禽流感病毒KMI/99(H9N2)HA和NA基因的序列分析

研究分别以自行设计的一对简并引物和另一对普通引物，经RT－CR，一次性成功地扩增禽流感病毒A／Chicken/Guangxi/KMI/99(H9N2）HA全和基因cDNA和NA全长基因cDNA，并将它们克隆于pGEM-T Easy的T－T窗口，首次报道了该毒株的HA全基因和NA全基因核苷酸序列及推导的氨基酸序列，测序结果显示，该毒株的HA基因全长为1683bp，编码560个氨基酸。NA全长为1398bp，编码466个氨基酸残基。研究发现其HAI羧基端的分子特征是：R－S－S－R。从分子水平推论。A/Chicken/Guangxi/KMI/99(H9N2)属于非高致病力毒株。研究还发现NA蛋白于63－65位缺失了T、E、I三个氨基酸。

Cloning and Sequencing of HA and NA gene of A/Chicken/Guangxi/99(H9N2)

RT-PCR was employed to amplify the cDNA of HA gene of CK/GX/KMI/99 with a pair of degenerate primers and the cDNA were cloned into the T-T windows of plasmid pGEM-T Easy.The inserts were sequenced,at first time,and the results revealed that the HA genes of CK/GX/KMI/99 had a long complete open reading frame and composed of 1 683 nucleotides,coding for 560 amino acids.The amino acid sequences of the HA connecting peptide revealed that CK/GX/KMI/99 had R-X-X-R(X,not basic amino acid)at the proteolytic cleavage site.The molecular basis of the HA gene of the strain was compatible with not highly pathogenicity.The cDNA of complete NA of N2 of CK/GX/KMI/99 was amplified with RT-PCR,and also was cloned into the T-T windows of pGEM-T Easy.The DNA were sequenced at the first time and the results demonstrated that the complete NA gene of N2 subtype was composed of 1 398 nucleotides in CK/GX/KMI/99 encoding 466 amino acids and there were 9 nucleotides deletion in the NA gene.