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以感染百合无症病毒(LSV)的百合叶片为试材,克隆LSV16 k D基因,连接到原核表达载体p ET-28a(+)上。将获得的重组质粒p ET-28a(+)+16 k D转化大肠杆菌BL21(DE3),经IPTG诱导得到了高效表达的16 k D蛋白,融合蛋白分子量约为20 k D。融合蛋白经过镍柱纯化后作为抗原免疫注射小鼠,制备得到16 k D蛋白抗血清。Western blot分析显示所制备的抗血清与诱导表达的融合蛋白发生特异性反应;通过ELISA检测和RT-PCR检测百合样品,证实制备的抗血清与LSV侵染的百合叶片发生了相同的特异性反应。结果表明,目的蛋白表达成功,所制备的抗血清具有特异性,可用于LSV的快速检测、免疫组织化学以及16 k D蛋白功能研究。
The LSV16 kD gene was cloned from lily leaves infected with LSV, and ligated into the prokaryotic expression vector p ET-28a (+). The recombinant plasmid p ET-28a (+) + 16 kD was transformed into E. coli BL21 (DE3), and the highly expressed 16 kD protein was induced by IPTG. The molecular weight of the fusion protein was about 20 kD. After the fusion protein was purified by nickel column, the mice were immunized as antigens to prepare 16 kD protein antiserum. Western blot analysis showed that the prepared antiserum specifically reacted with the fusion protein induced by expression; ELISA and RT-PCR detection of lily samples confirmed that the prepared antiserum produced the same specific reaction with LSV infected lily leaves . The results showed that the target protein was successfully expressed and the prepared antiserum was specific and could be used for rapid detection of LSV, immunohistochemistry and protein function of 16 kD.