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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (12): 177-181.doi: 10.11924/j.issn.1000-6850.2011-2810

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

李矮缩病毒外壳蛋白基因克隆及原核表达研究

陈立伟 宗晓娟 王文文 王甲威 魏海蓉 徐丽 严雪瑞 刘庆忠   

  • 收稿日期:2011-09-28 修回日期:2011-12-06 出版日期:2012-04-25 发布日期:2012-04-25
  • 基金资助:

    农业部948项目国外甜樱桃抗逆种质资源的引进创新及产业化（2011-Z40）;公益性行业（农业）科研专项樱桃产业主要障碍因素攻关研究（200903019）;山东省农业良种工程甜樱桃新品种和抗性砧木选育[鲁农良种字（2010）6号]

Cloning and Prokaryotic Expressing of Coat Protein Gene in Prunus Dwarf Virus

  • Received:2011-09-28 Revised:2011-12-06 Online:2012-04-25 Published:2012-04-25

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摘要：

为制备李矮缩病毒(Prunus dwarf virus, PDV)抗血清，克隆PDV外壳蛋白(CP)基因，构建原核表达载体，优化蛋白表达条件。以阳性感病甜樱桃叶片为试验材料，提取总RNA，根据PDV CP基因(L28145.1)设计特异引物，RT-PCR方法扩增，克隆、测序，构建原核表达载体pET30a-PDVCP，在大肠杆菌BL21(DE3)菌株表达。PDV CP基因（Genbank登录号：JF333587.1）全长657 bp，编码217个氨基酸，与GenBank其他PDV分离物CP基因核苷酸序列的同源性为89.2%~93.9%，推导的氨基酸序列同源性为97%~99%。根据完整CP基因核苷酸序列构建系统进化树显示：12个PDV分离物可分为3组，泰安分离物与巴西、土耳其等分离物属于Ⅰ组。成功构建原核表达载体pET30a-PDVCP，并在体外条件下诱导表达出融合蛋白。转pET30a-PDVCP载体的大肠杆菌BL21（DE3）菌株表达分子量约24 kDa的重组蛋白。该重组蛋白在30℃，1.0 mmol/L IPTG、诱导4 h表达量最大。

关键词: 农村居民, 农村居民, 收入差异, 研究

Abstract:

In order to preparate Prunus dwarf virus’ special antiserum, the coat protein gene of PDV was cloned, the prokaryotic expression vector was constructed and the expression condition was optimized. The sweet cherry leaves which is infected with PDV is the test material and the total RNA was extracted from them. According to the coat protein gene of PDV in Genbank (L28145.1), specific primers were designed to amplify the coding region of coat protein by RT-PCR, the PCR products were cloned and sequenced, the prokaryotic expression vector pET30a -PDVCP was constructed and the recombinant protein was expressed in E.coli BL21(DE3). Sequence analysis showed that the amplicons (NCBI: JF333587.1) included 657 bp nucleotides, encoding 217 amino acids and shared the similarity of 89.2%-93.9% at nucleotide acid level and the identity of 97%-99% at amino acid level with other PDV isolates. The phylogenetic tree constructed with the complete nucleotide sequences of the CP gene showed that PDV isolates were divided into 3 groups, Taian, Brazil, Turkey et al isolates belonged to groupⅠ. The prokaryotic expression vector pET30a-PDVCP was constructed successfully and the fusion protein was expressed by inducing in vitro. A specific recombinant protein of approximately 24 kDa was induced in the BL21 (DE3) which the prokaryotic expression vector pET30a-PDVCP was transformed into and the optimized expression condition was 30℃, 1.0 mmol/L IPTG for 4 hours.