表达H9亚型禽流感病毒HA基因重组鸡痘病毒疫苗的遗传稳定性

将表达H9亚型禽流感病毒HA基因重组鸡痘病毒疫苗连续传代至30代，取第10、20、30代重组病毒作为受检代次，进行外源基因片段的克隆与测序，以间接免疫荧光试验检验各代次的表达，并以第10、20、30代重组鸡痘病毒疫苗进行免疫保护效力试验。对各代次模板进行PCR反应，分别扩增出特异的1．7kb外源基因片段，酶切位点与原始代次相同；各代次外源基因序列与原始序列相比，仅有1个碱基发生变化，不涉及氨基酸的变化；免疫荧光试验显示各代次均有显著表达；各免疫组在免疫后第7、10、14、20天的HI抗体效价(log2)逐渐上升；攻毒后第5天各免疫组排毒率与对照组相比差异显著，各免疫组之间无显著差异。结果表明：此重组载体疫苗具有较好的遗传稳定性，经过30代的传代后外源插入基因及其表达未见变化，免疫保护效力亦与原代一致。

Genetic Stability of A Recombinant Fowlpox Virus with An Avian Influenza Virus H9 Hemaglutinin(HA) Gene Insert

A recombinant fowlpox virus vaccine containing H9 subtype avian influenza hemaglutinin(HA) gene was serially passaged to passage 30 on specific pathogen free(SPF) chicken embryo fibroblast monolayers.The HA genes from passage 10,20 and 30 were cloned by polymerase chain reaction(PCR) and sequenced.The expression of HA genes from passage 10,20 and 30 were identified by indirect immunofluorescence assay(IF).The three passages were administered to specific pathogen free(SPF) chickens at 5 days of age and challenged by parental strain of H9 subtype avian influenza virus three weeks later.Specific 1 7 kb fragments were cloned,identified and sequenced from the three passages respectively,and there was no change in the amino acid sequences.Specific fluorescence could be detected from plaques formed by each passage.Even though the mean hemagglutination inhibition antibody titer of each rFPV vaccinated group was lower than that of inactivated oil vaccine group before challenge,almost all three passage groups had more than 95% protection from virus shedding as oil vaccine group had at 5 days postchallenge while oil vaccine group had 88%,and there was no difference of protection among the different passage goups.These findings demonstrate that this recombinant fowlpox virus vaccine could provide enough protection against H9 subtype avian influenza virus and is genetically stable for at least 30 passages.