对虾病毒IHHNV介导的双启动子驱动的对虾CDK1基因表达载体的构建与表达研究

In response to the problems of division arrest and difficulty in continuous passage of shrimp cells cultured in vitro, this research team have previously developed a gene transfer and expression system based on the genome of shrimp infectious hypodermal and hematopoietic necrosis virus(Infectious hypodermal and hematopoietic necrosis virus, IHHNV), but it has not yet been applied to delivery the immortalization-related genes to shrimps and their cells. To achieve this, in this study, the GUS reporter gene in this system was replaced with the EGFP reporter gene, and shrimp immortalization-related gene: cyclin-dependent kinase 1(Cell cycle protein-dependent kinase, CDK1) was inserted and a viral expression vector: pUC19-IHHNV-PH-CDK1-P2A-EGFP was constructed. To improve the expression efficiency of the CDK1 gene in shrimp cells, the shrimp white spot syndrome virus promoter(White spot syndrome virus, WSV249) was further introduced and a dual-promoter viral expression vector: pUC19-IHHNV-PH-WSV249-CDK1-P2A-EGFP was also constructed. Using the insect virus polyhedrin protein(Polyhedrin, PH) gene as a promoter(PH promoter), the activity analysis of the PH promoter and WSV249 promoter were analyzed in Sf9 cells.The results showed that the WSV249 promoter could effectively drive gene expression in the Sf9 cells, but its activity was significantly lower than that of the PH promoter. Further, each of these two viral expression vectors was co-transfected with Bacmid-VP28 plasmid into the Sf9 cells, respectively. The experimental results showed that the successfully packaged mixed viruses could effectively infect the in vitro cultured shrimp hemolymph cells and hematopoietic cells, as well as the adult shrimp tissues. Notably, the mixed virus with dual promoters(PH-WSV249) exhibited a higher infection efficiency than PH promoter alone did. However, the survival ability of the infected shrimp cells was limited, thus no obvious proliferation-promoting effect was observed. This study suspects that this phenomenon is related to the toxic effect of the packaged IHHNV on cells. In short, this study has developed an efficient shrimp viral expression vector with verified gene transfer potential in shrimps and shrimp cells.