利用BmNPV-家蚕表达系统包装重组腺相关病毒2型(rAAV2)研究
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摘要
腺相关病毒(adeno-associated virus,AAV)是基因治疗中最常用的病毒载体之一,目前用于基因治疗的AAV多利用苜蓿银纹夜蛾核型多角体病毒表达系统(AcMNPV-sf9)包装,但较高的包装成本限制了AAV在基因治疗中的广泛应用。家蚕杆状病毒表达系统与AcMNPV-sf9系统相比,具有包装量更高、成本更低的优势,因此更适用于包装重组腺相关病毒(recombinant adeno-associated virus,rAAV)。首先,将AAV2功能基因cap和rep进行序列优化后合成,克隆到杆状病毒转移载体pVL1393上,将增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因和荧光素酶(luciferase,Luc)基因分别作为报告基因克隆到含有巨噬细胞病毒IE(cytomegalovirus-IE,CMV-IE)启动子的病毒转移载体pVL1393-ITRs-MCS上。随后,将构建好的转移载体分别与缺陷型家蚕杆状病毒re Bm Bac共转染Bm N细胞系,获得分别重组有cap、rep和报告基因的家蚕杆状病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)。再将纯化的重组病毒(re Bm-Cap2、re Bm-Rep2)与re Bm-EGFP、re Bm-Luc分别混合后感染家蚕,收获其表达产物,纯化得到含有目的基因的rAAV病毒。利用rAAV病毒感染哺乳动物细胞后,通过检测EGFP、Luc的表达状态来验证rAAV包装成功与否。结果显示,利用家蚕杆状病毒系统成功包装了rAAV2,并且在哺乳动物细胞中实现了报告基因的表达。
Adeno-associated virus( AAV) is the most commonly employed viral vector for gene therapy. The AAV currently used in gene therapy is mainly packaged with Autographa californica nucleocapsid polyhedrosis virus-Spodoptera frugiperda cell 9( Ac MNPV-sf9) system,however,the high packaging cost in Ac MNPV-sf9 system limits its widespread application. Due to the lower cost and higher packaging quantity of silkworm baculovirus expression system,in present study,silkworm baculovirus was employed to package recombinant AAV( rAAV). The AAV2 genes( cap and rep) were optimized and then cloned into baculovirus transfer vector p VL1393 separately. Utilizing enhanced green fluorescent protein( EGFP) gene and luciferase( Lus)gene as report genes,and cloned them into viral transfer vector p VL1393-ITRs-MCS,which contained cytomegalovirus-IE promoter. Then co-transfected them into Bm N cell line with the replication-inducible Bm Bacmid virus DNA,and harvested recombinant Bm NPV particles containing cap,rep and report genes. Afterwards,the purified recombinant viruses( re Bm-Cap2,re Bm-Rep2) were mixed with re Bm-EGFP and re Bm-Luc,respectively. Then silkworm larvae were infected by recombinant viruses,and collected their expression products to obtain and purify rAAV containing target genes. To determine successful packaging of rAAV2 in silkworm expression system,the expression of EGFP and Luc in mammalian cells was tested. The results revealed that rAAV2 was successfully packaged in silkworm baculovirus expression system,and the expression of the report geneswas achieved in mammalian cells.
Adeno-associated virus( AAV) is the most commonly employed viral vector for gene therapy. The AAV currently used in gene therapy is mainly packaged with Autographa californica nucleocapsid polyhedrosis virus-Spodoptera frugiperda cell 9( Ac MNPV-sf9) system,however,the high packaging cost in Ac MNPV-sf9 system limits its widespread application. Due to the lower cost and higher packaging quantity of silkworm baculovirus expression system,in present study,silkworm baculovirus was employed to package recombinant AAV( rAAV). The AAV2 genes( cap and rep) were optimized and then cloned into baculovirus transfer vector p VL1393 separately. Utilizing enhanced green fluorescent protein( EGFP) gene and luciferase( Lus)gene as report genes,and cloned them into viral transfer vector p VL1393-ITRs-MCS,which contained cytomegalovirus-IE promoter. Then co-transfected them into Bm N cell line with the replication-inducible Bm Bacmid virus DNA,and harvested recombinant Bm NPV particles containing cap,rep and report genes. Afterwards,the purified recombinant viruses( re Bm-Cap2,re Bm-Rep2) were mixed with re Bm-EGFP and re Bm-Luc,respectively. Then silkworm larvae were infected by recombinant viruses,and collected their expression products to obtain and purify rAAV containing target genes. To determine successful packaging of rAAV2 in silkworm expression system,the expression of EGFP and Luc in mammalian cells was tested. The results revealed that rAAV2 was successfully packaged in silkworm baculovirus expression system,and the expression of the report geneswas achieved in mammalian cells.
引文
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[16]Chen H.Intron splicing-mediated expression of AAV rep and cap genes and production of AAV vectors in insect cells[J].Mol.Ther.,2008,16(5):924-930.
[17]Vesuna F,Winnard J P,Raman V.Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase[J].Anal.Biochem.,2005,342(2):345-347.
[18]Liu X J,Li Y N,Hu X Y,et al..Gene delivery and gene expression in vertebrate using baculovirus Bombyx mori nucleopolyhedrovirus vector[J].Oncotarget,2017,8(62):106017-106025.
[19]King J A,Dubielzig R,Grimm D,et al..DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids[J].EMBO J.,2001,20(12):3282-3291.
[20]Weitzman M D,Linden M R.Adeno-associated virus:Methods and protocols[M].Berlin:Springer,2011,2-15.
[21]Usami A,Ishiyama S,Enomoto C,et al..Comparison of recombinant protein expression in a baculovirus system in insect cells(Sf9)and silkworm[J].J.Biochem.,2010,149(2):219-227.
[22]Galibert L,Merten O W,Jacob A.Baculovirus system for the expression of a gene therapy vector[P].FR:WO2013014400A1,2013-01-31.
[2]Kitts P A,Possee R D.A method for producing recombinant baculovirus expression vectors at high frequency[J].Biotechniques,1993,14(5):810-817.
[3]Tratschin J D,West M H P,Sandbank T,et al..A human parvovirus,adeno-associated virus as a eukaryotic vector:Transient expression and encapsidation of the prokaryotic gene for chloramphenicol acetyl transferase[J].Mol.Cell Biol.,1984,4:2072-2081.
[4]Bohenzky R A,Lefebvre R B,Berns K I.Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat[J].Virology,1988,166(2):316-327.
[5]Carter B.Adeno-associated virus and the development of adenoassociated virus vectors:A historical perspective[J].Mol.Ther.,2004,10(6):981-989.
[6]Booth M J,Mistry A,Li X,et al..Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids[J].Gene Ther.,2004,11(10):829-837.
[7]Chadeuf G,Salvetti A.Stable producer cell lines for adeno-associated virus(AAV)assembly[J].Cold Spring Harb.Protoc.,2010,doi:10.1101/pdb.prot5496.
[8]Urabe M,Ding C,Kotin R M.Insect cells as a factory to produce adeno-associated virus type 2 vectors[J].Hum.Gene Ther.,2002,13(16):1935-1943.
[9]Smith R H,Levy J R,Kotin R M.A simplified baculovirusAAV expression vector system coupled with one-step affinity purification yields high-titer r AAV stocks from insect cells[J].Mol.Ther.,2009,17(11):1888-1896.
[10]Yin X,Li Z,Li J,et al..Rabies virus nucleoprotein expressed in silkworm pupae at high-levels and evaluation of immune responses in mice[J].J.Biotechnol.,2013,163(3):333-338.
[11]张志芳,李轶女,易咏竹.表达多外源基因的昆虫生物反应器及其构建方法和应用[P].中国:CN102286534A,2011-12-21.
[12]Samulski R J,Chang L S,Shenk T.A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication[J].J.Virol.,1987,61(10):3096-3101.
[13]Wistuba A,Kern A,Weger S,et al..Subcellular compartmentalization of adeno-associated virus type 2 assembly[J].J.Virol.,1997,71(2):1341-1352.
[14]Summers M D,Smith G E.A manual of methods for baculovirus vectors and insect cell culture procedures[M].USA:Texas A&M University Press,1987.
[15]Qiu J,Pintel D J.Alternative polyadenylation of adeno-associated virus type 5 RNA within an internal intron is governed by the distance between the promoter and the intron and is inhibited by U1 small nuclear RNP binding to the intervening donor[J].J.Biol.Chem.,2004,279(15):14889-14898.
[16]Chen H.Intron splicing-mediated expression of AAV rep and cap genes and production of AAV vectors in insect cells[J].Mol.Ther.,2008,16(5):924-930.
[17]Vesuna F,Winnard J P,Raman V.Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase[J].Anal.Biochem.,2005,342(2):345-347.
[18]Liu X J,Li Y N,Hu X Y,et al..Gene delivery and gene expression in vertebrate using baculovirus Bombyx mori nucleopolyhedrovirus vector[J].Oncotarget,2017,8(62):106017-106025.
[19]King J A,Dubielzig R,Grimm D,et al..DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids[J].EMBO J.,2001,20(12):3282-3291.
[20]Weitzman M D,Linden M R.Adeno-associated virus:Methods and protocols[M].Berlin:Springer,2011,2-15.
[21]Usami A,Ishiyama S,Enomoto C,et al..Comparison of recombinant protein expression in a baculovirus system in insect cells(Sf9)and silkworm[J].J.Biochem.,2010,149(2):219-227.
[22]Galibert L,Merten O W,Jacob A.Baculovirus system for the expression of a gene therapy vector[P].FR:WO2013014400A1,2013-01-31.