水稻矮缩病毒最外层外壳蛋白基因(S_2)cDNA克隆、序列分析及其在大肠杆菌中的表达

MOLECULAR CLONING AND SEQUENCING OF OUTER CAPSIDE PROTEIN GENE OF RICE DWARF VIRUS AND ITS EXPRESSION IN ESCHERICHIA COLI

从水稻矮缩病毒（Ｒｉｃｅｄｗａｒｆｖｉｒｕｓ，ＲＤＶ）中国福建分离物中克隆分离了最外层外壳蛋白基因（Ｓ２）全长ｃＤＮＡ，并对其进行了序列分析，结果表明ＲＤＶＳ２ｃＤＮＡ全长３５１２ｂｐ，仅含一个３３４８ｂｐ的阅读框架，编码一个含有１１１６个氨基酸的蛋白（Ｐ２）。与基因库中已知基因序列比较，发现它与日本ＲＤＶＨ株系相应片段的核苷酸和氨基酸同源率分别为９４６％和９５４％，与轮状病毒ＶＰ２氨基酸序列有一定的同源性。Ｓ２核苷酸序列二级结构预测结果表明，５’端５０个核苷酸的二级结构为一个发夹结构和一个茎环结构。Ｐ２有４个富含亮氨酸的区域，位于Ｎ端亲水区域的１０个氨基酸（ＡＡ６９～７８）残基形成一个α螺旋，这些特点均与轮状病毒ＶＰ２的结构特征相似。ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹分析表明在大肠杆菌中分段高效表达了Ｓ２编码蛋白的Ｎ端和Ｃ端。

The S 2 full length cDNA of rice dwarf phytoreovirus w hich enocodes the viral outer capsid protein was cloned and its complete nuclotide sequence was determined. The results showed that S 2 is 3512bp long with a large open reading frame which encodes a proterin of 1116 amino acids. It shares 94.6% and 95.4% identity with RDV of Japanese H isolate in terms of nucleotide and amino acid sequences, respectively, and it also shows some homology with VP2 of rotavirus at the level of amino acid sequence. The search of deduced RDV S 2 amino acid seqence in Blast network found that there were 4 leucine rich motifs in P 2 protein, and ten amino acids within the hydrophibic region at amino terminus could form an α helix. Predicted secondary structure of S 2 cDNA indicated that a hairpin and a stem loop are present in the 5' end within 50bp, and a stem loop in the 3' end within 50bp. RDV S 2 partial and full length seqences were then cloned into expression vector pET 11d &pTrcHisC. SDS PAGE and Western blot proved that amino and carborn termini of P 2 were successfully expressed in E.coli .