共表达H_9亚型禽流行性感冒病毒血凝素基因和鸡Ⅱ型干扰素基因的重组鸡痘病毒及其免疫效力

Recombinant Fowlpox Virus Coexpressing HA from Subtype H_9N_2 of Avian Influenza Virus and Chicken Type Ⅱ Interferon and Its Protective Efficacy Against Homologous Challenge in Chickens

为了构建更为安全有效的抗低致病性H9亚型禽流行性感冒 (流感 )的基因工程疫苗 ,将包含有H9亚型禽流感病毒分离株的血凝素 (HA)基因、鸡Ⅱ型干扰素 (IFN Ⅱ )全长cDNA序列及调控其转录的鸡痘病毒早晚期启动子 (PE/L)的基因片段 ,定向插入鸡痘病毒转移载体 1175的痘苗病毒启动子P7.5的下游 ,得到HA和IFN Ⅱ基因分别处于P7.5及PE/L转录调控下的重组转移载体 1175HAIFN。以脂质体转染法将 1175HAIFN转染至已感染鸡痘病毒 2 82E4疫苗株 (wt FPV)的鸡胚成纤维细胞 (CEF)中。 1175HAIFN与wt FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV HA IFN Ⅱ。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rF PV HA IFN Ⅱ。以间接免疫荧光法和细胞病变抑制试验证实 ,纯化的rFPV HA IFN Ⅱ感染的CEF能以非融合的方式同时表达HA和具有抗VSV活性的鸡IFN Ⅱ。动物试验表明 ,rFPV HA IFN Ⅱ能显著抑制静脉攻毒后 1日龄SPF鸡及含抗FPV母源抗体的商品鸡从泄殖腔排毒 ,且能减轻单表达HA的重组鸡痘病毒 (rFPV HA)抑制 1日龄SPF雏鸡增重的副作用。

The hemagglutinin (HA) gene from subtype H 9N 2 avian influenza virus and the chicken type Ⅱ interferon (IFN-Ⅱ) gene were directionally inserted into transferring vector 1175,resulting in recombinant transferring vector 1175HAIFN.Then the recombinant transferring vector 1175HAIFN was used to transfect the chicken embryo fibroblasts (CEF),which was pre-infected with wild type fowlpox virus,to generate recombinant fowlpox virus coexpressing HA and IFN-Ⅱ(rFPV-HA-IFN-Ⅱ).By selection of blue plaques on the CEF,overlaid with agar containing X-gal,rFPV-HA-IFN-Ⅱ was obtained and purified.The in vitro expression of HA by rFPV-HA-IFN-Ⅱ was detected in the recombinant virus-infected CEF with indirect immunofluorescence and expression of IFN-Ⅱ was confirmed by detection of the suppressive effect of IFN-Ⅱ on the VSV-induced cytopathic effect.Experiments on chickens demonstrated that rFPV-HA-IFN-Ⅱ had the same protective efficacy to suppress SPF chickens or commercial broiler chickens from shedding challenged virus as inactivated vaccine and had the ability to reduce the adverse effect induced by vaccination stress of FPV.