【摘要】 目的原核表达棉铃虫Ⅳ型几丁质酶(HaCHT4)并制备其多克隆抗体。方法在大肠杆菌中表达组蛋白-HaCHT4融合蛋白(His-HaCHT4),镍柱纯化目的蛋白,经足垫加皮下注射免疫小鼠。4次免疫后,用ELISA检测抗His-HaCHT4多克隆抗体的效价,Western blot法检测其免疫特异性。结果成功纯化His-HaCHT4融合蛋白,小鼠抗His-HaCHT4的多克隆抗体的滴度高于1∶204 800。Western blot结果显示该抗体能与His-HaCHT4融合蛋白及棉铃虫体中的天然HaCHT4结合,但不能与黄粉虫的蛋白结合。结论成功原核表达和纯化了His-HaCHT4,制备了特异性的鼠多克隆抗体。更多还原

【Abstract】 Objective The group Ⅳ chitinase from Helicoverpa armigera( HaCHT4) was expressed in prokaryotic and itspolyclonal antibody against HaCHT4 was prepared. Methods The fusion protein His-HaCHT4 was expressed in E. coli andpurified the target protein by Ni-column,and immunized the mice by footpad and subcutaneous injection. The titer ofpolyclonal antibody against His-HaCHT4 was determined by ELISA after four immunizations. The immunological specificity ofthe anti His-HaCHT4 was assayed by Western blot analysis. Results Fusion protein His-HaCHT4 was purified successfullyand the titer of mouse anti His-HaCHT4 polyclonal antibody was higher than 1∶204 800. The results of Western blot showedthat the anti His-HaCHT4 polyclonal antibody was able to bind the fusion protein His-HaCHT4,and also specifically recognizethe natural HaCHT4 in Helicoverpa armigera,but not the protein in Tenebrio molitor. Conclusion His-HaCHT4 wassuccessfully expressed in prokaryotic and purified,and its mouse anti His-HaCHT4 polyclonal antibody was prepared.更多还原