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目的 选用植物细胞表达轮状病毒结构蛋白 ,拟构建有效植物细胞表达系统。方法 利用PT -PCR扩增A组轮状病毒外壳蛋白VP4基因片段 ,经双酶切后与植物表达载体连接 ,转化感受态细菌TG1。利用地高辛标记探针进行斑点杂交检测 ,筛选出阳性克隆 ,拟用该克隆转化马铃薯细胞 ,研制新型轮状病毒疫苗。本研究还利用 β -葡萄糖苷酸酶 (GUS)基因转化马铃薯细胞。结果 A组轮状病毒SA11株VP4基因产物大小与设计相同 ,为 96 0bp。在被检测的未知载体中 ,有四个显紫色斑 ,可判定其为带有VP4cDNA的转化载体。用肉眼或显微镜可观察到马铃薯组织细胞中的蓝色物质。结论在转基因植物组织器官中观察到GUS的活性 ,获得外源基因转化的条件。拟用本研究构建的表达载体转化马铃薯细胞 ,研制新型轮状病毒疫苗。
Objective To express plant rotavirus structural protein by plant cells and to construct an effective plant cell expression system. Methods The VP4 gene fragment of coat protein A of group A was amplified by PT-PCR. After double digestion, the fragment was connected with the plant expression vector and transformed into competent bacteria TG1. Digoxigenin-labeled probe was used to detect the dot blot hybridization, and the positive clones were screened out. The clone was transformed into potato cells and a new rotavirus vaccine was developed. In the present study, potato cells were also transformed using the β - glucuronidase (GUS) gene. Results The VP4 gene product of group A rotavirus strain SA11 had the same size and design as 96 0bp. Among the unknown vectors tested, there are four purple spots, which can be judged as the transformation vector with VP4cDNA. Blue material in potato cells can be observed with the naked eye or a microscope. Conclusion The activity of GUS was observed in the tissues and organs of transgenic plants and the conditions of foreign gene transformation were obtained. The proposed construction of the expression vector to transform potato cells, develop a new rotavirus vaccine.