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禽流感病毒H5N1 NS1蛋白是一种非结构蛋白,在病毒感染过程中发挥着重要的作用。构建基因截短的重组蛋白,可为进一步研究NS1不同结构域与宿主蛋白间的相互作用奠定基础。在成功克隆禽流感病毒H5N1全长NS1基因并测序的基础上,将部分截短基因序列克隆到表达载体pET28a(+)上,构建基因截短的重组表达质粒pET28a-NS1-RBD和pET28a-NS1-ED,转化大肠埃希菌BL21(DE3),阳性重组质粒经IPTG诱导表达后进行SDS-PAGE检测,获得预期蛋白的表达,然后利用Ni-NTA树脂蛋白纯化系统对重组蛋白进行纯化,并通过Western Blotting进一步确认NS1及截短体蛋白的表达。结果表明,实验成功构建禽流感病毒H5N1亚型的NS1蛋白截短体,并在大肠埃希菌中高效表达,这为进一步研究NS1蛋白不同结构域与宿主蛋白的相互作用提供了实验材料,为深入研究NS1蛋白的生物学功能奠定了坚实基础。
Avian influenza virus H5N1 NS1 protein is a non-structural protein that plays an important role in viral infection. Construction of gene-truncated recombinant proteins may lay the foundation for further study of the interaction between different domains of NS1 and host proteins. Based on the successful cloning and sequencing of the full-length NS1 gene of H5N1 strain, a partial truncated gene sequence was cloned into the expression vector pET28a (+) to construct the truncated recombinant expression plasmid pET28a-NS1-RBD and pET28a-NS1 -ED was transformed into Escherichia coli BL21 (DE3). The positive recombinant plasmids were induced by IPTG and detected by SDS-PAGE to obtain the expected protein expression. Then, the recombinant protein was purified by Ni-NTA resin protein purification system and passed Western Blotting further confirmed the expression of NS1 and truncated protein. The results showed that the NS1 protein truncated H5N1 subtype of bird flu virus was successfully constructed and highly expressed in Escherichia coli, which provided experimental materials for further study on the interaction between different domains of NS1 protein and host protein. In-depth study of the biological function of NS1 protein laid a solid foundation.