【作者】 吴庭鹤；

【导师】 魏萍；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2002， 硕士

【摘要】 副粘病毒科病毒的膜融合是由其表面糖蛋白，F蛋白介导的。新城疫病毒(NDV)感染宿主细胞，首先是引发病毒囊膜与细胞膜融合，随后导致病毒遗传物质释放入宿主细胞。深入研究病毒与细胞的融合机制有助于开发新型的抗病毒药物。现已证实，多数副粘病毒，包括其他囊膜病毒如HIV和RSV，F蛋白N-端和C-端各有一段七肽重复区，称为HR1和HR2。二者对病毒与细胞膜的融合过程起着重要作用。其他囊膜病毒的两个七肽重复序列可以形成一种异源三聚体结构，称为coiled-coil。它在病毒介导的融合过程中起关键作用。但仍不清楚NDV是否有与其它病毒相同的融合机制。 在本实验中，我们克隆、表达了融合形式的HR1LinkerHR2(HR1，2)蛋白，并将融合形式和除去融合蛋白的HR1，2纯化。化学交联实验和分子筛纯化结果表明，HR1，2可以形成同源三聚体结构。圆二色谱(CD)结果显示，HR1，2在208nm和222nm有明显的双负峰，具有明显的α螺旋结构特征，且其具有高度的热稳定性。这表明由HR1，2形成的同源三聚体具极稳定的。螺旋结构形式。NDV病毒也可能通过形成coiled coil形式介导融合。 NDV与其它囊膜病毒不同的是，在HR1和HR2序列之间间隔250个氨基酸。Ghosh等人已证实，HR1和HR2之间还有另外一个七肽重复序列区，称为HR3。单氨酸突变实验表明，HR3序列的单氨酸突变可以改变NDVF蛋白在引发合胞体形成实验中对HN蛋白的依赖性。为进一步研究HR3的结构和功能，我们克隆、表达了融合形式的HR1，3LinkerHR2(HR1，3，2)蛋白，并对His-tagged HR1，3，2进行了纯化。更多还原

【Abstract】 Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion protein. Infection by Newcastle Disease Virus (NDV) involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. Study of the molecular mechanism of membrane fusion is targeted for the development of new anti-viruses drugs. Two heptad repeat (HR) regions in the N-terminus and C-terminus of most paramyxoviruses F protein, includes of other enveloped viruses, such as HIV and respiratory syncytial virus (RSV), have been identified as important to fusion, named HRland HR2. The two heptad repeat regions can form coiled coil heterotrimer in other envelope viruses. The formation of coiled coil heterotrimer could play a key role in virus mediated fusion. But it is not clear if NDV has the same fusion mechanism as other envelope viruses. In this thesis, we have clone, expressed fusion form of HRlLinkerHR2 (HR.1,2) protein. And the fusion and free form of HR1,2 were purified. Chemical crosslinking assay and gel filtration confirmed that HR1,2 can form homeotrimer. The Circular Dichroism (CD) spectroscopy showed that HR1,2 has a -helix characteristic, exhibiting double minima at 208nm and 222nm, and has high thermal stability. All those indicated that the a -helix homeotrimer formed by HR1,2 of NDV F protein is the most stable form during fusion process and NDV mediate fusion with the same mechanism as other envelope viruses.Different to the other enveloped viruses, there are 250 amino acids between HR1 and HR2. Ghosh et al, have recently noted that NDV F protein sequences contain another heptad repeat region located between HR1 and HR2. Single point mutations assy showed that a single amino acid change in the HR3 sequences of NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation. To farther study the structure and function of HR3, we also have cloned, expressed the fusion form of HRl,3LinkerHR2 (HR1,3.2) protein. And, the fusion form of HR1,3,2 were purified.更多还原