高效表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及免疫效力试验

将禽流感病毒血凝素 H9A基因克隆入插入载体 p FG11S中 ,通过酶切鉴定获得了正向转移载体 p FG11SHA;将其与禽痘病毒疫苗株 (w FPV)共转染鸡成纤维细胞 (CEF) ,通过蓝白斑筛选纯化得到重组病毒 r FPV- Ps- HA;以间接免疫荧光法证实 HA基因得到了表达。将该病毒经颈部皮下免疫 1日龄 SPF鸡 ,免疫后 15 d以 H9亚型禽流感病毒 F株翅静脉攻毒 ,攻毒后第 5天采集泄殖腔棉拭子样品进行病毒分离。将此重组病毒与以痘苗病毒 P7.5启动子表达相同基因的重组病毒 r FPV- P7.5 - HA作比较 ,结果表明 ,r FPV- Ps- HA相对于 r FPV- P7.5 - HA明显抑制了病毒的排出 ;攻毒后第 2、5、7、9、11天分别对 r FPV- Ps- HA、油乳剂灭活苗免疫鸡进行泄殖腔、气管排毒规律的检测 ,发现疫苗组均能很好地抑制排毒 ,攻毒对照组泄殖腔的排毒率明显高于气管排毒率

Recombinant Fowlpox Virus Highly Expressing HA from Subtype H9N2 of Avian Influenza Virus and Its Protective Efficacies Against Homologous Challenge in Chickens

A stable recombinant fowl poxvirus(rFPV-Ps-HA) highly expressing the hemagglutinin protein of an avian influenza virus A/chicken/China/F/1998(H9N2) was constructed by inserting the coding sequence within the non-essential-region of fowlpox virus(FPV) by homologous recombination.The HA protein was expressed under control of a synthetic promoter Ps in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay,which is 3.5 times stronger than the P7.5 promoter of vaccina virus.To examine the protective efficacy of rFPV-Ps-HA,3 groups of 1-day-old SPF chickens were vaccinated with rFPV-Ps-HA,rFPV-P7.5-HA and inactived vaccine in oil emulsion by subcutaneous route into the neck skin at dosage of 10~5 PFU.All groups were challenged with 10~7 ELD50 AIV(H9N2 F strain) 16 days postvaccination.Cloacal swabs were taken from all chickens 5 days postchallenged for isolating challenged AIV.It is better for rFPV-Ps-HA to inhabit virus shedding than rFPV-P7.5-HA cloachal swabs and tracheal swabs were taken from all chickens 2/5/8/9/11 days postchallenged still for isolating challenged AIV.All the vaccined groups were found to inhibit the virus shedding;The rate of virus shedding is greater from cloaca than from tracheal.