表达H5亚型禽流感病毒HA基因和鸡IL-18基因重组禽痘病毒的构建  

Construction of recombinant fowlpox virus coexpressing HA from subtype H_5 of avain influenza virus and chicken interleukin-18

作　　者：陈红英[1] 黄青云[2] 崔保安[1] 李新生[1] 管倩[1] 

机构地区：[1]河南农业大学牧医工程学院 [2]华南农业大学兽医学院,广州510642

出　　处：《微生物学报》2008年第8期1025-1030,共6页Acta Microbiologica Sinica

基　　金：国家“十一五”科技支撑计划专项(2006BAD06A08)~~

摘　　要：【目的】获得共表达H5亚型AIVHA基因和鸡IL-18基因的重组禽痘病毒。【方法】将含痘病毒启动子LP2EP2的HA基因和鸡IL-18基因插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSYHA/IL-18。用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA和IL-18的重组禽痘病毒(rFPV-HA-IL-18)。在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组禽痘病毒又进行了多次蚀斑克隆。【结果】以重组禽痘病毒DNA为模板,利用HA基因和鸡IL-18基因引物进行PCR,分别扩增出1条约1.7kb带和1条0.6kb左右的带。以间接免疫荧光试验、T细胞转化试验和SPF雏鸡免疫接种证实重组禽痘病毒能表达HA和鸡IL-18,并初步证明鸡IL-18增强HA免疫作用。【结论】重组禽痘病毒能表达具有生物学活性的HA和鸡IL-18。[Objective] We developed recombinant fowlpox viruses （rFPV） coexpressing chicken IL-18 and H5 AIV HA. [Methods] Recombinant expression plasmid pSYHA/IL-18 was constructed by cloning chicken IL-18 into transfer plasmid containing HA gent and transfccted by lipofectamine on the chicken embryo fibroblasts cell （CEF） pre-infected with S-FPV-017. By selecting blue plaques on the CEF overlaid with agar containing X-gal, recombinants fowlpox virus rFPV-HA-IL-18 were obtained, and identified by PCR. [Results]The recombinant fowlpox viruses contained chicken IL-18 and HA gene and had stable genetic properties. The expression of HA was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against AIV. The expression of chicken IL-18 was detected by MTT in the recombinant virus-infected CEF fluid. The chickens vaccinated with recombinant fowlpox virus rFPV-HA-IL-18 and rFPV-HA had detectable hemagglutination inhibition （HI） antibody at 7 days post-vaccination, and HI antibody titers rose to peak at 14 days post-vaccination. No HI antibody was detected in the control or fowlpox virus immunized chickens before or after immunization. The chickens vaccinated with rFPV-HA-IL-18 had higher HI antibody titers than the chickens vaccinated with rFPV-HA. [Conclusion] Development of recombinant fowlpox virus （rFPV- HA-IL-18） had strong biological activity.

关 键 词：H5亚型禽流感病毒 HA基因 鸡IL-18 重组禽痘病毒 

分 类 号：S852.5[农业科学—基础兽医学]