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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35ku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∶16000;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。
Rabbit polyclonal antibody was prepared by using the expressed recombinant human cadherin N-terminal polypeptide fragment of Helicoverpa armigera (Hübner), and its resistance to Bt was identified. The gene fragment of midgut cadherin N-terminal polypeptide of cotton bollworm, Cad285, was amplified by RT-PCR and cloned into prokaryotic expression vector pET-30a. The recombinant plasmid was induced by IPTG in E.coli BL21 (DE3) 35ku fusion protein was obtained, and the inclusion body fusion protein was purified by denaturing, Ni-NTA column affinity purification and renaturation to obtain soluble protein. The purified protein was used to immunize New Zealand rabbits to prepare polyclonal antibody. The titer was higher than 1: 16000.Western blot analysis of midgut cadherin in the homozygous Bt antiseptic / susceptible strain of the house using the finally obtained polyclonal antibody showed that there was a significant difference between the susceptible and resistant strains , Indicating that it can be applied to the initial detection of Bt resistance.