西瓜花叶病毒2号外壳蛋白基因克隆及其在大肠杆菌中的表达

Cloning and Expression of Coat Protein Gene of Watermelon Mosaic Virus 2 in Escherichia coli

以郑州地区西瓜花叶病病叶分离物──西瓜花叶病毒２号（２－ＷＭＶ－）为毒源，从繁殖寄主西葫芦病叶中分离、鉴定了病毒。以病毒基因组ＲＮＡ为模板，逆转录合成ｃＤＮＡ，通过多聚酶链式反应（ＰＣＲ），从ｃＤＮＡ中扩增和克隆了病毒外壳蛋白（ＣＰ）基因，其中包括３’端非编码区，完成了全部核苷酸序列分析。ＣＰ基因全长１０９９个苷耷酸，其中编码区长９０３个核苷酸，编码３０１个氨基酸，３’端非编码区长１９６个核苷酸。Ｚ－ＷＭＶ－２ＣＰ基因的编码区比Ａ－ＷＭＶ－２，Ｕ－ＷＭＶ－２两株系的ＣＰ基因编码区分别长出６０个核耷酸，多编码２０个氨基酸。与两个株系相比，编码区的核昔酸序列同源性分别为８８．５％和８７．０％，氨基酸序列同源性分别为９０．０％和８８．７％，３，端非编码区的同源性分别为６８．３％和７１．８％。若不考虑Ｚ－ＷＭＶ－２多出的６０个核苷酸及２０个氨基酸，则上述同源性依次为９５．４％、９３．７％、９６．４％、９５．０％、８８．８％和９３．４％。构建了含ＷＭＶ－２ＣＰ基因的大肠杆菌表达载体，经聚丙烯酰胺凝胶电泳分析，表达出了与天然病毒外壳蛋白分子量相同的蛋白。

An isolate was separated from infected watermelon leaves and then identified the watermelon mosaicvirus 2.cDNA was synthesized by reverse transcription by using viral RNA as the template through PCR(poly-chain-reaction).The coat protein gene(CP),including 3' non-coding region,was amplified andcloned.The complete sequence of nucleotides was established.The full length of CP gene is 1099 nucleotidesincluding 903 nucleotides in the region coding,301 amino acids in each encode,and 196 nucleotides in the 3'untranslated region。The coding region of WMV-2CP gene is 60 nucleotides and 20 amino acids longer thanthat of A-WMV 2and U- WMV-2strain。Comparing the later strain,the homology nucleotide se-quence in the coding region are 88.5%and 87. 0%,respectively; the homology of the amino acid sequenceare 90.0%and 88.7%,respectively;and the homology of3'non-coding sequence are 68.3%and 71.8%,respectively.If the last 60 nucleotides and 20 amino acids in WMV-2CP gene are not taken into account,thehomology mentioned above will be 95.4%, 93.7%;96.4%,95.0%; 88.8%and 93.4%,respectively。Theexpression vector for WMV- 2CP gene in Escherichia coli was constructed.By the method of SDS- PAGEanalysis,it showed that the MW of the produced gene expressed in E. cori is equal to that of authentic viralcoat protein.