家蚕核型多角体病毒ORF4的研究
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摘要
根据NCBI同源比对,发现BmNPV ORF4(Bm4)与AcMNPV ORF11同源性最高,达96%,两者长度皆为1020bp。在29种已测序的杆状病毒中,还有另外三种杆状病毒中有Bm4的同系物,这三种病毒分别是Plutella xylostella multiplenucleopolyhedrovirus,Rachplusia ou multiple nucleopolyhedrovirus和Maruca vitrataMNPV。在Bm4的启动子区有早期启动子基序。然而,目前Bm4的功能完全未知。
为了研究Bm4基因产物在病毒感染环境下在细胞中的定位,我们用融合到Bm4 3'端的EGFP作为报告基因,以BmNPV杆粒作为载体来表达Bm4-EGFP。用共聚焦扫描显微镜观察发现:荧光信号主要集中在细胞核内,然而在细胞膜上也有较弱的信号。
通过用SnaBI和BamHI酶切pFastBacl,切除多角体启动子,置换上iel启动子和Bm4启动子,我们成功构建了在iel启动子和Bm4启动子驱动下表达外源基因的重组病毒,为构建Bm4缺失病毒的拯救病毒奠定了基础。同时,用我们构建的pFastbac-4p-EGFP(EGFP在Bm4启动子驱动下的重组转座载体)转染家蚕细胞发现:在没有病毒因子作用下,EGFP在Bm4启动子的驱动下就能表达,说明Bm4是一个立即早期基因。
为了缺失Bm4,构建了Bm4转移载体,并将其与BmNPV杆粒共转染家蚕细胞。所得病毒连续感染家蚕3遍,之后,纯化病毒一次。然后提取病毒DNA,并将其转化进感受态DH10B细菌。然后将感受态DH10B细菌涂到含有卡那抗生素、X-gal和IPTG的平板上。挑取蓝色菌落,用Bm4引物做PCR鉴定。鉴定出一个含有Bm4缺失的杆粒的菌落。提取Bm4缺失的杆粒,并将其转染进家蚕细胞,转染后5天,发现细胞有感染症状。收集上清再感染另一瓶细胞,发现有感染症状,说明Bm4在家蚕细胞中不是病毒繁殖的必需基因。
为了排出第二位点突变的可能,我们利用Bac-to-Bac/BmNPV杆状病毒表达系统,以及多角体启动子被iel启动子和Bm4启动子替换的重组转座载体,构建了在Bm4缺失杆粒的多角体基因座位表达Bm4的拯救病毒。为研究Bm4基因产物的具体功能奠定了基础。
总之,根据我们的实验结果,可知Bm4是一个其蛋白产物主要定位于核内的立即早期、非必需基因。
Using BLAST in NCBI, we found that open reading frame 4 of BmNPV (Bm4) and open reading frame 11 of AcMNPV share 96% identity in nucleotide sequence, and both of them are 1020 bp in size. Among the 29 sequenced baculoviruses, they have their homologues in three other baculoviruses including Plutella xylostella multiple nucleopolyhedrovirus, Rachiplusia ou multiple nucleopolyhedrovirus and Maruca vitrata MNPV. In the promoter region of Bm4, there is an early promoter motif. However, the function of Bm4 is completely unknown so far.
In order to study the subcellular localization of Bm4 product in the context of BmNPV infection, we used EGFP which is fused to 3' terminus of Bm4 as the reporter gene, and used BmNPV bacmid to express Bm4-EGFP fusion protein. Using Confocal scanning microscope, we found that the green fluorescent signal was localized primarily in the nucleus of Bm cells, however, a weaker signal was also observed in the membrane.
Polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter by digesting pFastBac1 with SnaBI and BamHI. With the modified pFastBac1, recombinant viruses were constructed expressing foreign genes under the control of ie1 promoter or Bm4 promoter, laying the foundation for the rescue experiment of Bm4-disrupted virus. Meanwhile, by transfecting Bm cells with the constructed pFastbac-4p-EGFP in which EGFP was under the control of Bm4 promoter, we found that EGFP was expressed without the help of viral factors, showing that Bm4 was an immediate early gene.
To disrupt Bm4, Bm4 transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells, and then three passages of viruses in Bm cells was carried out, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. Blue colonies grown on plate containing kanamycin, X-gai and IPTG, were selected to be identified by PCR with Bm4 primers. A colony harboring only Bm4-disrupted bacmid DNA was identified, and then Bm4-disrupted bacmid DNA was extracted and transfected into Bm cells. 5 days post infection, cytopathic effects were observed. Supernatant was removed from transfected cells and added to a second group of freshly plated Bm cells, cytopathic effects were also observed, suggesting that Bm4 is not an essential gene for viral growth in cultured Bm cells.
To exclude the possibility of second mutations, we constructed rescue viruses that express Bm4 at the polyhedrin locus of Bm4-disrupted bacmid by using Bac-to-Bac/BmNPV baculovirus expression system and the modified pFastBac1 in which the polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter, paving the way for studying the specific function of Bm4.
Taken together, the results showed that Bm4 was a non-esseantial immediate early gene whose product was primarily localized in the nucleus.
为了研究Bm4基因产物在病毒感染环境下在细胞中的定位,我们用融合到Bm4 3'端的EGFP作为报告基因,以BmNPV杆粒作为载体来表达Bm4-EGFP。用共聚焦扫描显微镜观察发现:荧光信号主要集中在细胞核内,然而在细胞膜上也有较弱的信号。
通过用SnaBI和BamHI酶切pFastBacl,切除多角体启动子,置换上iel启动子和Bm4启动子,我们成功构建了在iel启动子和Bm4启动子驱动下表达外源基因的重组病毒,为构建Bm4缺失病毒的拯救病毒奠定了基础。同时,用我们构建的pFastbac-4p-EGFP(EGFP在Bm4启动子驱动下的重组转座载体)转染家蚕细胞发现:在没有病毒因子作用下,EGFP在Bm4启动子的驱动下就能表达,说明Bm4是一个立即早期基因。
为了缺失Bm4,构建了Bm4转移载体,并将其与BmNPV杆粒共转染家蚕细胞。所得病毒连续感染家蚕3遍,之后,纯化病毒一次。然后提取病毒DNA,并将其转化进感受态DH10B细菌。然后将感受态DH10B细菌涂到含有卡那抗生素、X-gal和IPTG的平板上。挑取蓝色菌落,用Bm4引物做PCR鉴定。鉴定出一个含有Bm4缺失的杆粒的菌落。提取Bm4缺失的杆粒,并将其转染进家蚕细胞,转染后5天,发现细胞有感染症状。收集上清再感染另一瓶细胞,发现有感染症状,说明Bm4在家蚕细胞中不是病毒繁殖的必需基因。
为了排出第二位点突变的可能,我们利用Bac-to-Bac/BmNPV杆状病毒表达系统,以及多角体启动子被iel启动子和Bm4启动子替换的重组转座载体,构建了在Bm4缺失杆粒的多角体基因座位表达Bm4的拯救病毒。为研究Bm4基因产物的具体功能奠定了基础。
总之,根据我们的实验结果,可知Bm4是一个其蛋白产物主要定位于核内的立即早期、非必需基因。
Using BLAST in NCBI, we found that open reading frame 4 of BmNPV (Bm4) and open reading frame 11 of AcMNPV share 96% identity in nucleotide sequence, and both of them are 1020 bp in size. Among the 29 sequenced baculoviruses, they have their homologues in three other baculoviruses including Plutella xylostella multiple nucleopolyhedrovirus, Rachiplusia ou multiple nucleopolyhedrovirus and Maruca vitrata MNPV. In the promoter region of Bm4, there is an early promoter motif. However, the function of Bm4 is completely unknown so far.
In order to study the subcellular localization of Bm4 product in the context of BmNPV infection, we used EGFP which is fused to 3' terminus of Bm4 as the reporter gene, and used BmNPV bacmid to express Bm4-EGFP fusion protein. Using Confocal scanning microscope, we found that the green fluorescent signal was localized primarily in the nucleus of Bm cells, however, a weaker signal was also observed in the membrane.
Polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter by digesting pFastBac1 with SnaBI and BamHI. With the modified pFastBac1, recombinant viruses were constructed expressing foreign genes under the control of ie1 promoter or Bm4 promoter, laying the foundation for the rescue experiment of Bm4-disrupted virus. Meanwhile, by transfecting Bm cells with the constructed pFastbac-4p-EGFP in which EGFP was under the control of Bm4 promoter, we found that EGFP was expressed without the help of viral factors, showing that Bm4 was an immediate early gene.
To disrupt Bm4, Bm4 transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells, and then three passages of viruses in Bm cells was carried out, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. Blue colonies grown on plate containing kanamycin, X-gai and IPTG, were selected to be identified by PCR with Bm4 primers. A colony harboring only Bm4-disrupted bacmid DNA was identified, and then Bm4-disrupted bacmid DNA was extracted and transfected into Bm cells. 5 days post infection, cytopathic effects were observed. Supernatant was removed from transfected cells and added to a second group of freshly plated Bm cells, cytopathic effects were also observed, suggesting that Bm4 is not an essential gene for viral growth in cultured Bm cells.
To exclude the possibility of second mutations, we constructed rescue viruses that express Bm4 at the polyhedrin locus of Bm4-disrupted bacmid by using Bac-to-Bac/BmNPV baculovirus expression system and the modified pFastBac1 in which the polyhedrin promoter was replaced with ie1 promoter or Bm4 promoter, paving the way for studying the specific function of Bm4.
Taken together, the results showed that Bm4 was a non-esseantial immediate early gene whose product was primarily localized in the nucleus.
引文
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[3]吕鸿声著.昆虫病毒分子生物学[M].1998,北京:中国农业科学出版社,149-150.
[4]Lung OY.,Alvarez MC and Blissard GW.Ac23,an Envelope Fusion Protein Homolog in the Baculovirus Autographa californica Multicapsid Nucleopolyhedrovirus,Is a Viral Pathogenicity Factor.J VIROL,2003,77(1):328-339.
[5]Belyavskyi M,Braunagel ShC and Summers MD.The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.P Natal Acad Sci USA,1998,95(19):11205-11210.
[6]Jehlel JA,Blissard GW,Bonning BC,et al.On the classification and nomenclature of baculoviruses:A proposal for revision.Arch Virol,2006,151(7):1257-1266.
[7]Stewart TM,Huijskens I,Willis LG,et al.The Autographa californica Multiple Nucleopolyhedrovirus ieO-iel Gene Complex Is Essential for Wild-Type Virus Replication,but either IE0 or IE1 Can Support Virus Growth.J VIROL,2005,79(8):4619-4629.
[8]Xi QY,Wang JW,Deng RQ,et al.Characterization of AcMNPV with a deletion of me53 gene.Virus Genes,2007,34(2):223-232.
[9]Krappa R and Knebel-Morsdorf D.Identification of the very early transcribed baculovirus gene PE-38.J Virol,1991,65(2):805-812.
[10]Kool M,Ahrens CH,Goldbach RW,et al.Identification of Genes Involved in DNA Replication of the Autographa californica Baculovirus.P Natl Acad Sci USA,1994,91(23):11212-11216.
[11]Rapp JC,Wilson JA and Miller LK.Nineteen Baculovirus Open Reading Frames,Including LEF-12,Support Late Gene Expression.J Virol,1998,72(12):10197-10206.
[12]Chaabihi H,Ogliastro MH,Martin M,et al.Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells.J Virol,1993,67(5):2664-2671.
[13]Oers MM,Flipsen JTM,Reusken ChBEM,et al.Functional domains of the pl0 protein of Autographa californica nuclear polyhedrosis virus.J Gen Virol,1993,74(4):563-574
[14]O'Reilly DR and Miller LK.A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase.Science,1989,245:1110-1112.
[15]Slack JM,Kuzio J and Faulkner P.Characterization of v-cath,a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.J Gen Virol 1995,76(5):1091-1098.
[16]Kamita SG,Nagasaka K,Chua JW,et al.A baculovirus-encoded protein tyrosine phosphatase gene induces enhanced locomotory activity in a lepidopteran host.P Natl Acad Sci USA,2005,102(7):2584-2589.
[17]Hershberger PA,LaCount DJ and Friesen PD.The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.J Virol,1994,68(6):3467-3477.
[18]Takramah D,Seiffert BM,Schaller S,et al.Baculovirus P35 interacts with a subunit of human RNA polymerase Ⅱ and can enhance promoter activity in human cells.J Gen Virol,2003,84(11):3011-3019.
[19]McLachlin JR and Miller LK.Identification and characterization of vlf-1,a baculovirus gene involved in very late gene expression.J Virol,1994,68(12):7746-7756.
[20]Vanarsdall AL,Okano K and Rohrmann GF.Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing.J Virol,2006,80(4):1724-1733.
[21]袁哲明,游兰韶.重组昆虫杆状病毒杀虫剂研究进展.湖南农业大学学报(自然科学版),2001.27(6):494-497。
[22]梁昌镛,胡志红,陈新文.杆状病毒作为基因治疗载体的研究进展.生物化学与生物物理进展,2002,29(6):860-862。
[23]Davies AH.Baculophage":a new tool for protein display.Biotechnology(N Y),1995,13(10):1046.
[24]Rahman MM and Gopinathan KP.Bombyx mori nucleopolyhedrovirus-based surface display system for recombinant proteins.J Gen Virol,2003,84(8):2023-2031.
[25]Ernst W,Grabherr R,Wegner D,et al.Baculovirus surface display:construction and screening of a eukaryotic epitope library.Nucleic Acids Res,1998,26(7):1718-1723.
[26]Luckow VA,Lee SC,Barry GF,et al.Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.J Virol,1993,67(8):4566-4579.
[27]Miao Y,Zhang Y,Nakagaki K,et al.Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.APPL MICROBIOL BLOT,2006,71(2):192-199.
[28]Parks RJ,Krell PJ,Derbyshire JB,et al.Studies of fowlpox virus recombination in the generation of recombinant vaccines.Virus Res,1994,32(3):283-297.
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