›› 2017, Vol. 60 ›› Issue (9): 984-993.doi: 10.16380/j.kcxb.2017.09.002

• 研究论文 • 上一篇    下一篇


谭永安1, 2, 赵旭东1, 2, 肖留斌3, 孙洋3, 赵静3, 柏立新3, 郝德君1, 2,*   

  1. (1. 南京林业大学南方现代林业协同创新中心, 南京 210037; 2. 南京林业大学林学院, 南京 210037; 3. 江苏省农业科学院植物保护研究所, 南京 210014)
  • 出版日期:2017-09-20 发布日期:2017-09-20

Cloning and characterization of the nuclear receptor gene AlE75D in Apolygus lucorum (Hemiptera: Miridae)  

TAN Yong-An1,2, ZHAO Xu-Dong1,2, XIAO Liu-Bin3, SUN Yang3, ZHAO Jing3, BAI Li-Xin3, HAO De-Jun1,2,*   

  1. (1. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China; 3. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
  • Online:2017-09-20 Published:2017-09-20

摘要: 【目的】克隆绿盲蝽Apolygus lucorum核受体基因E75D全长,明确其表达谱,并获得该基因的重组蛋白及制备其单克隆抗体。【方法】利用RACE法克隆绿盲蝽核受体基因AlE75D全长;qRT-PCR法分析绿盲蝽不同日龄成虫(1-16 d)及7日龄雌成虫6种组织(脂肪体、飞行肌、卵巢、马氏管、中肠和表皮)中该基因的mRNA相对表达水平;利用NdeⅠ和XbaⅠ双酶切含有目的基因的pMD18-T载体后进行亚克隆,再利用IPTG诱导的重组质粒进行特异性表达重组蛋白,通过GST琼脂糖亲和层析和分子筛层析纯化靶蛋白;最后通过小鼠免疫、细胞融合及腹水制备重组蛋白的单克隆抗体,Western blot验证该抗体的特异性。【结果】从绿盲蝽中克隆获得蜕皮激素核受体基因AlE75D(GenBank登录号: KX912700),其开放阅读框长1 911 bp,编码636个氨基酸,预测分子量为75.68 kD;该基因编码蛋白具有E75D的典型特征,由A/B域(23 aa)、D域(85 aa)、E域(190 aa)和F域(338 aa)组成,但缺失C域;绿盲蝽AlE75D的氨基酸序列与半翅目蝽科的豆荚草盲蝽Lygus hesperus的E75D序列一致性最高(为98%)。AlE75D在整个绿盲蝽成虫期均有表达,在7日龄雌成虫中达到峰值;此外,AlE75D在雌成虫6个供试组织中也均有表达,并在脂肪体及卵巢中高表达。双酶切后的重组克隆载体可成功亚克隆到pCzn1载体上,IPTG可成功诱导pCzn1-AlE75D重组质粒特异性表达一个约75 kD的蛋白。用Ni-NTA琼脂糖树脂凝胶纯化重组蛋白,以纯化的重组蛋白为抗原,获得了一株能稳定传代并分泌抗AlE75D蛋白单克隆抗体的细胞株,该单克隆抗体可与绿盲蝽总蛋白及AlE75D重组蛋白特异性结合。【结论】AlE75D在绿盲蝽体内的表达谱表现出发育阶段特异性和组织特异性;本研究获得的其重组蛋白的单克隆抗体具有高特异性。结果为进一步在蛋白水平上分析该基因的功能奠定基础。

关键词: 绿盲蝽, AlE75D, 基因克隆, 表达谱, 原核表达, 单克隆抗体

Abstract: 【Aim】 To clone the nuclear receptor gene from the cotton mirid bug, Apolygus lucorum, analyze its expression profiles, obtain the recombinant protein and prepare the highly specific monoclonal antibody against E75D protein. 【Methods】 The full-length cDNA of the nuclear receptor gene was cloned using RT-PCR and RACE techniques. The relative expression levels of this gene in female adults of different ages (1-16 day-old) and six tissues (fat body, flight muscles, ovary, Malpighian tubules, midgut and cuticle) in 7 day-old female adults of A. lucorum were determined using qRT-PCR method. The T vector containing the target gene was digested with the restriction enzyme NdeI/XbaI and subcloned, and the recombinant plasmid was specifically expressed after induction by IPTG. The recombinant protein was purified by GST agarose affinity chromatography and molecular sieve chromatography. The monoclonal antibody was obtained by mice immunity, cell fusion and ascites preparation, and its specificity was determined by Western blotting. 【Results】 A nuclear receptor gene was cloned from A. lucorum and named AlE75D (GenBank accession no.: KX912700). Its ORF is 1 911 bp in length, encoding a polypeptide with a predicted molecular mass of 75.68 kD. Amino acid sequence comparison indicated that AlE75D has the classic characteristics of the nuclear hormone receptor, including a highly conserved ligand-independent A/B activation domain (23 a.a.), a hinge region (D domain, 85 a.a.), a ligand-binding domain (LBD) (E domain, 190 a.a.) and F domain (338 a.a.), but has no C domain. AlE75D showed the highest similarity to E75 from the hemipteran insect Lygus hesperus (98% amino acid sequence identity). AlE75D was found to be continuously expressed throughout the whole adult stage of A. lucorum, and its expression level reached the peak in the 7 day-old female adult. The transcription levels of AlE75D were higher in the fat body and ovary; however, only small amounts of mRNAs were detected in flight muscles and Malpighian tubules. The vector containing AlE75D gene was digested and linked to the vector pCzn1. The recombinant plasmid pCzn1-AlE75D expressed the target recombinant protein of 75 kD after induction by IPTG. The recombinant protein was purified by using Ni-NTA agarose and used as the antigen, and one cell line which could stably passage and secrete monoclonal antibody against AlE75D protein was obtained. The monoclonal antibody could specifically bind to the total protein of A. lucorum as well as the recombinant AlE75D. 【Conclusion】 The expression of the nuclear receptor gene AlE75D in A. lucorum showed developmental stage- and tissue-specificity. The obtained monoclonal antibody against AlE75D recombinant protein is highly specific. The results could provide the basis for analyzing the gene’s function at the protein level.  

Key words: Apolygus lucorum; AlE75D, gene cloning, expression profile, prokaryotic expression, monoclonal antibody