论文部分内容阅读
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。
In order to establish a rapid detection method of Lily mottle virus (LMoV), the coat protein (CP) gene of this virus was cloned from the lily leaf infected with LMoV by RT-PCR and ligated into prokaryotic expression vector pET28a (+), And introduced into E. coli Escherichia coli BL21 (DE3) to induce expression. The expressed recombinant protein was used as antigen to prepare the antisera of the virus. The results showed that the full length of LMoVCP was 822 bp, encoding 274 amino acids. The results of SDS-PAGE and Western blot showed that the target protein with a molecular weight of about 34 kD and HIS tag was induced by IPTG. The antiserum The results of indirect ELISA and Western blot showed that the titer was 1:51 200, which showed high specificity and could be used to detect LMOV lily. The results of the detection were consistent with those of RT-PCR.