Song Shanshan, Ge Jingping, Li Mei, Gao Dongni, Jin Liying, An Qi, Ping Wenxiang, Lou Zhuangwei
Key Laboratory of Microbiology, College of Life Science, Heilongjiang University, Harbin 150080, China.
Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):586-95.
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.
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.
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 10 mmol/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.
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.
杆状病毒因其在哺乳动物细胞中不复制而被认为是一种安全的载体候选物。通过引入细胞特异性启动子、水泡性口炎病毒糖蛋白（VSV-G）包膜结构域（GED）和不同的功能调控元件，可以改善其对不同细胞的嗜性和转导效率。优化后的假型重组杆状病毒能够在鸡原代细胞中表达增强型绿色荧光蛋白（eGFP）基因，这为开发工程化家禽疫苗提供了一种新方法。
构建含有巨细胞病毒（CMV）启动子、VSV-GED、土拨鼠肝炎病毒转录后调控元件（WPRE）和反向末端重复序列（ITR）的假型重组杆状病毒。将携带eGFP报告基因的重组杆状病毒转染鸡原代细胞，并分析不同杆状病毒介导的eGFP蛋白表达水平。
感染后12小时检测到eGFP的表达。通过插入VSV-GED，假型重组杆状病毒的转导效率从36%提高到48.2%。携带WPRE元件的重组杆状病毒中eGFP的表达效果与添加10 mmol/L丁酸钠时相似。然而，WPRE元件对细胞无毒。在72小时内，含有ITR的重组杆状病毒中eGFP的表达强度逐渐增加。
VSV-GED元件可提高转导效率，WPRE可增加杆状病毒在鸡原代细胞中介导的报告基因eGFP的表达水平。含有ITR元件的重组杆状病毒可延长eGFP的表达时间。
