H5N1亚型禽流感病毒NS1基因在昆虫细胞中的表达

将H5N1亚型禽流感病毒（AIV）NS1基因插入到杆状病毒转移载体pFastBac1中，获得重组转移载体pFastBac1- NS1。将pFastBac1- NS1转化到DH10Bac感受态细胞中，筛选到重组转座子rBacmid-NS1。在脂质体转染试剂介导下将rBacmid-NS1转染对数生长期的Sf9昆虫细胞获得重组杆状病毒rBV-NS1。rBV-NS1感染Sf9细胞后，通过SDS-PAGE、Western blot和ELISA分析表明：获得了分子量为26ku的特异性NS1蛋白；并且该蛋白可与H5N1 AIV攻毒鸭的血清发生特异性免疫反应，而不能与H5N1AIV灭活疫苗免疫鸭的血清发生反应。试验结果表明：NS1在Sf9昆虫细胞中获得了高效表达，具有与天然蛋白相似的免疫活性，并可以作为区分免疫及自然感染个体的鉴别诊断抗原。本实验为建立禽流感病毒自然感染家禽与禽流感灭活苗免疫家禽的鉴别诊断方法奠定基础。

The NS1 gene of H5N1 Avian Influenza Virus(AIV) Expressed in Insect Cells

The NS1 gene of H5N1 subtype of highly pathogenic influenza virus was subcloned into transfer plasmid pFastBac1, then the recombinant transfer plasmid pFastBac1- NS1 was transformed into DH10Bac competent cells. The recombinant transposition rBacmid- NS1 was obstained by screening of white plaque and was identified by PCR. After the rBacmid- NS1 transfected into Sf 9 cells, the recombinant baculovirus rBV- NS1 was harvested. The expressed NS1 protein was analyzed by SDS-PAGE and Western blot. The specific protein band of 26kDa was obtained, and it can react with the sera of infected duck but can not react with that of immunized duck. So it was suggested that the NS1 protein can take as diagnosis antigen to differentiate the infected birds from immunized ones.