多型别HPV L2片段的融合表达及免疫效果研究

发布时间：2018-03-20 08:38

本文选题：人乳头瘤病毒　切入点：次要衣壳蛋白　出处：《中国疾病预防控制中心》2009年硕士论文　论文类型：学位论文

【摘要】：人乳头瘤病毒(HPV)感染可以诱发人体不同部位发生疣或乳头状的瘤样病变,是危害人类健康的重要病原体之一。根据其致癌性的不同,可分为低危型和高危型。其中高危型主要有HPV16、18、31、33、39、45、51、52、56、58、59、68、73、82共15个型别,高危型HPV的感染与食管癌、喉癌、舌癌的发生有关,与宫颈癌发生的关系尤为密切。宫颈癌是危及全球妇女健康的严重疾病,在世界范围内的发病率居女性恶性肿瘤的第二位。现行的宫颈粘膜涂片筛查可实现宫颈癌的早期发现,但是筛查的假阴性率高而且费用大难以普及。同时对于中晚期宫颈癌的治疗手术和化疗后的效果并不理想,复发率高,花费也大。因此在发展中国家研发一种HPV预防性疫苗是从根本上预防HPV感染,控制宫颈癌发病率的一种有效的方法。 HPV疫苗可分为预防性疫苗和治疗性疫苗。目前研究成功的是针对HPV主要衣壳蛋白L1的VLPs预防性疫苗,已经上市的HPV预防性疫苗有默克(Merk)公司的HPV预防性疫苗Gardasil,为HPV6、11、16和18型的VLP组成的四价疫苗和葛兰素史克(GSK)公司的HPV预防性疫苗Cervarix,为HPV16和18型的VLP组成的两价疫苗,在人群中的抗体阳转率达到100%,4.5年保护率为100%,但由于存在价格昂贵,缺乏交叉保护作用等缺点限制了其在全球的广泛使用。作为一种理想的HPV疫苗应是价格低廉、具有较好的交叉保护活性,覆盖尽可能多的HPV高危型别。上述内容已成为第二代HPV预防性疫苗的研究目标。L2是HPV的次要衣壳蛋白,对于衣壳的形成不是必要的,但已有的研究表明其N端可诱发产生具有交叉中和活性的抗体。本试验的主要研究目的就是以L2蛋白为靶抗原,探索研制一种低廉、广谱的HPV预防性疫苗。本论文的设计思路是：首先根据我国宫颈癌患者中HPV感染率的流行病学调查资料选择HPV16、HPV18和HPV58为研究型别,以L2可诱发产生交叉中和抗体的N端为靶抗原,在原核表达系统中表达HPV16、18、58不同长度的L2蛋白,分别筛选不同型别HPV L2中诱发中和抗体滴度高的片段,然后将入选的个型别片段融合,进行原核表达、纯化,检测其诱发的中和抗体及交叉中和抗体情况,为探讨研制一种价格低廉、广谱的HPV预防性疫苗提供实验依据。主要研究结果如下：第一、构建了分别表达HPV16、18和58型不同长度L2蛋白的十一种原核表达质粒。上述十一个质粒在IPTG诱导下均能表达相应的目的蛋白,并对这十一种蛋白western-blot鉴定正确后进行纯化,得到十一种纯化的蛋白,纯度在90%。左右；纯化蛋白免疫小鼠后血清ELISA结果显示,三种HPV型别不同长度的L2蛋白均能诱发产生高滴度的特异性抗体,均在1：100000以上；用包装的HPV16、18和58型假病毒颗粒,建立了HPV中和实验技术平台,用该方法检测各纯化蛋白免疫血清中和抗体,筛选出不同型别HPV最佳L2片段,它们分别是：HPV16L2(11-50aa), HPV18L2(11-200aa)和HPV58L2(11-150aa),针对同型假病毒的中和抗体滴度分别为1：640、1：1280和1：1280。第二、将筛选出来的不同型别HPV L2蛋白片段进行了两种方式的融合,分别为18-16-58和5816-18,构建了表达HPV16、18和58型L2融合蛋白的两种pET-9a原核表达质粒；上述两个个质粒在IPTG诱导下均能表达相应的目的蛋白,对上述蛋白进行western-blot鉴定正确并进行纯化,纯度在90%左右；体外中和实验结果显示,18-16-58L2融合蛋白诱发抗体对HPV16、18和58型假病毒的中和滴度分别为1：3200、1：3200和1：6400优于58-16-18L2融合蛋白诱发的中和抗体水平(提示蛋白间不同的组合方式,可能对其表位的暴露有影响),而且能够诱发产生针对HPV6、45和52型假病毒的交叉中和抗体,抗体滴度分别为1：640、1：1280和1：640；18-16-58L2融合蛋白与各型别单价蛋白相比,免疫原性加强,对异型假病毒的中和抗体水平有了明显的提高,具备广谱性。综上所述,本研究分别筛选出HPV16、18和58型的能够诱发较高中和活性的L2片段,并将筛选出的三种型别L2片段构建融合蛋白,对其中和活性进行了初步研究,对HPV广谱中和抗体疫苗进行了有意义的探索,为深入进行这类疫苗的研究提供了有价值的实验资料。
[Abstract]:Human papilloma virus (HPV) infection can induce tumor like lesions in different parts of human body wart or nipple, is one of the important pathogens of harm to human health. According to its carcinogenicity is different, can be divided into low-risk and high-risk types. Among high risk type HPV16,18,31,33,39,45,51,52,56,58,59,68,73,82 mainly consists of 15 types of high-risk HPV infection. With esophageal cancer, laryngeal cancer, tongue cancer occurrence, especially in close relationship with the occurrence of cervical cancer. Cervical cancer is a serious disease endangering the health of women worldwide, the incidence rate in the world within the scope of the malignant tumor in women second. Early detection of cervical mucosa smear screening for cervical cancer can be present, but false negative screening the high rate and high cost. At the same time difficult to spread for the treatment of surgery and chemotherapy in advanced cervical cancer after the effect is not ideal, the recurrence rate is high, cost is also large. So in the development The development of a HPV prophylactic vaccine in China is an effective way to prevent HPV infection and control the incidence of cervical cancer.
HPV vaccine can be divided into prophylactic vaccines and therapeutic vaccines. The present study is successful for the HPV major capsid protein L1 VLPs vaccine has been listed on the HPV preventive vaccine Merck (Merk), HPV vaccine Gardasil, HPV6,11,16 and type 18 VLP composed of tetravalent vaccine and GlaxoSmithKline (GSK), HPV Cervarix vaccine, two valent vaccine for HPV16 and 18 VLP. The antibody positive among the conversion rate reached 100%, 4.5 years of protection rate was 100%, but because of the expensive price, lack of cross protection deficiency limits its widely used in the world. As a kind of ideal HPV vaccine should be inexpensive, has cross protection good activity, coverage of high-risk HPV types as much as possible. The content has become the research target of.L2 second generation HPV preventive vaccine is HPV minor capsid protein, for clothing The formation of the shell is not necessary. However, previous studies have shown that the N terminal can induce the production of antibodies with cross neutralization activity. The main purpose of this experiment is to explore a cheap and broad-spectrum HPV preventive vaccine based on L2 protein as target antigen.
The design idea of this thesis is: firstly, according to the epidemiological data of HPV infection rate in Chinese patients with cervical cancer in HPV16, HPV18 and HPV58 for the study on type L2 inducing cross neutralizing antibody N terminal target antigen in prokaryotic expression of HPV16,18,58 of different length L2 protein screening system, respectively the high titer of neutralizing antibody fragments induced by different types of HPV L2, and then selected a type of fusion fragment, prokaryotic expression, purification, and detection of neutralizing antibodies induced by the cross neutralizing antibody, to explore the development of a low price, to provide experimental basis for prevention of vaccine against HPV. The main results are as follows:
First, eleven kinds of expression were constructed prokaryotic expression plasmid of HPV16,18 and 58 different length L2 protein. The eleven plasmids were able to express the corresponding protein induced by IPTG, and the eleven Western-blot protein was purified after correct identification, eleven kinds of purified protein, the purity was about 90%.; the results showed that serum ELISA immunize mice with the purified protein, L2 protein of three genotypes of HPV with different length can induce high titer of specific antibody, were more than 1:100000; the package of HPV16,18 and type 58 virus particles, and established the HPV experimental technology platform, the purification and detection of serum antibody in the immune protein methods, selected different types of HPV L2 fragments, they are: HPV16L2 (11-50aa), HPV18L2 (11-200aa) and HPV58L2 (11-150aa), and according to the same pseudotyped virus antibody titers were 1 640,1:1280 and 1:1280.
Second, will be screened out different types of HPV L2 protein fragments were fused in two ways, 18-16-58 and 5816-18 respectively, constructed the prokaryotic expression plasmid of two pET-9a HPV16,18 and 58 L2 fusion protein; the two one can express the corresponding plasmid protein induced by IPTG, the protein Western-blot was purified and identified, the purity was about 90%; in vitro neutralization test results showed that 18-16-58L2 fusion protein induced by antibodies to HPV16,18 and type 58 virus neutralization titers were neutralizing antibody levels of 1:3200,1:3200 and 1:6400 is better than that of 58-16-18L2 fusion protein induced by different ways (hint, combination of protein may have an impact on the exposed position the table, but also can induce) cross neutralizing antibodies against HPV6,45 and type 52 virus, antibody titers were 1:640,1:1280 and 1:640 The 18-16-58L2 fusion protein is more immunogenic than all types of monovalent proteins, and the neutralizing antibody level of the heterotypic fake virus has been significantly improved.
In summary, this study selected HPV16,18 and type 58 can induce a relatively high activity of L2 fragments, three types of L2 fragments and will be screened to construct fusion protein, and the activity of a preliminary study, made a meaningful exploration on HPV broad-spectrum neutralizing antibody vaccine provides valuable experimental data for an in-depth study of this type of vaccine.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

【参考文献】