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

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

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。

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.