禽流感病毒A/Chicken/Mudanjiang/0823/00(H9N2) 株NS1基因的克隆及序列分析

用PCR方法,成功扩增出国内分离株禽流感病毒H9N2型非结构蛋白(NS1)基因cDNA,并将该片段连接到pMD 18-T载体上,转化TGI感受态细胞,在含氨苄青霉素的LB平板上筛选阳性克隆,提取质粒,经限制性内切酶鉴定,对目的片段进行测序,结果获得了NS1基因全长,约700bp.该片段的核酸序列与已知A/Chick/HongKong/1074/99(H9N2)、A/Chick/HongKong/1073/99(H9N2)毒株序列进行同源性比较,同源性皆为94.37%,氨基酸序列同源性分别为93.91%和93.48%.

Cloning and sequence of NS1 gene of avian influenza virus A/CHICKEN/Mudanjiang/0823/00(H9N2)

The cDNA of NS1 gene of avian influenza virus (H9N2) was amplified by PCR. The fragment was inserted into the pMD18-T vector. Then the TG1 competent cells were transformed with the recombinant plasimid. Consequently, the recombinant plasimid was amplified and extracted from the transformants and digested by BamHI or XholI to test the inserting of DNA fragment. Sequencing the NS1 fragment of the positive clone showed that the full length NS1 gene (about 700bp) was cloned. When comparing with A/chick/Hongkong/1074/99(H9N2) and A/chick/Hongkong/1074/99(H9N2), there is homology of 93.43 % at nucleotide acid level and homology of 93.91 % and 93.48% respectively at deduced amio acid level.