We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant t...We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35.展开更多
Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficie...Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently.The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter.MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells.Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate,so baculovirus-mediated p35 expression had no adverse effect on cell proliferation.In addition,baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D,UV or serum-free media.These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.展开更多
利用去血清方法建立 L s细胞凋亡的实验体系 ,证明 L s NPV p 35基因在 L s细胞中瞬时表达可以抑制L s细胞由于无血清造成的凋亡 .构建了在哺乳动物中高效表达 L s NPV p 35基因的载体 p Rc/ RSV- L p35 ,并导入Vero细胞瞬时表达 ,发现 ...利用去血清方法建立 L s细胞凋亡的实验体系 ,证明 L s NPV p 35基因在 L s细胞中瞬时表达可以抑制L s细胞由于无血清造成的凋亡 .构建了在哺乳动物中高效表达 L s NPV p 35基因的载体 p Rc/ RSV- L p35 ,并导入Vero细胞瞬时表达 ,发现 L s NPV p35基因产物也可以抑制 Vero细胞由于病毒感染造成的凋亡 .用含 L s NPVp35的质粒与 v Ac Anh(Ac NPV p35基因缺失病毒 ) DNA共转染 Sf- 9细胞 ,可以使 v Ac Anh DNA在 Sf- 9细胞中形成多角体 ,并且传代后多角体不扩增 ,表明 L s NPV p35基因与 Ac NPV p 35基因具有功能互补性 .展开更多
文摘We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35.
基金Foundation items:National Nature Science Foundations of China (30325002,30670077)Innovative Foundations Wuhan Institute of Virology,CAS (020208)
文摘Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently.The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter.MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells.Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate,so baculovirus-mediated p35 expression had no adverse effect on cell proliferation.In addition,baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D,UV or serum-free media.These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.
文摘利用去血清方法建立 L s细胞凋亡的实验体系 ,证明 L s NPV p 35基因在 L s细胞中瞬时表达可以抑制L s细胞由于无血清造成的凋亡 .构建了在哺乳动物中高效表达 L s NPV p 35基因的载体 p Rc/ RSV- L p35 ,并导入Vero细胞瞬时表达 ,发现 L s NPV p35基因产物也可以抑制 Vero细胞由于病毒感染造成的凋亡 .用含 L s NPVp35的质粒与 v Ac Anh(Ac NPV p35基因缺失病毒 ) DNA共转染 Sf- 9细胞 ,可以使 v Ac Anh DNA在 Sf- 9细胞中形成多角体 ,并且传代后多角体不扩增 ,表明 L s NPV p35基因与 Ac NPV p 35基因具有功能互补性 .
