人乳头瘤病毒（HPV）功能化自组装

Genitotropic human papillomavirus (HPV) infections are considered the most common sexually transmitted infection in the Chinese. The major manifestations of anogenital HPV include genital warts (condyloma acuminatum) and anogenital intraepithelial neoplasia. The presence of HPV DNA has been reported in 99.7% of cervical carcinomas worldwide, indicating that HPV infection is a necessary cause of this cancer and that this disease can be prevented by prophylactic HPV vaccination. Approximately 35 of the 100 subtypes of HPV are specific for the anogenital epithelium and have varying potentials for malignant transformation. Of the 15 oncogenic genital HPV types, HPV16 is the most common, followed by HPV18 and HPV45 (contributing 50%, 20%, and 10%, respectively, of cervical cancer cases worldwide).Human papillomaviruses (HPVs), are small non-enveloped DNA viruses and consists of a 8 kb circular dsDNA genome packaged into a 60 nm icosahedral capsid. The capsid is comprised of 72 pentamers of the major capsid protein L1 and up to 72 molecules of the minor capsid protein L2, localized along the inner surface of the capsid, within the central cavities beneath the L1 pentamers. Biochemical and structural studies show that mature virions exist in an oxidized state with adjacent L1 pentamers crosslinked together by disulfide bonds, stabilizing the capsid structure. The L2 protein is critical for HPV16 infection and is a multifunctional protein, having roles in genome encapsidation, L1 interaction and capsid stabilization, and endosomal escape of virions. L2 protein from incoming particles has been shown to colocalize with viral genomes and nuclear promyelocytic leukemia protein (PML) bodies, and L2 may function to escort the viral genome into the host cell nucleus, perhaps through direct interactions with various nuclear import receptors. Because of its simple structure and the duality of complex functions, and can self-assemble into VLPs, chemists and biologists use HPV to understand virus propagation mechanisms and the important model to simulation of virus self-assembly. This application is based on the deeply understanding of the key elements of the single structural unit of the HPV virus protein self-assembly. And it is the first time to bring up the idea that design an artificial transformation of HPV structural unit of proteins L1, L2, and even the introduction of the fragments of other proteins, self-assemble, and form a new feature of the VLPs. The aim is to reveal the viral protein structure, from the nature of the internal relations of the self-assembly and its function of biological macromolecules, to open up a new path as the simulated assembly of simple organisms, also the theoretical basis for the effective prevention or uniform virus.

人乳头瘤病毒（HPV）是球形无囊膜双链DNA病毒，病毒衣壳由结构蛋白L1和L2组成。L1蛋白自身能形成五聚体（Pentamer），并由72个五聚体自组装（Self-assembly）成类病毒颗粒(Virus-like Particles,VLPs)。因其具有简单结构和复杂功能的两重性，以及可自组装成VLPs的特点，HPV成为当前化学家和生物学家了解、认识和模拟病毒繁衍机制及自组装体的重要模型。本申请在部分的理解HPV病毒蛋白质的单一结构单元自组装关键要素的基础上，首次提出人为地去设计、改造HPV自组装结构单元蛋白L1、L2，甚至引入其它蛋白质的片段，使之形成一种崭新的功能化的VLPs，并进行体外可控自组装，揭示其中相关分子机理、组装过程和动态机制。希望能从本质上揭示病毒蛋白结构、生物大分子自组装体与其功能的内在联系，为简单生物体的模拟组装开辟一条新路径，也为有效预防或制服病毒奠定理论基础。

人乳头瘤病毒（human papillomavirus，HPV）是乳多空瘤病毒科多瘤病毒亚科的一个无包膜的小型双链环状DNA病毒，它是乳头瘤病毒属中的成员之一。HPV主要侵染人体器官的表皮组织（如皮肤、口、咽喉、食道、生殖器等部位），可引起鳞状上皮增生，高致病亚型可引发恶性肿瘤，并可引起女性宫颈癌的发生。.L1蛋白自身能形成五聚体（Pentamer），并由72个五聚体自组装（Self-assembly）成类病毒颗粒(Virus-like Particles,VLPs)。而且L1体现的HPV的形貌特征免疫原性以及稳定性等的特征。通过利用融合表达载体pGEX-6p-1，进行GST融合蛋白的原核体系表达以分析L1蛋白突变体的结构。研究发现对466位残基精氨酸（Arg）突变为组氨酸（His）使得L1蛋白无法形成五聚体的高级结构，因此以此突变体为结构基元进行改造，此蛋白在N端含有GST标签，在C端含有His标签。并进行构建与表达、纯化。这样在HPV组装的过程中，使外源蛋白序列对组装的影响降低至最小。另外在体系中同时添加的His标签与Ni2+离子具有特异性结合的相互作用，从而可以引入Ni2+离子进行HPV的VLPs组装与调控，进行线性组装。结果可以发现，突变型HPV蛋白溶液中加入Ni2+后形成了直径为100~1000nm不等粒径大小的颗粒。二价Ni2+可以对突变型HPV蛋白的线性组装进行调控。