共表达禽流感病毒HA和NA基因重组禽痘病毒的遗传稳定性

将表达禽流感病毒H5HA及N1NA基因的重组禽痘病毒rFPV HA NA连续传代至 2 5代 ,取第 5、15及 2 5代重组病毒作为受检代次 ,进行外源基因的PCR扩增与测序 ,同时比较这 3个代次重组禽痘病毒的免疫效力。结果对各代次重组病毒DNA的模板进行PCR扩增 ,均能够获得HA和NA两个目的片段 ;经测序证明两个外源基因在细胞传代过程中没有发生氨基酸水平的改变。动物试验结果表明 ,该重组病毒的毒力在细胞传代过程中没有发生变化 ,接种试验鸡后在鸡体内能够检测到外源基因的存在 ,各免疫组在免疫 2周后的抗体效价平均为 6 5log2 ,完全抵抗了高致病力禽流感病毒的攻击。以上结果表明此重组病毒具有较好的遗传稳定性 ,经过 2 5代的传代后 ,所插入的外源基因及其表达产物的免疫原性未见变化 ,重组病毒的免疫效力相当稳定。

Genetic Stability of A Recombinant Fowlpox Virus Co-expressing H5 HA and N1 NA Gene of Avian Influenza Virus

A recombinant fowlpox virus (rFPV) containing H5 HA and N1 NA gene was serially passaged to passage 25 on specific-pathogen-free (SPF) chicken embryo fibroblast monolayer. The HA gene and NA gene from passage 5,15 and 25 were amplified by polymerase chain reaction (PCR) and sequenced. SPF chickens were vaccinated with the recombinant viruses of three passages. The results demonstrated that specific fragments of HA and NA were cloned, identified and sequenced from the three passages, respectively, and there were no change in the amino acid sequences. The virulence and immune efficacy of the recombinant virus was not changed with the passage of the virus. These three passages of recombinant virus could provide complete protection against challenge with homologous HPAIV strain and is genetically stable for at least 25 passages.