ORFⅡ gene of Banana streak virus GuangDong isolate(BSV-GD) was amplified from a BSV-GD recombinant plasmid by PCR,and the gene was expressed by being cloned into prokaryote expression vector pET-28b(+).The fusion protein was about 16.5 kDa in size and was soluble with SDS-PAGE analysis.The purified protein was obtained by using the histidine labeling kit of N-terminus of protein.The antiserum was obtained by immunizing healthy rabbits with the purified protein.Western blot and ELISA analysis showed that the special antiserum of BSV possessed high titer,which was tested as 1∶51 200.The study was a base for further research on BSV including ORFⅡ gene function and virus detection.
以采自广东省水稻瘤矮病的典型病株为材料,通过RT-PCR法扩增和克隆了水稻瘤矮病毒(Rice gall dwarf virus,RGDV)S11组分核苷酸序列全长。序列分析结果表明,RGDV广东分离物S11(登录号EF532326)核苷酸序列全长1168bp,含有一个1068bp的开放阅读框,编码一条355个氨基酸的多肽,推测分子量约40kD,与泰国分离物基因结构基本一致,其核苷酸与氨基酸序列相似性分别为94.3%和98.8%。将广东分离物S11基因克隆至pBV221温敏表达载体上,在大肠杆菌DH5α中实现了高效表达。以诱导蛋白为抗原免疫家兔获得了抗血清。利用ELISA法测定抗血清的效价为1∶4096,Western blot分析结果表明,抗血清能与S11蛋白发生特异血清学反应。这可为进一步研究S11蛋白的结构和功能,揭示其抑制基因沉默的作用机制提供参考。
