构建具有巨细胞病毒(CMV)启动子的杆状病毒高效表达载体并利用其在鸡胚原代细胞中表达eGFP基因

Construction of baculovirus vector with cytomegaoviyns promoter to express eGFP in primary chicken embryo cells

摘要:

摘要:【目的】杆状病毒载体在哺乳动物细胞中不复制，具有极高的生物安全性，而通过引入细胞特异性启动子、“病毒假型化”、添加不同功能调控元件等优化手段，杆状病毒可转导的细胞类型明显增多、转导效率明显增高。优化的重组杆状病毒可以在鸡细胞中表达，确定重组杆状病毒在鸡细胞中高效表达的条件，为禽类基因工程疫苗的开发奠定了坚实的基础。【方法】本研究以增强型绿色荧光蛋白( Enhanced Green Fluorescent Protein，eGFP)为报告基因，构建含巨细胞病毒(Cytomegaoviyns，CMV)启动子启动的重组转座载体，并且通过病毒假型化(TM/CTD of VSV-GED)、添加转录后调控元件(Woodchuck hepatitis virus posttranscriptional regulatory element，WPRE)和腺病毒反向重复序列(Inverted terminal repeats，ITRs)等手段优化重组杆状病毒表达载体，最终利用重组杆状病毒对鸡胚原代细胞的侵染进行eGFP 的表达，比较分析不同重组载体对蛋白表达水平的影响。【结果】eGFP表达结果显示，12 h便可在倒置荧光显微镜下检测出eGFP的表达，随着时间的延长，eGFP表达效率先增加后降低。经过VSVGED病毒假型化的重组杆状病毒转导效率从36%增加至48.2%。添加转录后调控元件WPRE的重组杆状病毒介导eGFP的表达效果与添加10mmol/L丁酸盐法基本相同，但对细胞几乎没有毒性。从72 h内的eGFP 表达强度看，只有添加ITRs元件的重组杆状病毒的表达强度随时间的延长而极缓慢增加。【结论】eGFP表达结果显示，利用VSVGED病毒 假型化手段可以提高杆状病毒转导鸡胚原代细胞的效率;添加转录后调控元件WPRE的重组杆状病毒介导eGFP的表达效果与添加丁酸盐法基本相同，可以增强杆状病毒介导报告基因在鸡胚原代细胞中的表达水平;添加ITRs元件的重组杆状病毒具有延长eGFP 表达时间的作用。

Abstract:

Abstract:［Objective］Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells．The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter，VSV-GED and different functional regulatory elements． The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells，which provides us a new approach to develop engineered poultry vaccines．［Method］ The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor，VSV-GED，woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs)．The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells，and the eGFP protein expression levels mediated by different baculoviruses were analyzed．［Results］The expression of eGFP was detected at 12 hours after infection．The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED． The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10mmol/L butyrate． However，the WPRE elements are nontoxic to cells． Within 72 hours，the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually．［Conclusion］The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells． The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.