芜菁花叶病毒长春分离物p3基因的克隆、序列分析及P3蛋白抗血清的制备

Cloning,sequencing of p3 gene of Turnip mosaic virus isolates in Changchun and preparation of P3 protein antiserum

用RT-PCR方法从长春感染芜菁花叶病毒（ Turnip mosaic virus,TuMV）的十字花科蔬菜中扩增获得该病毒的p3基因,并对其序列进行了比较分析。结果表明,本研究所获得的10个TuMV分离物p3基因含1 065个核苷酸,其序列一致率为98.8%～99.6%,与GenBank中其他15个TuMV分离物核苷酸一致率为80.9%～99.4%。根据p3基因核苷酸序列构建的系统进化树显示：25个TuMV分离物可分为4个组,本研究得到的10个TuMV分离物均属于basal-BR组。将TuMV JCR06分离物p3基因N端663 bp片段克隆至原核表达载体pET-28a（＋）,并在大肠杆菌BL21（DE3） pLysS中表达出分子量约为28 kDa的融合蛋白。以纯化的融合蛋白为抗原免疫家兔,制备了P3蛋白的特异性抗血清。以TuMV侵染的萝卜为抗原,间接ELISA测定抗血清的效价为1∶2 048。Western blotting分析表明,制备的抗血清能与诱导表达的融合蛋白发生特异性反应。

Ten Turnip mosaic virus （TuMV） isolates were collected from infected cruciferous vegetables in Changchun. The p3 genes of these 10 isolates consisted of 1 065 nucleotides, with identities of 98.8%-99.6% at the nucleotide level. These p3 genes shared nucleotide identities of 80.9%-99.4% with other 15 TuMV isolates available in the GenBank. In the phylogenetic tree constructed with the complete nucleotide sequences of the p3 genes, the 25 TuMV isolates were divided into 4 groups, and the 10 TuMV isolates characterized in this resarch belonged to group basal-BR. The N-terminnal 663 nucleotides of p3 gene in isolate JCR06 was cloned into expression vector pET-28a（＋） and transferred into E. coli BL21（DE3） pLysS. SDS-PAGE showed that the N-terminnal fragment of p3 gene was expressed as a 28 kDa fusion protein with IPTG induction. Rabbit was immunized with purified fusion protein to obtain the antiserum with high specificity. The titer was 1∶2 048 determined by indirect ELISA test to detect TuMV in radish. Western blotting showed that the prepared antiserum could specifically bind to fusion protein.