香蕉束顶病毒海口分离物Rep基因的克隆、原核表达、抗血清制备及检测

［目的］ 对香蕉束顶病毒(Banana bunchy top virus，BBTV)海口分离物起始蛋白(replication initiation proteins, Rep)基因进行克隆、原核表达、抗血清制备，为BBTV有效的检测及分子流行病学等研究奠定基础。［方法］ 以海口地区染病香蕉幼嫩假茎和叶片的总DNA为模板，通过PCR技术克隆BBTV海口分离物的起始蛋白基因，连接到表达载体并进行大肠杆菌原核表达，制备高效价特异性抗血清。［结果］ 应用PCR方法从染毒香蕉幼嫩假茎和叶片总DNA中扩增复制rep基因，回收目的片段后经酶切，获得了含BBTV Rep基因的重组质粒pET32b Rep。将重组质粒转化E.coli BL21(DE3)，经不同的时间、温度及IPTG浓度优化，12% SDS PAGE电泳分析，在20 ℃、0.1 mmol/L IPTG条件诱导4h，最终获得大量的可溶性融合蛋白。将可溶性蛋白上清液经Ni2+ NTA亲和层析柱纯化，得到高纯度的融合蛋白。用纯化后的融合蛋白免疫家兔，获得了BBTV Rep蛋白抗血清。以融合蛋白做抗原，间接ELISA法测定抗血清效价大于125 000。以田间样品做抗原时，结果表明抗血清最佳工作浓度为1∶1 000。Western blot鉴定结果表明抗血清能与融合蛋白特异性结合。［结论］ 利用Rep基因制备的特异性抗血清在BBTV病毒粒体的组装机制研究及病毒病诊断上具有重要的应用价值。

Cloning and prokaryotic expression of rep gene of the Haikou isolate of Banana bunchy top virus and preparation of polyclonal antiserum against Rep

Objective] This study was aimed to clone and express the rep gene of Banana bunchy top virus Haikou isolate,and to prepare polyclonal antiserum against Rep,which may provide the basis for effective detection of BBTV and molecular epidemiological research.Method] The replication initiation proteins(Rep) gene was cloned from total DNA isolated from tender pseudostems and leaves of banana plants infected with the virus by polymerase chain reaction(PCR).The rep gene was then ligated with expression vector for...