柑橘溃疡病菌抗铜相关基因copA和copB的克隆及原核表达

Cloning and prokaryotic expression of the copper-resistance related genes copA and copB of Xanthomonas citri subsp. citri

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。

Copper-resistance related genes copA and copB were amplified by using the DNA as template extracted from Xanthomonas citri subsp.citri.The target products were cloned,and sequencing results shows that sizes of copA and copB with 1 782 bp and 1 095 bp.The prokaryotic expression vectors pET-copA and pET-copB for these two genes were constructed.Both copA and copB were effectively expressed as fusion proteins in transformed Escherichia coli strain BL21（DE3） under the induction of IPTG.The expressed proteins were gel-purified and used to raise antisera in rabbits.The titers of antisera against recombinant proteins of copA and copB were 1∶6 400 in indirect-ELISA.The expressed products were subjected to Western blot analysis with prepared antisera.The results showed that the raised antisera reacted strongly with the antigenic proteins.These results indicated that the raised antisera could recognize specifically the recombinant proteins of copA and copB.