小菜蛾顆粒體病毒晚期啟動(dòng)子在非受納細(xì)胞系中的表達(dá)活性分析
發(fā)布時(shí)間：2018-08-20 12:44
【摘要】：桿狀病毒是一類由囊膜包被桿狀核衣殼的、含單一雙鏈環(huán)狀超螺旋基因組的病毒。小菜蛾顆粒體病毒(Plutella xylo5tellagranulovirus,PlxyGV)屬于桿狀病毒中的β-桿狀病毒屬。由于缺少合適的受納細(xì)胞系,有關(guān)PlxyGV的分子生物學(xué)研究進(jìn)展一直較為緩慢。本文通過小菜蛾顆粒體病毒的晚期啟動(dòng)子在非受納細(xì)胞系中的表達(dá)活性分析,進(jìn)一步研究制約PlxyGV離體復(fù)制的原因。(1)通過基因克隆,構(gòu)建由PlxyGV的晚期啟動(dòng)子(pxPan、pxPe18、pxPe25、pxPgp41、pxPp6.9、pxPvp39、pxPpk1、pxPgran、pxPorf21 與 pxPorf50)分別控制報(bào)告基因luciferase的10個(gè)報(bào)告質(zhì)粒。通過瞬時(shí)表達(dá)實(shí)驗(yàn)我們發(fā)現(xiàn),PlxyGV基因組不能在被轉(zhuǎn)染的Sf9細(xì)胞中啟動(dòng)晚期與極晚期基因表達(dá)。同時(shí)我們也注意到,PlxyGV的晚期啟動(dòng)子報(bào)告質(zhì)粒與AcMNPV共轉(zhuǎn)染時(shí)均產(chǎn)生較高水平的熒光素酶活性。(2)通過基因克隆,構(gòu)建與PlxyGV報(bào)告質(zhì)粒對(duì)應(yīng)的由AcMNPV的晚期啟動(dòng)子(acPan、acPe18、acPe25、acPgp41、acPp6.9、acPvp39、acPpk1、acPpolh、acPp10)分別控制報(bào)告基因luciferase的9個(gè)報(bào)告質(zhì)粒。通過瞬時(shí)表達(dá)實(shí)驗(yàn),我們發(fā)現(xiàn)PlxyGV晚期基因報(bào)告質(zhì)粒與AcMNPV DNA共轉(zhuǎn)染細(xì)胞中的熒光素酶活性與對(duì)應(yīng)AcMNPV晚期基因報(bào)告質(zhì)粒和AcMNPV DNA共轉(zhuǎn)染細(xì)胞中的熒光素酶活性接近甚至更高。(3)利用Bac-to-Bac系統(tǒng)將由PlxyGV和AcMNPV晚期啟動(dòng)子控制的luc基因分別轉(zhuǎn)座到AcMNPV bacmid bMON14272上構(gòu)建相應(yīng)的晚期基因報(bào)告病毒。通過轉(zhuǎn)染后感染和實(shí)時(shí)表達(dá)分析,我們發(fā)現(xiàn):在PlxyGV的非受納細(xì)胞系Sf9細(xì)胞中,插入AcMNPV基因組的PlxyGV晚期啟動(dòng)子都能夠被激活并驅(qū)動(dòng)報(bào)告基因表達(dá)。其中,pxPgp41(或 pxPgp41L)與 acPgp41、pxPvp39(或 pxPvp39L)與 acPvp39、pxPe18與acPe18(或acPe18L)、pxPorf50與acPp10四組中,雖然對(duì)應(yīng)啟動(dòng)子所含核心基元序列TAAG的數(shù)目可能相等,但它們的表達(dá)活性隨時(shí)間變化的趨勢(shì)差異顯著。在pxPan與acPan、pxPpk1與acPpk1兩組中,對(duì)應(yīng)啟動(dòng)子在報(bào)告病毒感染細(xì)胞中的表達(dá)活性高低有明顯差異,但它們隨時(shí)間變化的趨勢(shì)卻基本相同。而acPp6.9與pxPp6.9、acPe25 與 pxPe25、acPpolh 與 pxPgran、pxPorf21 與 acPp10 四組中對(duì)應(yīng)啟動(dòng)子在重組病毒感染的細(xì)胞中的表達(dá)活性隨時(shí)間的變化基本相同。(4)通過λ-red同源重組的方法從AcMNPV基因組中分別敲除ie1、p35、lef8或vp39基因構(gòu)成重組病毒vAci~(leIKO)、vA~(cp35KO)、vAc~(lef8KO)和vAc~(vp39KO)。同時(shí),利用Bac-To-Bac系統(tǒng)將含pF-~(pxPp6.9-luc)與pF-~(pxPgran-luc)的報(bào)告質(zhì)粒分別轉(zhuǎn)座到PlxyGV bacmid上,構(gòu)成報(bào)告病毒vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)。通過瞬時(shí)表達(dá)實(shí)驗(yàn),研究不同 AcMNPV 突變體(vAc~(ie1KO)、vA~(cp35KO)、vAc~(lef8KO)、vA~(vp39KO))對(duì)報(bào)告病毒中PlxyGV的晚期基因表達(dá)的影響。結(jié)果表明:在報(bào)告病毒vPx~(pxPp6.9-1uc)和vPx~(pxPgran-luc)分別與vAc~(vp39KO)共轉(zhuǎn)染的Sf9細(xì)胞中均可檢測(cè)到高水平的熒光素酶活性;在vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)分別與vAc~(ie1KO)共轉(zhuǎn)染的細(xì)胞中熒光素酶活性僅略高于vPx~(pxPp6.9-luc)和vPx~(pxPgran-luc)單獨(dú)轉(zhuǎn)染細(xì)胞的熒光素酶活性。vPx~(pxPp6.9-luc)與vAc~(lef8KO)或vA~(cp35KO)共轉(zhuǎn)染細(xì)胞中的熒光素酶活性分別是其與vAc~(vp39KO)共轉(zhuǎn)染細(xì)胞熒光素酶活性的0.98%和34.49%;而vPx~(pxPgran-luc)與 vAc~(lef8KO)或vA~(cp35KO)共轉(zhuǎn)染細(xì)胞的熒光素酶活性分別其與vAc~(vp39KO)共轉(zhuǎn)染細(xì)胞熒光素酶活性的0.29%和28.49%。這些結(jié)果說明:在被轉(zhuǎn)染Sf9細(xì)胞中,由PlxyGV晚期啟動(dòng)子控制的報(bào)告基因的表達(dá)主要依賴于AcMNPV編碼的RNA聚合酶;PlxyGV自身的晚期表達(dá)因子可能沒有有效表達(dá)。(5)為研究AcMNPV基因組對(duì)PlxyGV DNA復(fù)制的影響,用不同的PlxyGV bacmids單獨(dú)或與AcMNPV的突變體共轉(zhuǎn)染Sf9或Hi5細(xì)胞。從被轉(zhuǎn)染細(xì)胞中提取的病毒DNA經(jīng)DpnI處理后,以gp41為標(biāo)的,通過PCR檢測(cè)復(fù)制的PlxyGV DNA。無論是在Sf9細(xì)胞中還是在Hi5細(xì)胞中,PlxyGV的基因組均可以進(jìn)行復(fù)制;AcMNPV的三種拯救因子ie1、p35和gp64可以增強(qiáng)其基因組的復(fù)制;在vAc~(lef8KO)或vAC~(vp39KO)存在的情況下,PlxyGV基因組的復(fù)制可進(jìn)一步得到加強(qiáng)。
[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).
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：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).
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S476.13
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