H5N1亚型禽流感病毒NS1基因截短体的克隆与原核表达  

Cloning and Procaryotic Expression of the NS1 Gene Truncated Body of Avian Influenza Virus H5N1 Subtype

作　　者：郑方亮[1,2] 朱春玉[1,2] 商玲玲[1,2] 马杰良[1,2] 孙婷婷[1,2] 艾海新[1,2] 朱俊峰[1,2] 段艳婷[1,2] 朱应[1,2] 刘宏生[1,2] 

机构地区：[1]辽宁大学生命科学院辽宁省高校动物资源与疫病防治重点实验室,辽宁沈阳110036 [2]辽宁省生物大分子计算模拟与信息处理工程技术研究中心,辽宁沈阳110036

出　　处：《微生物学杂志》2012年第3期1-6,共6页Journal of Microbiology

基　　金：病毒学国家重点实验室开放研究基金(2010009);辽宁省教育厅重点实验室项目(2009S043);辽宁大学青年科研基金(LDQN-2011-06)

摘　　要：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。The NS1 protein of H5NI avian influenza virus （AIV） is the non-structure protein playing an important role during the viral infection process. The construction of the gene truncated recombinant protein could lay a founda- tion for further study on interaction between different domain of NS1 and host proteins. Based on successful cloning and sequencing of NSI full length gene of AIV HSN1, part of the truncated NS1 gene fragments were cloned into pro- karyotic expression vector pET28a （ ＋ ） , constructing recombinant expression vector pET28a-NSI-RBD and pET28a- NS1-ED, which were then transformed into E. colt BL21 （DE3） and the positive recombinant plasmid was verified with SDS-PAGE after expression induced by IPTG, thus gained the expected protein expression. The recombinant pro- teins were purified by Ni-NTA resin purification system. And further confirmed the expression of NS1 truncated body protein through Western Blotting. The results showed that the NS1 truncated body proteins of AIV were successfully constructed and highly effective expressed in E. colt. This provides experimental material for further study on the interaetion between host protein and different structural domain of NS1 protein, and laid a solid foundation for intensive study on the biological function of NSI protein.

关 键 词：禽流感病毒H5N1 NS1 截短体 原核表达 纯化 

分 类 号：Q93[生物学—微生物学]