HPV58型衣壳蛋白L1和L2基因的克隆表达及病毒样颗粒的装配与免疫学分析

发布时间：2017-12-31 21:00

本文关键词：HPV58型衣壳蛋白L1和L2基因的克隆表达及病毒样颗粒的装配与免疫学分析　出处：《吉林大学》2005年博士论文　论文类型：学位论文

【摘要】：高危型HPV 感染与宫颈癌的发病密切相关,其中16 和58 型是我国最常见的型别。为降低高危型HPV 感染的发生率和宫颈癌的发病率,制备预防HPV 感染的疫苗势在必行。由于HPV 难以通过体外组织培养或感染动物而进行繁殖,而且天然的HPV 感染病灶中获得的HPV 颗粒量很少,不足以用于疫苗制备。分子生物学研究发现HPV 结构蛋白的Ll 蛋白或L1 +L2 蛋白在真核细胞大量表达可以自发组装成病毒样颗粒(virus-like particles, VLP) 是目前公认的最具前景的HPV 疫苗。但这种VLP 的中和活性具备型特异性,即一种型的VLP接种后只能对同型HPV 的感染起预防作用,而对其他型不起作用,因此有必要制备多型VLP。目前尚无关于HPV58 的VLP 的详细报道,针对我国HPV58感染高发的特点,本实验对HPV58-VLP 进行了详细的研究。本实验首先应用基因重组技术构建了表达HPV58L1 和HPV58L1L2 共表达的重组杆状病毒载体,感染昆虫细胞后大量表达L1、L2 蛋白并进行纯化,形成由58L1单独构成的VLP和L1L2共同构成的VLP。用它们分别免疫小鼠,制备相应的抗血清。通过ELISA 和Western-blot 方法证明了58VLP 具备良好的免疫原性,由它制备的抗血清含有针对L1 的高滴度抗体。另外,应用HPV58L1L2-VLP 重新包装pSV/?-gal 质粒制备HPV58 假病毒,通过抗血清对假病毒感染COS-1 细胞的阻断实验证实了抗血清具备特异性中和活性。关键词:人乳头瘤病毒58型主要衣壳蛋白次要衣壳蛋白病毒样颗粒杆状病毒昆虫细胞表达系统
[Abstract]:High risk HPV infection is closely related to the incidence of cervical cancer, of which 16 and 58 are the most common types in China. In order to reduce the incidence of high-risk HPV infection and cervical cancer incidence. It is imperative to prepare a vaccine to prevent HPV infection. Because HPV is difficult to propagate through tissue culture in vitro or infected animals, and the quantity of HPV particles in the natural HPV infected lesions is very small. Molecular biology has found that the expression of Ll protein or L1 L2 protein of HPV structural protein in eukaryotic cells can spontaneously assemble into virus-like particles (. Virus-like particles. VLP is the most promising vaccine for HPV, but its neutralization activity is type-specific. That is, one type of VLP inoculation can only prevent the infection of the same type of HPV, but not the other type. Therefore, it is necessary to prepare polytype VLP. At present, there is no detailed report on the VLP of HPV58, aiming at the high incidence of HPV58 infection in China. In this experiment, HPV58-VLP is studied in detail. In this experiment, the recombinant baculovirus vector expressing HPV58L1 and HPV58L1L2 was constructed by gene recombination technique. After infecting insect cells, a large number of L1 was expressed. The L2 protein was purified to form a VLP composed of 58L1 alone and L1L2. Mice were immunized with them. The corresponding antiserum was prepared and the immunogenicity of 58VLP was proved by ELISA and Western-blot. The antiserum prepared by it contains a high titer antibody against L1. In addition, HPV58L1L2-VLP was used to repackage pSVP? HPV58 pseudovirus was prepared by -gal plasmid. The specific neutralization activity of the antiserum against pseudovirus-infected COS-1 cells was confirmed by the blocking test of the antiserum. Human papillomavirus 58 main capsid protein minor capsid protein virus like baculovirus insect cell expression system
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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