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为了进一步研究水稻瘤矮病毒(Rice gall dwarf virus,RGDV)P12的功能,将RGDV-GX P12编码区亚克隆至原核表达载体,并导入大肠杆菌诱导表达;重组的P12融合蛋白经Ni-NTA His·Bind Resins纯化后用于免疫小鼠制备抗血清,并进行Western blotting分析。结果显示,约41 kD大小的RGDV P12融合蛋白可在大肠杆菌中高效表达;利用该重组蛋白所制备的抗血清可特异地与融合表达和非融合的P12蛋白发生强烈的免疫学反应。利用该特异性抗血清在病毒粒子样品中检测不到P12蛋白,而在病株总蛋白样品中可检测到1条与预期大小一致的明显条带,表明RGDV P12是一种非结构蛋白。
In order to further study the function of P12 in rice gall dwarf virus (RGDV), the coding region of RGDV-GX P12 was subcloned into the prokaryotic expression vector and induced into E. coli to induce expression. The recombinant P12 fusion protein was subcloned into Ni-NTA His Bind Resins purified for immunization of mice to prepare antiserum, and Western blotting analysis. The results showed that about 41 kD size RGDV P12 fusion protein can be highly expressed in E. coli; antisera prepared using the recombinant protein can be specifically fused with the fusion protein P12 protein and a strong immunological reaction. Using this specific antiserum, no P12 protein was detected in the virion samples, and a clear band corresponding to the expected size was detected in the total protein samples of the diseased plants, indicating that RGDV P12 is a non-structural protein.