GⅡ.4型诺如病毒GZ121株P蛋白的表达及其结合HBGA受体特征  

Expression of norovirus G Ⅱ. 4 GZ121 strain P protein and analysis of its binding activity with HBGAs receptor

作　　者：张绪富[1] 吴娴波[2] 戴迎春[2] 

机构地区：[1]南方医科大学中医药学院, 广州510515 [2]南方医科大学公共卫生与热带医学学院流行病学系,广州510515

出　　处：《中华微生物学和免疫学杂志》2013年第4期270-275,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助（No.30700716,No.30901992）;高等学校博士学科点专项科研基金新教师类（20104433120015）;广东省医学科研基金（B2010174）志谢：美国辛辛那提儿童医院医学研究中心JiangXi教授、TanMing助理教授提供部分实验试剂和技术指导,特此感谢!

摘　　要：目的建立我国流行优势株GⅡ-4型诺如病毒（GZ121）P蛋白（包括P颗粒和P二聚体）的原核表达系统，并明确其结合HBGAs受体的能力和方式。方法从GⅡ．4型诺如病毒GZ121株基因组中克隆P区域基因片段，通过构建P区域氨基酸序列进化树，确定其基因簇。构建含铰链Hinge-P和不含铰链P-CDCRGDCFC的pGEX-4T-1原核表达质粒，将构建的原核表达载体导入大肠杆菌（Ecoli）BL21，用0．6mmol／LIPTG（isoProPyhhio-β-D-galaetoside）过夜诱导P颗粒（不含铰链）和P二聚体（含铰链）在B1221中表达。目的重组蛋白经溶血酶酶切和FPLC鉴定分析，用EIA法检测P颗粒和P二聚体与HBGAs的结合特征。结果GZl21株诺如病毒为GⅡ．4基因型2004簇（GⅡ．4／2004cluster）。SDS．PAGE分析确定P颗粒和P二聚体相对分子质量（Mr）约为36×10^3，大小与报道相符。重组P颗粒经FPLC证实，其在M，约为830×10^3处形成特异的P颗粒波峰，Westernblot技术证实了重组P蛋白的特异性。EIA分析结果表明，GZ121株P蛋白与A、B、O型唾液均能结合，与非分泌型唾液不结合，而P颗粒结合HBGAs受体能力是P二聚体的80～100倍，与96簇VA387P颗粒相比，其结合O型能力增强，而结合A型能力稍弱。结论我国优势流行株GII．4型诺如病毒P蛋白的表达成功及其与HBGAs受体结合模型的建立，为今后研究我国诺如病毒的流行进化规律及疫苗的开发研究奠定了实验基础。Objective To construct a prokaryotic expression system of norovirus （NoV） G Ⅱ -.4 strain P protein （ P particle and P dimer） and to explore its binding activity and patterns with HBGAs receptor. Methods P domain sequence of GZI21 NoV ORF2 gene was cloned and its phylogenic tree was constructed to/dentify the gene cluster. The pGEX-4T-1-based expression plasmids were constructed respectively by inserting P domain gene fragments with hinge and P-CDCRGDCFC without hinge, and then transformed into BL21 to express fusion proteins, which was induced with 0.6 mmol/L IPTG at 22℃ overnight. P proteins were purified by thrombin cutting and characterized by FPLC. The binding patterns of NoV P protein to HBGAs antigens were analyzed by EIA. Results P region gene of GZ121 belonged to genotype G I1.4/2004 cluster. SDS-PAGE analysis showed the relative molecular weight of P particle and P dimer was about 36×10^3, which was consistent with other reports. The peak appeared at 830×10^3 confirmed the formation of P particle by FPLC. The expression of P protein was further confirmed by Western blot. The EIA results showed that GZ121 P protein could bind to sa- liva of A-group, B-group and O-group secretors, but not to nonsecretor. The binding affinity of P particle was 80-100 times higher than that of P dimer. Compared with VA387 P particle, it showed stronger binding affinity to O-group, but weaker to A-group. Conclusion The NoV G I1-4 GZ121 P proteins including P particle and P dimer were successfully expressed and HBGAs receptor binding assays were established, This pave the way for further studies on the evolution dynamics of NoV G II. 4 strains and the development of NoV vaccines.

关 键 词：诺如病毒 原核表达系统 P颗粒 P二聚体 HBGAs受体 

分 类 号：R3[医药卫生—基础医学]