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以柑橘溃疡病菌DNA为模板,对抗铜相关基因copA和copB进行了PCR扩增和克隆,获得了大小分别为1 782 bp和1 095 bp的目标片段。构建了这2个基因的原核表达载体(pET-copA和pET-copB),并在大肠杆菌[Escherichia coliBL21(DE3)]中成功诱导表达。利用原核表达的融合蛋白免疫大耳白兔,制备了抗copA和copB原核表达蛋白的多克隆抗体。用间接ELISA法测定了所制备的多克隆抗体的效价均为1∶6 400。用所制备的抗体分别对copA和copB的原核表达产物进行Western blot分析的结果显示,在相应位置产生了较强的免疫反应条带,表明所制备抗体能特异性地与相应的抗原发生免疫反应。
Using the DNA of citrus canker as a template, copA and copB of copper-related genes were amplified by PCR and cloned. The target fragments of 1 782 bp and 1 095 bp in size were obtained. The prokaryotic expression vectors (pET-copA and pET-copB) of these two genes were constructed and successfully induced in Escherichia coli BL21 (DE3). Polyclonal antibodies against prokaryotic expression of copA and copB were prepared by immunization of large white rabbits with the prokaryotic expression fusion protein. The titer of the prepared polyclonal antibodies was determined to be 1: 400 by indirect ELISA. Western blot analysis of prokaryotic expression products of copA and copB using the prepared antibodies showed that strong immunoreactive bands were generated at the corresponding positions, indicating that the prepared antibodies can specifically immunoreact with the corresponding antigens .