棉铃虫钙粘蛋白N端多肽多克隆抗体制备及对Bt抗性的初步检测
Preparation of polyclonal antibody of N\|terminal peptide of cadherin of Helicoverpa armigera and primary detection of Btresistance.
黄晶晶1**,韩斌1,常菊花2,沈文飚1***,沈晋良2
点击：1896次 下载：4次
DOI：
作者单位：1南京农业大学生命科学学院南京210095； 2南京农业大学植物保护学院南京210095
中文关键词：棉铃虫，钙粘蛋白，原核表达，多克隆抗体, Bt抗性
英文关键词：Helicoverpa armigera, Ecadherin, prokaryotic expression, polyclonal antibody, Bt resistance
中文摘要：
用重组表达的棉铃虫Helicoverpa armigera（Hübner)中肠钙粘蛋白N端多肽片段制备兔多克隆抗体，并利用其对Bt抗性进行鉴定。通过RT-PCR方法对棉铃虫中肠钙粘蛋白N端多肽的基因片段Cad285进行PCR扩增，将其克隆到pET-30a原核表达载体中，在大肠杆菌BL21（DE3）中经IPTG诱导表达，得到35 ku 的重组融和蛋白，融合表达的包涵体经过变性、Ni-NTA柱亲和纯化、复性等方法处理包涵体，获得可溶性纯化蛋白，用纯化后蛋白免疫新西兰兔制备多克隆抗体， ELISA检测其效价高于1∶16 000；利用最终获得的多克隆抗体对室内纯合Bt抗/感品系的棉铃虫中肠钙粘蛋白进行Western blot分析，结果显示敏感和抗性品系之间有明显差异，表明其能够应用对Bt抗性进行初步检测。
英文摘要： Rabbit polyclonal antibodies were prepared with recombinant N\|terminal peptide of Helicoverpa armigera (Hübner) cadherin, and were used for identification of Bt\|resistance. N\|terminal peptide fragment encoding gene Cad285 was amplified by RT\|PCR from midgut of H. armigera and cloned to prokaryotic expression vector pET\|30a, then induced expressing in E. coli BL21 with IPTG. Recombinant protein (~ 35ku) was highly expressed and existed as inclusion bodies. The inclusion bodies were purified with denaturing, purifying by Ni\|NTA and refolding. The purified recombinant Cad285 was used to immunize rabbit for preparing polyclonal antibody with higher titer than 1∶16 000 measured by ELISA. Finally, a laboratory strains of H. armigera was detected by western blot with the polyclonal antibody. The results showed a significant difference between the sensitive and resistant strains.