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

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。

Preparation of polyclonal antibody of N-terminal peptide of cadherin of Helicoverpa armigera and primary detection of Bt-resistance

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 1：16000 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.