利用BmNPV-家蚕表达系统包装重组腺相关病毒2型(rAAV2)研究

Package of Recombinant Adeno-associated Virus Type 2 Based on BmNPV-silkworm Expression System

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.