百合斑驳病毒CP与CI基因的融合表达、多抗制备及其应用

采用RT-PCR方法从甘肃地区引种的切花百合上克隆了百合斑驳病毒(Lily mottle virus,LMoV)的外壳蛋白(coatprotein,CP)基因与细胞质内含体(cytoplasmic inclusions,CI)基因的3′端600 bp片段,连接到原核表达载体pET-28a(+)上,构建了融合表达的重组质粒pET-28a-CP-CI200。重组质粒转化大肠杆菌BL21成功表达了融合蛋白CP-CI200。经镍柱亲和层析获得纯化的融合蛋白,进一步用其免疫家兔制备了多克隆抗体。Western Blot结果显示,该多抗与感染LMoV的百合叶片中的病毒CP与CI均发生特异性反应,而对健康百合叶片无反应。用该多抗建立双抗夹心酶联免疫吸附法(DAS-ELISA)检测百合叶片样品,相对于RT-PCR方法其灵敏度和特异性均达到了93.3%。研究结果为快速、准确检测百合斑驳病毒的试剂盒研制奠定了基础。

Production and application of antisera to expression product of CP and CI fusion gene of Lily mottle virus

The cDNA fragments of CP gene and the 3'-terminal of CI gene of Lily mottle virus (LMoV) were amplified by RT-PCR from imported cutflower lilies in Gansu Province. The amplified PCR fragments were then inserted into the vector pET-28a (+), and the recombinant plasmid pET-28a-CP-CI200 was constructed. After transformation of the recombinant pET-28a-CP-CI200 to E. coli BL21 strain, the fusion protein was induced expressed. The purified protein was obtained by Ni²⁺ affinity chromatography. After renatu-ration, the protein was used to raise polyclonal antibodies in rabbit. Western blot analysis showed that the antisera were specifically reacted to viral CP and CI of LmoV-infected lily leaf but not to healthy leaf. A DAS-ELISA method was developed with the polyclonal antibodies. Sixty lily leaf samples were detected by both DAS-ELISA and RT-PCR method respectively. The results revealed that compared with RT-PCR method, DAS-ELISA had a specifity of 93.3% and sensitivity of 93.3%.