表达H5N1亚型禽流感病毒HA基因重组腺病毒的遗传稳定性分析及滴度测定

研究表达H5N1亚型禽流感病毒HA基因重组腺病毒pAd-H5的遗传稳定性及重组病毒的滴度测定。将重组腺病毒pAd-H5在293细胞上连续传代20次，取第5、10、15和20代的重组病毒采用PCR 方法扩增禽流感病毒HA基因，并进行基因序列测定分析；用标记为GFP的快速测定法计算出20代次时重组病毒的滴度。从各代重组病毒DNA 中均扩增出了约1 700bp的目的条带，与HA基因片段长度一致，基因序列分析表明：第5 、10 、15 代重组病毒中的HA基因序列与原始转移载体序列完全一致，第20代重组病毒插入基因有1处发生了点突变(即HA基因417位A→G)，但其编码的氨基酸未发生变化(即同义突变)，表明表达的目的蛋白抗原表位未发生变化；计算出的重组病毒滴度为108.875pfu/0.1ml。重组腺病毒pAd-H5在293细胞上连续传代20次，具有良好的遗传稳定性，重组腺病毒的病毒滴度相对较高。

Genetic Stability and Titer Determination of Recombinant Adenovirus Expressing HA Gene of Strain H5N1 of Avian Influenza Virus

The focus is to evaluate the genetic stability and titer determination of the recombinant adenovirus (pAd-H5) expressing HA gene of H5N1 Avian Influenza virus (AIV). The recombinant adenovirus pAd-H5 had been serially passaged for 20 passages in 293 cell line. The passages of 5,10,15 and 20 were chosen to be identified by PCR and sequence; Using GFP rapid monitoring method to test the viral titer of pAd-H5, passage 20. Furthermore, the analysis supported that HA gene was amplified correctly, without any changes on the nucleotide in passages of 5, 10 and 15. While there was only one point mutation on HA gene (417 A→G) in passage 20, but this point mutation did not lead to amino acid changes (samesense mutation). The titer of pAd H5 in 293 cell line identified by GFP rapid monitoring method was 108.75pfu/0.1ml. All the results showed that the pAd-H5 has good genetic stability within 20 passages, and the titer was relativly high.