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经PCR获得了中国番茄黄化曲叶病毒(TYLCV-CHI)外壳蛋白(CP)基因,并克隆到pGEM-7Zf(+)载体上.序列分析表明,TYLCV-CHI感染番茄或烟草时,CP基因的核苷酸序列发生变化,并且在烟草上随感染时间的延长,突变频率增高.连接到含有编码tRNAarg4基因argU的表达载体pSBET后,TYLCV-CHI CP基因在大肠杆菌中以包含体形式得到大量表达(约33 ku).以表达的蛋白作抗原免疫家兔,制备了抗TYLCV-CHI CP的抗血清.ELISA实验表明,该血清与抗原特异反应,血清效价为1:3000.Western印迹实验还表明,该血清除了与感染TYLCV-CHI的番茄、烟草有特异免疫反应外,还与感染南瓜曲叶病、烟草曲叶病的叶片有免疫反应,表明这些病毒之间有明显的血清学关系.
The CP gene of TYLCV-CHI was obtained by PCR and cloned into pGEM-7Zf (+) vector.The sequence analysis showed that when TYLCV-CHI infected tomato or tobacco, CP gene , And the frequency of mutation increased with the extension of infection time in tobacco.After being linked to the expression vector pSBET containing the argU encoding the tRNAarg4 gene, the TYLCV-CHI CP gene obtained a large amount of inclusion bodies in Escherichia coli (About 33 ku), and the antiserum against TYLCV-CHI CP was prepared by immunizing rabbits with the expressed protein as an antigen.The ELISA assay showed that the serum reacted specifically with the antigen and the serum titer was 1: 3000. Western blotting assay The results also showed that this serum, in addition to the specific immune response to TYLCV-CHI-infected tomato and tobacco, was also immunoreactive with the leaves infected with pumpkin leaf and tobacco leaf curl, indicating a clear serological relationship between these viruses .