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以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板，通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上，得到重组质粒ｐＧＢＹ５６。序列分析结果表明，内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ，其中包含３个开放阅读框架，分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比，其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上，构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明，２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后，可特异地表达２５ｋＤ蛋白
The full-length cDNA of BNYVV RNA3 was obtained by reverse transcription-PCR amplification using the total RNA of the Inner Mongolia isolate of beet necrotic vein virus (BNYVV) as a template. This was cloned into pGEM-7Zf (+) to obtain recombinant plasmid pGBY56. Sequence analysis showed that the RNA3 genome of Inner Mongolia isolate had a total length of 1775nt and contained three open reading frames encoding 25kD protein, 4.6kD protein and a 59 amino acid N protein, respectively. The homologies of the nucleotide sequences were 96.4%, 96.8% and 97.3%, respectively, compared with those of French F2 isolate, German G1 isolate and Japanese S isolate. The 25kD protein coding gene was cloned into pJW2, and a prokaryotic expression vector was constructed. The results of SDS-PAGE and Western blotting showed that the 25kD protein was expressed in E.coli. After induced by temperature (42 ℃) in E. coli BL21 (DE3), it can specifically express 25kD protein