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 H5 of avain influenza virus and chicken interleukin-18

摘要:

【目的】获得共表达H5亚型AIV HA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1.7 kb带和1条0.6 kb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。

Abstract:

[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 gene and transfected 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.