endosymbionts to N. lugens ovary. These results lay the foundation for further studies on the biological function of Endo A and Endo B in N. lugens.

Key words: Nilaparvata lugens, endophilin, gene cloning, polyclonal antibody, protein localization, ovary