汉坦病毒疫苗株Z10与Z37重组核蛋白NP的表达纯化及其特异结合基因组末端反向重复序列的功能研究

汉坦病毒是引起肾综合征出血热(HFRS)和汉坦病毒型肺炎综合征(HPS)的主要病原体.其基因组由三节段的单股负链RNA组成,即S、M与L基因片段.汉坦病毒基因组的一个重要特点是每个基因片段的两个末端都有一段长18个核苷酸的高度保守的反向重复序列,互补可形成双链发夹结构,并且这一特点为不同型病毒所共有.为了研究该基因组末端保守的反向重复序列的功能,首先构建了汉坦病毒中国疫苗株Z10(汉滩型)及Z37(汉城型)的核蛋白原核表达载体,并在大肠杆菌中高效表达.经NI-NAT亲和柱和HiPrep16/10 DEAE离子交换柱液相色谱(FPLC)二步提纯,获得高纯度的重组核蛋白,并分别以胰蛋白酶消化后,用Western blotting进行区分和鉴定.以T4 DNA激酶同位素标记一对人工合成互补的18个核苷酸反向重复序列,制备双链探针.然后将该探针与纯化的Z10、Z37株的核蛋白NP进行非变性凝胶电泳迁移改变实验(EMSA)后发现,重组的Z10、Z37株的核蛋白NP,在体外均可特异地结合其基因组末端反向重复序列形成的双链探针.该结果表明,汉坦病毒基因组末端的反向重复序列是核蛋白重要结合位点,这对理解汉坦病毒核蛋白功能以及病毒复制过程中病毒粒子的包装机制有重要的意义.

Expression, Purification and Characterization of Nucleocapsid Protein of Hantavirus Chinese Vaccine Strains Z10 and Z37:Nucleocapsid Protein Specifically Binds to the Inverted Repeat Sequences Conserved at Terminals of Hantavirus Genome in vitro

Hantavirus,the causative agent of human hemorrhagic fever with renal syndrome(HFRS),possesses a tripartite,single-stranded RNA genome,with segments designated as small(S),medium(M)and large(L)encoding the viral nucleocapsid protein,envelope glycoproteins(G1 and G2)and polymerase protein,respectively.A unique structural feature of the viral genome is having about 18-nucleotide highly conserved inverted repeat sequences presenting at the terminals of each gene segment in different types of Hantavirus.This inverted repeat sequence at the ends of each gene segment can form a special hairpin structure when it got complemented.However,the function of the hairpin structure has not been clearly known.In this study,we expressed in E.coli the nucleocapsid proteins(NP)of two Chinese Hantavirus vaccine strains Z10(Hantaan type)and Z37(Seoul type),isolated from a HFRS patient and Rattus norvegicus, respectively.Using a UNICORE~ (TM) FPLC system,the His-tagged recombinant nucleocapsid proteins were then purified with Ni-NAT affinity chromatography and DEAE ion exchange chromatography.The purified recombinant NPs were then examined and distinguished by trypsin incomplete digestion,followed by Western blotting using anti-His monoclonal antibody.Electrophoresis mobility shift assay(EMSA)showed that both NPs of Z10 and Z37 hantaviruses bound specifically to the double-stranded DNA probe possessing the 18-nucleotide inverted repeat sequences in vitro. These data suggest that the inverted repeat sequence at the ends of each gene segment could be an important target site for nucleocapsid protein,and therefore plays an important role during Hantavirus packaging to assemble the viral apparatus into a complete viral particle.