带TAP标签的流感病毒8质粒反向遗传包装系统的初步构建

目的构建带TAP标签流感病毒8质粒反向遗传包装系统，以期应用于流感病毒感染宿主细胞的蛋白分子机理的研究。方法两步RT—PCR方法扩增流感病毒8基因，人工合成TAP基因序列，并采用3次PCR的方法融合TAP基因和NP基因。将流感病毒8基因和NP—TAP基因克隆于反向遗传载体PIVVⅡ-1，阳性克隆用PCR和序列测定进行鉴定。结果经PCR和序列分析鉴定，筛选到了带TAP标签的流感病毒8质粒反向遗传阳性克隆。结论成功构建了带TAP标签的流感病毒8质粒反向遗传包装系统，为进一步研究流感病毒感染宿主细胞的蛋白分子机理奠定了基础。

Construction of 8-plasmid reverse genetics packaging system carrying tandem affinity purification tag for influenza virus

Objective To construct a 8 -plasmid reverse genetics packaging system carrying tandem affinity purification tag for influenza virus which can be used to further study protein molecular mechanism of influenza virus infection. Methods Full- length 8 gene of influenza virus were amplified by 2 step RT -PCR, using 3 step PCR method to construct the fusion gene （ NP - TAP） through a chimeric primer. The 8 gene and NP - TAP gene were cloned into reverse genetics vector PIVV Ⅱ - 1 by CaCl2 transforming method and the positive colony was testified by PCR and sequence analysis, respectively. Results Eight full length gene of influenza virus and NP - TAP were correctly inserted into reverse genetics vector PIVV Ⅱ - 1 by PCR and sequencing assay. Conclusion The 8 - plasmid reverse genetics packaging system carrying tandem affinity purification tag for influenza virus was successfully constructed.