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

中国人. 摘　　要：［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. ＋-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。

英语. [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 _Escherichia coli_ prokaryotic expression to prepare the antiserum of Rep of BBTV. [Result] The _rep _gene was amplified from total DNA isolated from tender pseudostems and leaves of banana plants infected with the virus by polymerase chain _reaction_ （PCR）. The target product was extracted, and digested with _Xho_Ⅰand _Nco_Ⅰ. The _rep_ gene was inserted into prokaryotic expression vector pET_32b（＋） of the same digestion, and the recombinant plasmid pET32b_Rep was identified by digestion. The recombinant plasmid was introduced into _E. coli_ strain BL21 （DE3）, and induced with IPTG at different times, different temperatures, different IPTG concentrations. As shown in 12% SDS_PAGE, large amounts of soluble fused proteins were induced at 20 ℃ with 0.1 mmol/L IPTG for 4 hours. The supernatant of fused protein was purified by Ni. _＋_-NTA agarose affinity chromatography, and high purity of fused protein solution was obtained. The fused protein was injected into a rabbit for 3 times to prepare the antiserum of Rep of BBTV. The titer of the antiserum was higher than 1〖DK1〗∶125 000, determined by indirect enzyme_linked immunosorbent assay （ ID_ELISA）, and the best working concentration of 1〖DK1〗∶1 000 was determined in field testing. Western blot analysis showed a specific binding between the antiserum and the fused protein. [Conclusion] Specific antiserum prepared from _rep_ gene may highly valuable in BBTV assembly mechanism research and the disease diagnosis.