共表达H5和H9亚型禽流行性感冒病毒血凝素基因的重组禽痘病毒及其免疫效力

为了构建更为安全有效能同时抵抗高致病性H5亚型和低致病忡H9亚型禽流行性感冒(禽流感)病毒的基因工程疫苗，将H5和H9亚型禽流感病毒分离株的血凝素(HA)基因，分别由鸡痘病毒早晚期启动子PS和PE／L调控其转求，定向插入鸡痘病毒转移载体p11s中，获得H5A和H9A基因分别处于PS及PE／L启动子转录调控下的重组转移载体p11SH5H9。以FuGene^TM6转染法将p11SH5H9转染至已感染鸡痘病毒282E4疫苗株(wt-FPV)的鸡胚成纤维细胞(CEF)中。p11SH5H9与wt—FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV11SH5H9。通过在含X-gal的营养琼脂上连续挑选蓝色病毒蚀斑获得并纯化rFPV-11SH5H9。以间接免疫荧光法试验证实，纯化的rFPV-11SH5H9感染的CEF能同时表达H5A和H9A。初步的动物试验表明，用10^5PFU的rFPV-11SH5H9免疫无特定病原体(SPF)鸡，免疫后血凝抑制(HI)抗体监测阳性率均为100％(8／8)；该重组病毒能显著抑制H9亚型AIV滴鼻、点眼后7日龄SPF鸡从气管和泄殖腔排毒，同时也能抵抗H5亚型AIV肌肉注射后对7日龄SPF鸡致死性攻击，保护率均为100％，显示出一定的应用前景。

Recombinant Fowlpox Virus Coexpressing HA from Subtype H5N1 and Subtype H9N2 of Avain Influenza Virus and Its Protective Efficacy

The hemagglutinin(HA) genes from subtype H5N1 and H9N2 avain influenza viruses were directly inserted into transferring vector p11S, resulting in production of recombinant transferring vector p11SH5H9.Then the recombinant transferring vector p11SH5H9 was used to transfect the chicken embryo fibroblasts(CEF), which was pre-infected with wildtype fowlpox virus,to generate recombinant fowlpox virus coexpressing H5A and H9A (rFPV-11SH5H9). By selection of blue plaques on the CEF,overlaid with agar containing X-gal, rFPV-11SH5H9 was obtained and purified.The in vitro expression of HA by rFPV-11SH5H9 was detected in recombinant virus-infected CEF by indirect immunofluorescence assay.Experiments on chickens demonstrated that rFPV-11SH5H9 had the same protective efficacy to suppress SPF chickens from shedding challenged virus as that of subtype H9N2 inactivated vaccine and to protect SPF chickens against lethal challenge with homologous subtype H5N1 avain influenza virus. The results showed that the rFPV-11SH5H9 has a good applied foreground.