小菜蛾颗粒体病毒晚期启动子在非受纳细胞系中的表达活性分析
发布时间：2018-08-20 12:44
【摘要】：杆状病毒是一类由囊膜包被杆状核衣壳的、含单一双链环状超螺旋基因组的病毒。小菜蛾颗粒体病毒(Plutella xylo5tellagranulovirus,PlxyGV)属于杆状病毒中的β-杆状病毒属。由于缺少合适的受纳细胞系,有关PlxyGV的分子生物学研究进展一直较为缓慢。本文通过小菜蛾颗粒体病毒的晚期启动子在非受纳细胞系中的表达活性分析,进一步研究制约PlxyGV离体复制的原因。(1)通过基因克隆,构建由PlxyGV的晚期启动子(pxPan、pxPe18、pxPe25、pxPgp41、pxPp6.9、pxPvp39、pxPpk1、pxPgran、pxPorf21 与 pxPorf50)分别控制报告基因luciferase的10个报告质粒。通过瞬时表达实验我们发现,PlxyGV基因组不能在被转染的Sf9细胞中启动晚期与极晚期基因表达。同时我们也注意到,PlxyGV的晚期启动子报告质粒与AcMNPV共转染时均产生较高水平的荧光素酶活性。(2)通过基因克隆,构建与PlxyGV报告质粒对应的由AcMNPV的晚期启动子(acPan、acPe18、acPe25、acPgp41、acPp6.9、acPvp39、acPpk1、acPpolh、acPp10)分别控制报告基因luciferase的9个报告质粒。通过瞬时表达实验,我们发现PlxyGV晚期基因报告质粒与AcMNPV DNA共转染细胞中的荧光素酶活性与对应AcMNPV晚期基因报告质粒和AcMNPV DNA共转染细胞中的荧光素酶活性接近甚至更高。(3)利用Bac-to-Bac系统将由PlxyGV和AcMNPV晚期启动子控制的luc基因分别转座到AcMNPV bacmid bMON14272上构建相应的晚期基因报告病毒。通过转染后感染和实时表达分析,我们发现:在PlxyGV的非受纳细胞系Sf9细胞中,插入AcMNPV基因组的PlxyGV晚期启动子都能够被激活并驱动报告基因表达。其中,pxPgp41(或 pxPgp41L)与 acPgp41、pxPvp39(或 pxPvp39L)与 acPvp39、pxPe18与acPe18(或acPe18L)、pxPorf50与acPp10四组中,虽然对应启动子所含核心基元序列TAAG的数目可能相等,但它们的表达活性随时间变化的趋势差异显著。在pxPan与acPan、pxPpk1与acPpk1两组中,对应启动子在报告病毒感染细胞中的表达活性高低有明显差异,但它们随时间变化的趋势却基本相同。而acPp6.9与pxPp6.9、acPe25 与 pxPe25、acPpolh 与 pxPgran、pxPorf21 与 acPp10 四组中对应启动子在重组病毒感染的细胞中的表达活性随时间的变化基本相同。(4)通过λ-red同源重组的方法从AcMNPV基因组中分别敲除ie1、p35、lef8或vp39基因构成重组病毒vAci~(leIKO)、vA~(cp35KO)、vAc~(lef8KO)和vAc~(vp39KO)。同时,利用Bac-To-Bac系统将含pF-~(pxPp6.9-luc)与pF-~(pxPgran-luc)的报告质粒分别转座到PlxyGV bacmid上,构成报告病毒vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)。通过瞬时表达实验,研究不同 AcMNPV 突变体(vAc~(ie1KO)、vA~(cp35KO)、vAc~(lef8KO)、vA~(vp39KO))对报告病毒中PlxyGV的晚期基因表达的影响。结果表明:在报告病毒vPx~(pxPp6.9-1uc)和vPx~(pxPgran-luc)分别与vAc~(vp39KO)共转染的Sf9细胞中均可检测到高水平的荧光素酶活性;在vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)分别与vAc~(ie1KO)共转染的细胞中荧光素酶活性仅略高于vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)单独转染细胞的荧光素酶活性。vPx~(pxPp6.9-luc)与vAc~(lef8KO)或vA~(cp35KO)共转染细胞中的荧光素酶活性分别是其与vAc~(vp39KO)共转染细胞荧光素酶活性的0.98%和34.49%;而vPx~(pxPgran-luc)与 vAc~(lef8KO)或vA~(cp35KO)共转染细胞的荧光素酶活性分别其与vAc~(vp39KO)共转染细胞荧光素酶活性的0.29%和28.49%。这些结果说明:在被转染Sf9细胞中,由PlxyGV晚期启动子控制的报告基因的表达主要依赖于AcMNPV编码的RNA聚合酶;PlxyGV自身的晚期表达因子可能没有有效表达。(5)为研究AcMNPV基因组对PlxyGV DNA复制的影响,用不同的PlxyGV bacmids单独或与AcMNPV的突变体共转染Sf9或Hi5细胞。从被转染细胞中提取的病毒DNA经DpnI处理后,以gp41为标的,通过PCR检测复制的PlxyGV DNA。无论是在Sf9细胞中还是在Hi5细胞中,PlxyGV的基因组均可以进行复制;AcMNPV的三种拯救因子ie1、p35和gp64可以增强其基因组的复制;在vAc~(lef8KO)或vAC~(vp39KO)存在的情况下,PlxyGV基因组的复制可进一步得到加强。
[Abstract]:Plutella xylo 5 tellagranulovirus (PlxyGV) belongs to the baculovirus genus Beta-baculovirus, which belongs to the baculovirus genus. Due to the lack of suitable acceptor cell lines, the molecular biology of PlxyGV has been relatively advanced. In this study, the expression of PlxyGV late promoters in non-acceptor cell lines was analyzed to further study the reasons that restrict PlxyGV replication in vitro. (1) The late promoters (pxPan, pxPe18, pxPe25, pxPgp41, pxPp6.9, pxPvp39, pxPpk1, pxPgran, pxPorf21 and pxPorf50) of PlxyGV were constructed by gene cloning. We found that the PlxyGV genome could not initiate late and very late gene expression in the transfected Sf9 cells. At the same time, we also noticed that the late promoter of PlxyGV reported high levels of luciferase activity when co-transfected with AcMNPV. (2) Nine reporter plasmids of luciferase gene were constructed by cloning and transfection of AcMNPV late promoter (acPan, acPe18, acPe25, acPgp41, acPppp6.9, acPvp39, acPpk1, acPpolh, acPpp10) corresponding to PlxyGV reporter plasmid. The luciferase activity in the cells was close to or even higher than that in the cells co-transfected with AcMNPV late gene reporter plasmid and AcMNPV DNA. (3) The Luc gene controlled by PlxyGV and AcMNPV late promoter was translocated to AcMNPV bacmid bMON14272 by Bac-to-Bac system to construct the corresponding late gene reporter virus. Through post-transfection infection and real-time expression analysis, we found that the late promoters of PxyGV inserted into AcMNPV genome were able to activate and drive reporter gene expression in PlxyGV non-acceptance cell line Sf9. Among them, pxPgp41 (or pxPgp41L) and acPgp41, pxPvp39 (or pxPvp39L) and acPvp39, pxPe18 and Peac18 (or Peac18L), pxPorf50. Although the number of TAAG in the corresponding promoter may be the same as that in acPp10, the expression activity of TAAG in the corresponding promoter varies significantly with time. The expression activity of the corresponding promoters in the four groups of ACPp6.9 and pxPp6.9, acPe25 and pxPe25, acPpolh and pxPgran, pxPorf21 and acPp10 was similar with time. (4) The expression of ie1, p35, lef8 or vp39 was knocked out from the genome of AcMNPV by the method of lambda-Red homologous recombination. The recombinant viruses vAci ~ (leIKO), vA ~ (cp35KO), vAc ~ (lef8KO), vAc ~ (lef8KO) and vAc ~ (vpp39KO) were constructed by transposing the reporter plasmidcontaining pF ~ (pxPppxPpppppp6.9-luc) and pF ~ (pxPgran-luc) into PlxyGV bacmid, vAc ~ (pxPx Px 6.9-pppppp6.9-luc) and vAcpx Px ~ (ppx Px Pluc ~ (ppx PPX Pluc) into the Bac-To-Bac system, and the AcMNPV process The effects of variants (vAc ~ (ie1KO), vA ~ (cp35KO), vAc ~ (lef8KO), vAc ~ (lef8KO), vA ~ (vp39KO) and vA ~ (vp39KO) on the expression of late gene of PlxyGV in the reporter virus were studied. The results showed that high levels of luluciferase activity were detected in Sf9 cells co-transfected with vAc ~ (vppppp39KO), vPx ~ (ppxpppxppxppppppppx ~ (pxpppppppp6.9-1uc) and vPx ~ (pxpxPgran-luc) co-transxPg Luciferase activity of ran-luc co-transfected with vAc ~ (ie1KO) was only slightly higher than that of vPx ~ (pxPp6.9-luc) and vPx ~ (pxPgran-luc) co-transfected cells. Luciferase activity of vPx ~ (pxPp6.9-luc) co-transfected with vAc ~ (lef8KO) or vA ~ (cp35KO) was higher than that of vPx ~ (p39KO) co-transfected cells, respectively. The activity of luciferase in vPx ~ (pxPgran-luc) and vAc ~ (lef8KO) or vA ~ (cp35KO) co-transfected cells was 0.98% and 34.49% respectively, and that of luciferase in vAc ~ (vp39KO) co-transfected cells was 0.29% and 28.49% respectively. These results indicated that the expression of reporter gene controlled by PlxyGV late promoter in Sf9 cells was mainly dependent on AcMN ~ (vp39KO). In order to study the effect of AcMNPV genome on PlxyGV DNA replication, different PlxyGV bacmids were transfected into Sf9 or Hi5 cells alone or with mutants of AcMNPV. Viral DNA extracted from the transfected cells was treated with DpnI, and gp41 was used as the target through P The replication of PlxyGV DNA was detected by CR. The genome of PlxyGV could be replicated either in Sf9 or Hi5 cells; the three rescue factors of AcMNPV, ie1, p35 and gp64, could enhance the replication of PlxyGV genome; and the replication of PlxyGV genome could be further enhanced in the presence of vAc ~ (lef8KO) or vAC ~ (vp39KO).
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.13
[Abstract]:Plutella xylo 5 tellagranulovirus (PlxyGV) belongs to the baculovirus genus Beta-baculovirus, which belongs to the baculovirus genus. Due to the lack of suitable acceptor cell lines, the molecular biology of PlxyGV has been relatively advanced. In this study, the expression of PlxyGV late promoters in non-acceptor cell lines was analyzed to further study the reasons that restrict PlxyGV replication in vitro. (1) The late promoters (pxPan, pxPe18, pxPe25, pxPgp41, pxPp6.9, pxPvp39, pxPpk1, pxPgran, pxPorf21 and pxPorf50) of PlxyGV were constructed by gene cloning. We found that the PlxyGV genome could not initiate late and very late gene expression in the transfected Sf9 cells. At the same time, we also noticed that the late promoter of PlxyGV reported high levels of luciferase activity when co-transfected with AcMNPV. (2) Nine reporter plasmids of luciferase gene were constructed by cloning and transfection of AcMNPV late promoter (acPan, acPe18, acPe25, acPgp41, acPppp6.9, acPvp39, acPpk1, acPpolh, acPpp10) corresponding to PlxyGV reporter plasmid. The luciferase activity in the cells was close to or even higher than that in the cells co-transfected with AcMNPV late gene reporter plasmid and AcMNPV DNA. (3) The Luc gene controlled by PlxyGV and AcMNPV late promoter was translocated to AcMNPV bacmid bMON14272 by Bac-to-Bac system to construct the corresponding late gene reporter virus. Through post-transfection infection and real-time expression analysis, we found that the late promoters of PxyGV inserted into AcMNPV genome were able to activate and drive reporter gene expression in PlxyGV non-acceptance cell line Sf9. Among them, pxPgp41 (or pxPgp41L) and acPgp41, pxPvp39 (or pxPvp39L) and acPvp39, pxPe18 and Peac18 (or Peac18L), pxPorf50. Although the number of TAAG in the corresponding promoter may be the same as that in acPp10, the expression activity of TAAG in the corresponding promoter varies significantly with time. The expression activity of the corresponding promoters in the four groups of ACPp6.9 and pxPp6.9, acPe25 and pxPe25, acPpolh and pxPgran, pxPorf21 and acPp10 was similar with time. (4) The expression of ie1, p35, lef8 or vp39 was knocked out from the genome of AcMNPV by the method of lambda-Red homologous recombination. The recombinant viruses vAci ~ (leIKO), vA ~ (cp35KO), vAc ~ (lef8KO), vAc ~ (lef8KO) and vAc ~ (vpp39KO) were constructed by transposing the reporter plasmidcontaining pF ~ (pxPppxPpppppp6.9-luc) and pF ~ (pxPgran-luc) into PlxyGV bacmid, vAc ~ (pxPx Px 6.9-pppppp6.9-luc) and vAcpx Px ~ (ppx Px Pluc ~ (ppx PPX Pluc) into the Bac-To-Bac system, and the AcMNPV process The effects of variants (vAc ~ (ie1KO), vA ~ (cp35KO), vAc ~ (lef8KO), vAc ~ (lef8KO), vA ~ (vp39KO) and vA ~ (vp39KO) on the expression of late gene of PlxyGV in the reporter virus were studied. The results showed that high levels of luluciferase activity were detected in Sf9 cells co-transfected with vAc ~ (vppppp39KO), vPx ~ (ppxpppxppxppppppppx ~ (pxpppppppp6.9-1uc) and vPx ~ (pxpxPgran-luc) co-transxPg Luciferase activity of ran-luc co-transfected with vAc ~ (ie1KO) was only slightly higher than that of vPx ~ (pxPp6.9-luc) and vPx ~ (pxPgran-luc) co-transfected cells. Luciferase activity of vPx ~ (pxPp6.9-luc) co-transfected with vAc ~ (lef8KO) or vA ~ (cp35KO) was higher than that of vPx ~ (p39KO) co-transfected cells, respectively. The activity of luciferase in vPx ~ (pxPgran-luc) and vAc ~ (lef8KO) or vA ~ (cp35KO) co-transfected cells was 0.98% and 34.49% respectively, and that of luciferase in vAc ~ (vp39KO) co-transfected cells was 0.29% and 28.49% respectively. These results indicated that the expression of reporter gene controlled by PlxyGV late promoter in Sf9 cells was mainly dependent on AcMN ~ (vp39KO). In order to study the effect of AcMNPV genome on PlxyGV DNA replication, different PlxyGV bacmids were transfected into Sf9 or Hi5 cells alone or with mutants of AcMNPV. Viral DNA extracted from the transfected cells was treated with DpnI, and gp41 was used as the target through P The replication of PlxyGV DNA was detected by CR. The genome of PlxyGV could be replicated either in Sf9 or Hi5 cells; the three rescue factors of AcMNPV, ie1, p35 and gp64, could enhance the replication of PlxyGV genome; and the replication of PlxyGV genome could be further enhanced in the presence of vAc ~ (lef8KO) or vAC ~ (vp39KO).
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.13
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