【摘要】 现已发现杆状病毒可进入某些培养的哺乳动物细胞 ,这提示可将杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。对杆状病毒转移载体的改造及对哺乳动物细胞的基因转移方式进行了进一步的研究。以绿色荧光蛋白基因为报告基因 ,利用Bac to Bac系统构建了分别含有正向和反向CMV启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达 ,但表达效率较低。用重组杆状病毒感染后Sf9细胞的培养上清直接与HepG2细胞作用 ,以流式细胞术检测基因转移效率及荧光表达强度 ,发现这两种病毒在相同的感染复数下对HepG2细胞具有相似的基因转移及表达效率。同时 ,利用流式细胞术进一步研究了直接使用重组杆状病毒感染 4d后Sf9细胞的培养上清对哺乳动物细胞进行基因转移的方法。通过对HepG2细胞的实验结果显示 ,将带毒Sf9细胞培养上清 (1.2× 10 7PFU mL)用哺乳动物细胞培养基 1倍稀释后 ,37℃下孵育靶细胞 12h(moi=5 0 ) ,可达到较高的基因转移及表达效率 ,同时不会对细胞造成明显损伤。将重组杆状病毒与脂质体和逆转录病毒这两种系统对HepG2及CV1细胞的基因转移效率进行了比较 ,结果发现在同样未经浓缩等特殊处理的条件下重组杆状病毒对这两种细胞的基因转移效率是最高的。因此可以认为 ,更多还原

【Abstract】 The baculovirus insect cell expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, reports have described that recombinant baculoviruses can transduce a broad spectrum of primary and established mammalian cells, which shows the baculoviruses could serve as a new gene transfer vehicle for mammalian cells. In this report, we further research the modification of baculovirus vector and the way to deliver exogenous gene into mammalian cells. On the base of Bac to Bac baculovirus insect cell expression system, two recombinant baculoviruses(BacV CMV EGFPA, BacV CMV EGFPB) were constructed containing different direction of CMV promoters which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells with relatively low efficiency. The culture supernatant of Sf9 cells which have been infected by the recombinant baculoviruses for four days were collected and the titers of the viruses in culture supernatant were determined by plaque assay on Sf9 cells. The HepG2 cells, an human hepatocellular carcinoma cell line, were directly incubated with the collected culture supernatant which contains the recombinant baculoviruses for 8 hours in 37℃ CO 2 incubator (moi=100). Twenty four hours post transduction the efficiencies of gene transfer and expression were analyzed by flow cytometry (FCM) which detect the green fluorescence of individual cells. Results show that these two recombinant baculoviruses have similar gene transfer and expression efficiency in HepG2 cells, which means the direction of CMV promoters has no effects on reporter gene expression. The optimal transduction conditions of incubating the mammalian cells with the culture supernatant of Sf9 cells infected by recombinant baculoviruses for four days were determined by FCM assay in HepG2 cells. The HepG2 cells inoculated in 24 well plate (5×10 4/well) were incubated with the culture supernatant (BacV CMV EGFPA, 1.2×10 7pfu/mL) serially diluted by DMEM culture medium containing 10% FBS and the transduction times ranged from 1 to 24 hours. Twenty four hours post transduction the efficiencies of gene transfer and expression were analyzed by FCM. Results show that incubating the target cells with the 1:1 diluted culture supernatant (moi=50) for 12 hours in 37℃ CO 2 incubator would achieve the highest infection and expression efficiency with the least impairment on cell viability. We compared the gene transfer and expression efficiency of recombinant baculovirus in HepG2 and CV1 cells with lipofectAMINE and recombinant retrovirus system, results show that under the similar conditions the recombinant baculovirus could achieve the highest gene transfer and expression efficiency than the other two systems. So we can draw a conclusion that directly incubating the mammalian cells with the culture supernantant of the infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign gene in mammalian cells.更多还原