Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes.
Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes.
复制标题
DOI：
10.1371/journal.pone.0067875
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Britton P
中科院分区：
文献类型：
--
作者：
Bentley K;Armesto M;Britton P
禽冠状病毒传染性支气管炎病毒（IBV）是引起家禽呼吸道传染性支气管炎的病原，常规免疫是控制传染性支气管炎的有效手段。为了探索IBV作为疫苗载体的潜在用途，利用反向遗传学系统来产生表达报告基因增强的绿色荧光蛋白（eGFP）或人源化海肾荧光素酶（hRluc）的感染性重组IBV（rIBV）。在用eGFP或hRluc替换基因5或基因间区域（IR），或用hRluc替换ORF 3a和3b后获得感染性rIBV。用IBV密码子优化版本的hRluc基因替换基因5也导致感染性rIBV的成功拯救。通过荧光显微镜或荧光素酶活性测定证实了报告基因表达，所有成功拯救的rIBV感染原代鸡肾（CK）细胞后。通过在CK细胞上连续传代分析rIBV的遗传稳定性。重组IBV的稳定性根据被替换的基因组区域而变化，其中报告基因在替换基因5后保持至至少第8代（P8），P7用于替换IR，P5用于替换ORF 3a和3b。当替换基因5时，hRluc基因的密码子优化导致基因组稳定性增加，与标准hRluc的P8相比，hRluc表达稳定至P10。以0.01的MOI重复传代表达hRluc的rIBV证明了稳定性的增加，其中hRluc表达在替换基因5后稳定至至少P12。这项研究表明，异源基因可以被纳入，并从一系列的IBV基因组位置表达和辅助基因5的替代提供了一个有前途的目标，实现IBV作为其他禽病原体的疫苗载体的潜力。
The avian coronavirus infectious bronchitis virus (IBV) is the causative agent of the respiratory disease infectious bronchitis of domestic fowl, and is controlled by routine vaccination. To explore the potential use of IBV as a vaccine vector a reverse genetics system was utilised to generate infectious recombinant IBVs (rIBVs) expressing the reporter genes enhanced green fluorescent protein (eGFP) or humanised Renilla luciferase (hRluc). Infectious rIBVs were obtained following the replacement of Gene 5 or the intergenic region (IR) with eGFP or hRluc, or the replacement of ORFs 3a and 3b with hRluc. The replacement of Gene 5 with an IBV codon-optimised version of the hRluc gene also resulted in successful rescue of infectious rIBV. Reporter gene expression was confirmed by fluorescence microscopy, or luciferase activity assays, for all successfully rescued rIBVs following infection of primary chick kidney (CK) cells. The genetic stability of rIBVs was analysed by serial passage on CK cells. Recombinant IBV stability varied depending on the genome region being replaced, with the reporter genes maintained up to at least passage 8 (P8) following replacement of Gene 5, P7 for replacement of the IR and P5 for replacement of ORFs 3a and 3b. Codon-optimisation of the hRluc gene, when replacing Gene 5, resulted in an increase in genome stability, with hRluc expression stable up to P10 compared to P8 for standard hRluc. Repeated passaging of rIBVs expressing hRluc at an MOI of 0.01 demonstrated an increase in stability, with hRluc expression stable up to at least P12 following the replacement of Gene 5. This study has demonstrated that heterologous genes can be incorporated into, and expressed from a range of IBV genome locations and that replacement of accessory Gene 5 offers a promising target for realising the potential of IBV as a vaccine vector for other avian pathogens.
登录
查看更多内容
影响因子：
3.7
作者：
Armesto M;Cavanagh D;Britton P
通讯作者：
Britton P
影响因子：
5.5
作者：
Cavanagh D;Casais R;Armesto M;Hodgson T;Izadkhasti S;Davies M;Lin F;Tarpey I;Britton P
通讯作者：
Britton P
影响因子：
5.4
作者：
Casais, R;Dove, B;Britton, P
通讯作者：
Britton, P
DOI：
10.1073/pnas.97.10.5516
发表时间：
2000-05-09
影响因子：
11.1
作者：
Almazán, F;González, JM;Enjuanes, L
通讯作者：
Enjuanes, L
DOI：
10.1073/pnas.0703584104
发表时间：
2007-06-05
影响因子：
11.1
作者：
DiNapoli, Joshua M.;Kotelkin, Alexander;Bukreyev, Alexander
通讯作者：
Bukreyev, Alexander