甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达  

SEQUENCE ANALYSIS OF RNA3 FROM BEET NECROTIC YELLOW VEIN VIRUS NM ISOLATE AND EXPRESSION OF 25kD PROTEIN GENE IN E.COLI

作　　者：李大伟[1,2] 于嘉林[1,2] 韩成贵 邢怡明[1,2] 刘仪 陈受宜[1,2] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室 [2]中国科学院遗传所

出　　处：《病毒学报》1998年第2期165-171,共7页Chinese Journal of Virology

基　　金：国家自然科学基金

摘　　要：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。The RNA was extracted from purified beet necrotic yellow vein virus(BNYVV)isolated from Inner Mongolia(NM) of China.The first strand of cDNA was synthesized from viral RNA template using reverse transcription,and a 1.8kb fragment was obtained after 30 PCR amplification cycles.The target fragment was cloned into pGEM-7Zf(+)and its whole sequence has been analyzed.The result shows that the RNA3 genome of BNYVV NM isolate has 1775nts in length and it contains 3 open reading frames(ORF),which encode two proteins with molecular weights of 25kD,4.6kD and a polypeptide with 59 amino acids in length (N protein).Comparing with published F2,G1 and S isolates,it shares 96.4%、96.8% and 97.3% nucleotide homology respectively. The temperature-inducible expression vector containing the gene encoding 25kD protein was constructed by cloning this gene into pJW2 and transformed into E.coli BL21(DE3).The results of SDS-PAGE and Western blotting showed that the expression of specific 25kD protein was achieved by temperature induction(42℃).

关 键 词：甜菜 坏死 黄脉病毒 RNA3基因组 序列分析 

分 类 号：S435.663[农业科学—农业昆虫与害虫防治]