EF1α启动的高效重组杆状病毒载体的构建及其表达效果

Construction of recombinant baculovirus vectors started by EF1α

摘要:

摘要:【目的】通过选用哺乳动物细胞特异性启动子—人类延伸因子1α启动子(Elonggation factor 1αpromoter，EF1-α)、利用病毒假型化工具—截断型水疱型口膜炎病毒G 蛋白(Truncated Vessicular stomatitis virus protein G，VSV-GED)、添加转录后调控元件(Woodchuck hepatitis virus post-transcriptional regulatory element，WPRE)和腺病毒反向重复序列(Inverted terminal repeats，ITRs)，来提高杆状病毒的转导效率及外源基因的表达水平。【方法】从pFB-VSV-GED-WPRE出发构建含EF1α启动子的2个重组杆状病毒转移载体pWK 和pWK-ITR。再以增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein，eGFP)基因为报告基因插入EF1α启动子的下游，构建重组转移载体pWK-eGFP和pWK-ITR-eGFP以及阴性对照pWK (－)-eGFP。将构建好的重组质粒转染Sf9 昆虫细胞获得重组杆状病毒，侵染少突神经胶质细胞(OL细胞)后，利用倒置荧光显微镜观察绿色荧光蛋白的表达情况。【结果】含有WPRE和ITRs的病毒BV-WK-eGFP、BVWK-ITR-eGFP荧光表达率明显高于阴性对照，且ITRs 能够有效延长eGFP的表达时间，比对照组BV-WK(－)-eGFP延长了72 h。经VSV-GED假型化的杆状病毒BV-WK-eGFP、BV-WK-ITR-eGFP转导OL细胞的时间明显缩短，比阴性对照BV-WK(－)-eGFP减少了24 h，并且转导效率分别高出阴性对照25.7%与36.5%。【结论】实验证明了VSV-GED能够有效提高杆状病毒的转导效率，WPRE能够显著增强外源基因的表达效率，ITRs延长外源基因的表达时间。为探究改进型重组杆状病毒在脊椎动物细胞表达外源基因及新型重组活载体疫苗的研发奠定了基础。

Abstract:

Abstract:【Objective】To improve the transduction efficiency of baculovirus and exogenous gene expression level，we chose a mammalian cell-specific promoter-human extension factor 1α promoter ( EF1-α)，used virus pseudotyped tools-truncated vessicular stomatitis virus protein G (VSV-GED )，added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs)．【Method】We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE．The recombinant transfer vectors pWK-eGFP，pWK-ITＲ-eGFP and pWK (－)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1α promoter． Constructed recombinant plasmid transfected Sf9 insect cells，and observed the expression of green fluorescent protein by using the inverted fluorescence microscope．【Results】The fluorescence expression rate of BV-WK-eGFP，BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control，ITRs can effectively extend the expression time of eGFP，the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BVWK(－)-eGFP．The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP，BV-WK-ITR-eGFP was obviously shorten in OL cells，and reduced 24 hours compared to the negative control BV-WK(－)-eGFP，transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK(－)-eGFP，respectively．【Conclusion】The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus，WPRE could enhance the expression efficiency of the exogenous gene significantly，and ITRs extend the expression time．The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine．