杆状病毒对不同哺乳动物细胞基因转移及表达效率的研究

研究已证实杆状病毒可进入某些哺乳动物细胞，这提示了可将重组杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。利用已构建的重组杆状病毒BacV-CMV-EGFPA，以含病毒的Sf9细胞培养上清同时孵育多种哺乳动物细胞，利用流式细胞术检测报告基因在不同细胞株中的转移效率及表达效率。共使用了20种哺乳动物细胞株，其中有12种人类组织细胞，7种小鼠组织细胞，1种猴组织细胞。实验结果显示携带CMV启动子的重组杆状病毒可有效进入多数哺乳动物细胞，其中对人、猴来源细胞的基因转移效率显著高于对鼠源细胞，对贴壁细胞的基因转移效率显著高于对悬浮细胞。同时，通过脂质体LipofectAMINE将携带有CMV启动子和EGFP基因的哺乳动物细胞表达质粒pCDNA3-1-EGFP分别转染部分特别是被认为杆状病毒进入能力较低的细胞株，结果显示CMV启动子在这些细胞中均可有效引导EGFP基因的表达，因此认为携带了CMV启动子的重组杆状病毒对不同哺乳动物细胞基因转移效率能基本反映出杆状病毒对不同种哺乳动物细胞的进入能力。通过综合比较携带CMV启动子的杆状病毒对不同种属及组织来源细胞的基因转移及表达效率，可看出杆状病毒对灵长类动物贴壁细胞的基因转移及表达效果是较好的，而对小鼠来源的细胞及悬浮培养细胞却并不十分理想，这表明将重组杆状病毒作为一种对哺乳动物细胞的基因转移工具，仍有其局限性，不一定对所有的细胞都合适，在应用时应予以充分考虑。

Mammalian Gene-transfer and Expression Efficiencies of Baculovirus BacV-CMV-EGFPA

It has been reported that baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. We have previously constructed recombinant baculovirus BacV-CMV-EGFPA and have proven that mammalian cells could be effectively infected by the recombinant baculovirus. In this report, we studied the efficiency of baculovirus to deliver exogenous gene into twenty mammalian cells, including twelve human cell lines (WI-38, Hela, HepG2, 293, PLC/PRF/5, 143B, MCF-7, BGC-223, DMS 114, CNE, Raji, LCL-cm), seven murine cell lines (BNL 1ME A.7R.1, CHO-K1, L-929, JC, PT67, NIH3T3, P815) and one monkey cell line (CV1). Results showed that most mammalian cell lines could be transduced by the recombinant baculovirus, the transduction efficiencies of the human and monkey cell lines were markedly higher than that of murine cell lines, and the transduction efficiencies in adherent culture cell lines higher than that of suspend culture cell lines, implying that the infection efficiency of the baculovirus may be correlative with the organism used and the growth properties of the cell lines. The plasmid pcDNA3.1-EGFP, which contains the CMV promoter and EGFP reporter gene, was next transfected by LipofectAMINE into a number of mammalian cells, especially those cells that were low in the baculovirus transfection. Results showed that the CMV promoter could effectively direct the expression of the reporter gene in these mammalian cells. Therefore the gene-expression efficiencies in different mammalian cell lines by the recombinant baculovirus which contains the same CMV promoter were dictated by the ability of the baculovirus to enter the cell lines. This study suggested that the recombinant baculovirus vector is more suitable for gene expression in primate adherent culture cells than in murine cells and suspend culture cells.