芜菁花叶病毒外壳蛋白基因的序列分析及其在大肠杆菌中的表达

SEQUENCE ANALYSIS AND EXPRESSION IN E.COLI OF TURNIP MOSAIC VIRUS COAT PROTEIN GENE

从杭州郊区感病的甘蓝型油菜上分离到一种致病力较强的芜菁花叶病毒（Ｔｕｒｎｉｐｍｏｓａｉｃｖｉｒｕｓ，ＴｕＭＶ）分离物。大量提取病毒并纯化后，电镜观察病毒粒子约７４０ｎｍ×１２ｎｍ。以寡聚（ｄＴ）１５为引物合成ｃＤＮＡ，并根据外壳蛋白（ＣＰ）基因两端序列设计两个诱变引物进行ＰＣＲ扩增，得到一个特异性ＤＮＡ片段。将该片段克隆到ｐＵＣ１８中，经Ｓａｎｇｅｒ双脱氧法测序，结果表明该ＣＰ基因全长为８６７ｂｐ。与前人报道的两种芜青花叶病毒ＣＰ基因的核苷酸序列的同源性分别为８９．５％和９６．７％。，氨基酸序列的同源性分别为９４．５％和９７．９％。将ＣＰ基因亚克隆到原核表达载体ｐＫＫ２３３－２中，用ＩＰＴＧ诱导含插入片段表达载体的大肠杆菌，Ｗｅｓｔｅｍ印迹检测证明，大肠杆菌能表达病毒的ＣＰ。

ne virulent isolate of turnip mosaic virus was isolated from infected Brassica rape inHangzhou suburbs.the virus was purified by sucrose gradient centrifugation.The purifiedvirus particles observed by electronic microscope were about 740nm×12nm.The cDNA was synthesized with oligo(dT)15 and PCR was carried out with two mutagenesis primers flankingthe coat protein gene.One specific DNA segment was amplified.It was cloned into pUC18.The sequence of the coat protein gene was 867 nucleotides long determined by Sanger dideoxymethod.It was compared with two reported TuMV coat protein gene sequences.Thehomology of nucleotide sequences between them were 89.5% and 96.7% respectively and thehomology of amino acid sequences were 94.5% and 97.9% respectively.The coat protein genewas subcloned into the prokaryotic expression vector pKK233-2.E.coli containing theexpression vector with the inserted fragment was induced by IPTG.It was proved that thecoat protein gene was expressed in E.coli by Western blotting.