百合斑驳病毒外壳蛋白基因原核表达、抗血清制备及其应用

为了建立百合斑驳病毒(Lily mottle virus,LMoV)的快速检测方法,采用RT-PCR方法从感染LMoV的百合叶片中克隆该病毒的外壳蛋白(coat protein,CP)基因,然后连接到原核表达载体pET28a(+)上,导入大肠杆菌Escherichia coliBL21(DE3)并诱导表达,以表达的重组蛋白为抗原制备该病毒的抗血清。结果显示:LMoVCP全长为822 bp,编码274个氨基酸;SDS-PAGE及Western blot检测结果表明,经IPTG诱导得到了分子量约为34 kD带有HIS标签的目的蛋白;用该蛋白制备的抗血清经间接ELISA和Western blot检测结果显示,其效价为1∶51 200,具有较高的特异性,可用于感染LMoV百合的检测,其检测结果与RT-PCR检测结果一致。

Prokaryotic expression of coat protein gene of Lily mottle virus and preparation, application of its antiserum

In order to establish a rapid method of detecting Lily mottle virus (LMoV), the coat protein (CP) gene of LMoV cloned from the virus-infected lily with RT-PCR method, then constructed into the prokaryotic expression vector pET28a(+), and transformed into Escherichia coli BL21(DE3). The recombinant protein obtained from the induced-expression was used as an antigen to prepare antiserum for the virus. The sequence analysis showed that the complete nucleotide sequence of LMoV CP was 822 bp, and encoded 274 amino acids. SDS-PAGE and Western blot analysis showed that a 34 kD target protein with HIS tag was obtained from prokaryotic expression induced by IPTG. Indirect ELISA, and western blot detection of the titer of the antiserum was 1:512 000 with high specificity to LMoV. The target protein could be used for LMoV detection in lily, and its detection result was consistent with RT-PCR.