人类乳头瘤病毒HPV58亚型L1重组蛋白的原核表达与鉴定

发布时间：2018-03-01 10:27

  本文关键词： 人类乳头瘤病毒 L1重组蛋白 可溶性表达 纯化 鉴定　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：宫颈癌是常见的妇科恶性肿瘤之一，，全球每年约有50万新增病例，发病率在女性恶性肿瘤中居第二位，仅次于乳腺癌。人类乳头瘤病毒HPV（Human Pa-pillomavirus，HPV）属乳头瘤病毒科乳头瘤病毒属，为无包膜DNA病毒，主要引起人类皮肤粘膜的增生性病变。大量分子流行病学和分子生物学研究已经证明，高危型人类乳头瘤病毒诱发宫颈癌，HPV58型在我国宫颈癌妇女中是继HPV16和18之后的第三重要型别，由于HPV具有严格的种属特异性且其增殖严格依赖宿主细胞的分化状态，使得其难以在体外培养，使得传统的灭活及减毒疫苗难以进展，所以研究的重点走向了基因工程疫苗，HPV L1蛋白形成的病毒杨颗粒VLP（virus-like particle）是HPV疫苗的主要形式,在HPV VLP疫苗的研制中，如何以简便高效的制备结构完整、免疫原性强的VLP是所有问题的关键。 本研究利用重组表达质粒pGex-6p-1-HPV58-L1转入大肠杆菌E.coli BL21表达HPV58L1蛋白,通过对诱导剂IPTG的浓度、诱导时间以及诱导温度等条件的优化，筛选出最佳条件；由于HPV58L1蛋白的空间结构复杂性等因素，使表达的目的蛋白易形成包涵体，我们通过包涵体复性的办法，使目的蛋白的生物活性得以恢复，同时对目的蛋白的可溶性表达进行了探索，转入伴侣分子，同时通过不同温度的诱导，得到的最佳条件是15℃低温诱导表达的重组蛋白可溶性最好，并进行过柱纯化目的蛋白，western blotting鉴定，结果显示表达的HPV58L1蛋白能HPV16亚型的单克隆抗体发生特异性反应，进行HPV58VLP的生物活性分析，为HPV新型疫苗的研制奠定一定得基础。
[Abstract]:Cervical cancer is one of the most common gynecological malignancies. There are about 500,000 new cases in the world every year. The incidence of cervical cancer is the second most common in female malignant tumors after breast cancer. Human papillomavirus (HPV(Human) belongs to papillomavirus family. It is a non-encapsulated DNA virus that mainly causes proliferative lesions of human skin and mucous membrane. A large number of molecular epidemiology and molecular biology studies have proved that, High risk human papillomavirus induced cervical carcinoma type HPV58 is the third most important type after HPV16 and 18 in cervical cancer women in China. Because HPV has strict species-specificity and its proliferation is strictly dependent on the differentiation of host cells. This makes it difficult to be cultured in vitro and makes it difficult for traditional inactivated and attenuated vaccines to progress. Therefore, the focus of the research is towards VLP(virus-like particle of virus poplar formed by HPVL1 protein, which is the main form of HPV vaccine. In the development of HPV VLP vaccine, how to prepare VLP with simple and efficient structure and strong immunogenicity is the key to all problems. In this study, the recombinant expression plasmid pGex-6p-1-HPV58-L1 was transferred into E. coli BL21 to express HPV58L1 protein. The optimal conditions were obtained by optimizing the concentration, induction time and induction temperature of the inducer IPTG. Because of the complexity of the spatial structure of HPV58L1 protein, the expressed target protein is easy to form inclusion body, so that the biological activity of the target protein can be restored by renaturation of inclusion body. At the same time, the soluble expression of the target protein was explored and transferred to chaperone molecule. At the same time, the best conditions were obtained by inducing the recombinant protein at 15 鈩

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