H5N1型禽流感病毒核蛋白C端缺失削弱核蛋白间的相互作用

目的：克隆H5N1型禽流感病毒的核蛋白（NP）基因，将其定向插入双分子荧光载体，在细胞水平验证NP-NP的相互作用，进而确定NP-NP相互作用的关键区域。方法：根据双分子荧光互补（BiFC）实验的载体和NP基因序列设计引物，将NP的结构基因定向克隆到荧光载体上，得到重组荧光载体pBiFC-YC155-NP、pBiFC-YN155-NP、pBiFC-YC173-NP和pB-iFC-YN173-NP，瞬时转染293FT细胞，研究NP-NP相互作用；进一步对NP的C端进行缺失突变，然后定向克隆到pBiFC-YN173载体，令其分别与pBiFC-YC155-NP共转染293FT细胞，确定NP的C端在NP-NP相互作用过程中的地位。结果：构建了NP基因的BiFC载体pBiFC-YC155-NP、pBiFC-YN155-NP、pBiFC-YC173-NP和pBiFC-YN173-NP；将pBiFC-YC155-NP和pBiFC-YN173-NP、pBiFC-YC173-NP和pBiFC-YN173-NP共转染293FT细胞后出现了荧光；缺失实验表明，NP的C端是NP在体内相互作用形成寡聚体所必需的。结论：验证了H5N1型禽流感病毒NP在体内的相互作用，并初步证明NP的C端是NP形成寡聚体的必需片段，为进一步研究NP的作用奠定了基础。

Carboxy-Terminal Deletion of H5N1 Avian Influenza Virus Nucleoprotein Decreased Their Interaction

Objective： To study the interaction between nucleoprotein （NP） of avian influenza virus H5N1 subtype at cellular level and determine the key domain of NP in NP-NP interaction. Methods： The gene encoding NP was cloned by PCR and then constructed into bimolecular fluorescence complemented vectors, and the recombinant vectors pBiFC- YC155-NP, pBiFC-YN155-NP, pBiFC-YC173-NP and pBiFC-YN173-NP were obtained. After transient transfection in 293FT cells, the interaction between NP was well learned through analysis of fluorescence. In order to investigate the role of C terminal of NP in that interaction, the C terminal of NP was deleted, followed by bimolecular fluorescence complementation（BiFC） analysis mentioned before. Results： pBiFC-YC155-NP, pBiFC-YN155-NP, pBiFC-YC173-NP, pBiFC-YN173-NP needed by BiFC system were successfully constructed, and strong fluorescence was detected after cotransfection of those vectors in 293FT. The C terminal deletion experiment indicated that it was required by the interaction and oligomer formation of NP. Conclusion： Our work testified the interaction of NP in vivo, and preliminarily proved that C terminal fragment is essential to the construction of NP complex, all of which laid a foundation for further research.