李矮缩病毒外壳蛋白基因克隆及原核表达研究

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

为制备李矮缩病毒（prunusdwarfvims，PDv）抗血清，克隆PDV外壳蛋O（ce）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PDVCP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凹基因（GenBank登录号：JF333587．1）全长657bp，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．如Ⅵ≯载体的大肠杆菌BL21（DE3）菌株表达分子量约24kDa的重组蛋白。该重组蛋白在30℃，1．0mmol／LIPTG、诱导4h表达量最大。

Abstract： In order to prepare 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 leaf which was infected with PDV was 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 -PDVCPwas 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 group I . The prokaryotic expression vector pET3Oa-PDVCPwas 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 pET3Oa-PDVCP was transformed into and the optimized expression condition was 30℃, 1.0 mmol/L IPTG for 4 hours.