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

 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。

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

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.