摘要
针对体外培养刀额新对虾(Metapenaeus ensis)细胞存在分裂停滞和难以连续传代的问题,本研究团队已建立了一种基于传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)基因组的基因转移与表达系统(pUC19-IHHNV-PH-GUS和Bacmid-VP28),但该系统尚未应用于介导永生性转化相关基因在对虾及其细胞中的转移。鉴于此,本研究将该系统中的GUS报告基因替换为EGFP报告基因,并插入对虾永生性转化相关基因——细胞周期蛋白依赖性激酶(Cell cycle protein-dependent kinase,CDK1),构建了病毒表达载体pUC19-IHHNV-PH-CDK1-P2A-EGFP。同时,为提高CDK1基因在对虾细胞中的表达效率,进一步引入对虾白斑综合征病毒(White spot syndrome virus,WSSV)的WSV249启动子,构建了双启动子病毒表达载体pUC19-IHHNV-PH-WSV249-CDK1-P2A-EGFP。利用昆虫病毒多角体蛋白(Polyhedrin,PH)基因作为启动子(PH启动子),通过Sf9细胞对PH启动子和WSV249启动子的活性进行分析,结果表明,WSV249启动子可以在Sf9细胞中有效驱动基因的表达,但其活性显著低于PH启动子。进一步,将上述2种病毒表达载体分别与Bacmid-VP28质粒共转染至Sf9细胞中,从而发现所包装出的混合病毒能够有效感染对虾体外培养血淋巴细胞和造血组织细胞及对虾成体组织,且含双启动子PH-WSV249的混合病毒的感染效率更高。然而病毒感染后的对虾细胞存活能力有限,未在其中观察到明显的促进细胞增殖现象,本研究怀疑该现象与包装出的IHHNV会对细胞产生毒害作用有关。总之,本研究优化并构建了高效的对虾基因病毒表达载体,并验证了其在对虾及其细胞中的基因转移潜力。
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.
作者
肖蕊
王金武
郭华荣
Xiao Rui;Wang Jinwu;Guo Huarong(Key Laboratory of Marine Genetics and Breeding,Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao 266003,China;Institute of Evolution and Marine Biodiversity,Ocean University of China,Qingdao 266003,China)
出处
《中国海洋大学学报(自然科学版)》
北大核心
2026年第1期153-164,共12页
Periodical of Ocean University of China
基金
国家自然科学基金项目(32273116)
山东省重点研究发展计划项目(2023CXGC010710)资助。
