【摘要】 将H5亚型禽流感病毒(AIV)血凝素(HA)基因和鸡白介素(chiIL-2)基因分别插入到鸡痘病毒表达载体p12LS的启动子Ps和PE/L下游,使这两个基因反向串联,构建携带AIV HA基因和chiIL-2基因的重组鸡痘病毒转移载体质粒p12LSH5AIL2。将重组鸡痘病毒转移载体质粒转染至已感染野生型鸡痘病毒282E4株的鸡胚成纤维细胞(CEF)中,经蓝斑筛选获得纯化的重组鸡痘病毒rFPV-H5AIL2。间接免疫荧光试验表明重组鸡痘病毒能够表达H5亚型AIV HA;RT-PCR和间接免疫荧光试验证实重组鸡痘病毒可表达chiIL-2。SPF鸡和商品蛋鸡的免疫攻毒保护试验表明,经rFPV-H5AIL2免疫的试验鸡的抗体水平显著高于经rFPV-H5A免疫的试验鸡;在SPF鸡中rFPV-H5AIL2免疫组和rPFV-H5A免疫组的攻毒保护率和排毒率相当,但在商品鸡中rFPV-H5AIL2免疫组的攻毒保护率显著高于rFPV-H5A免疫组,而排毒率显著低于后者。本研究为研制新型禽流感重组鸡痘病毒疫苗奠定了基础。更多还原

【Abstract】 The hemagglutinin(HA) gene from H5N1 avian influenza virus and the chicken interleukin 2(chiIL-2) gene were inserted into a expressing vector p12LS to construct a recombinant transferring vector p12LSH5AIL2,in which HA gene under the control of the promoter Ps was in inverse tandem connection with the chiIL-2 gene under the control of the promoter PE/L.The p12LSH5AIL2 was then used to transfect the chicken embryo fibroblasts(CEF) pre-infected with a wild-type fowlpox virus 282E4 strain,to generate a recombinant fowlpox virus coexpressing the inserted HA and chiIL2 genes(rFPV-H5AIL2).The rFPV-H5AIL2 was obtained and purified by blue plaque screening on the CEF.The in vitro expression of HA gene by rFPV-H5AIL2 was detected in the recombinant fowlpox virus-infected CEFs with an indirect immunofluorescence assay,and the expression of the chiIL2 gene by rFPV-H5AIL2 was confirmed by detection of the chiIL2 mRNA by RT-PCR and by detection of chiIL2 by the indirect immunofluorescence assay.Experiments on SPF and commercial chickens demonstrated that the titer for HI antibodies induced by the rFPV-H5AIL2 was significantly higher than that by the rFPV-HA.The group immunized with the rFPV-H5AIL2 exhibited the similar ratios of protective efficacy and virus shedding as the group immunized with the rFPV-HA in SPF chicken.However,in commercial chicken,the group immunized with the rFPV-H5AIL2 generated significantly higher protection against H5N1 avian influenza virus challenge and lower virus shedding than the group immunized with the rFPV-HA.This study paved the way for further development of a new AIV recombinant vaccine.更多还原