重组H5N1亚型禽流感病毒NA基因腺病毒的滴度测定及遗传稳定性研究

Virus Titration and Genetic Stability of Recombinant Adenovirus Expressing NA Gene of Strain H5N1 of Avian Influenza Virus

为了研究表达H5N1亚型禽流感病毒NA基因重组腺病毒pAd-N1的遗传稳定性及重组病毒的滴度,将重组腺病毒pAd-N1在293细胞上连续传代20次,取第5、10、15、20代的重组病毒,采用PCR方法扩增禽流感病毒NA基因,并进行基因序列分析;用绿色荧光蛋白做标记计算出20代次时重组病毒的滴度。结果显示:从各代重组病毒DNA中均扩增出了约1 400 bp的目的条带,序列分析结果表明,第5、10、15代重组病毒中的NA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2处发生了点突变(516位A→G,1 323位T→C),但其编码的氨基酸未发生变化,即蛋白抗原表位未发生变化;采用快速测定法计算出重组腺病毒的滴度为109.5pfu/mL。

To evaluate the genetic stability and determine the virus titer of recombinant adenovirus expressing NA gene of strain H5N1 avian influenza virus（pAd-N1）,pAd-N1 was serially passaged 20 times in 293 cells.The passage 5,10,15 and 20 were chose for identification by PCR amplification and DNA sequencing.The virus titer of passage 20 was determined by GFP rapid monitoring method.From all of these pAd-N1,NA gene can be amplified and no changes on the nucleotide sequences of passages 5,10,and 15 was found,except two points mutation happened in the NA gene of passages 20（516 A→G and 1323 T→C）.Point mutations did not cause amino acid changes.The titer of pAd-N1 in 293 cells determined by rapid monitoring method was 109.5pfu/mL.All the results showed that pAd-N1 has good genetic stability within 20 passages and the titer was relatively high.