构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。

Construction of BV-T7 hybrid expression system based on baculovirus to express target gene eGFP in mammalian and chicken cells

[Objective]To investigate whether the recombinant baculovirus BV-T7 hybrid expression system can be effectively transduced into chicken cells in vitro,and whether it can express the foreign genes(eGFP).[Method] We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells and chicken cells.Two recombinant baculoviruses were constructed,one carrying cDNA of bacteriophage T7 RNA polymerase,with a nuclear localization signal,under the control of a mammalian promoter and the other expressing eGFP gene under the control of T7 promoter.The constructed recombinant baculoviruses co-infected mammalian oligodendrocyte cells,as well as chicken embryo fibroblasts cells and chicken embryo skeletal muscle cells.[Results] The eGFP activity was detected in mammalian cell lines and embryo fibroblasts cells and chicken embryo skeletal muscle cells.The recombinant baculovirus transduction efficiency of oligodendrocyte cells was 59.5%,and in CEF cells and myoblast cells the transduction efficiencies were 23.2% and 33.1%.[Conclusion] BV-T7 hybrid expression could be expressed T7 RNAP in mammalian cells and avian cells.