本文選題：人乳頭瘤病毒　切入點(diǎn)：次要衣殼蛋白　出處：《中國疾病預(yù)防控制中心》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：人乳頭瘤病毒(HPV)感染可以誘發(fā)人體不同部位發(fā)生疣或乳頭狀的瘤樣病變,是危害人類健康的重要病原體之一。根據(jù)其致癌性的不同,可分為低危型和高危型。其中高危型主要有HPV16、18、31、33、39、45、51、52、56、58、59、68、73、82共15個(gè)型別,高危型HPV的感染與食管癌、喉癌、舌癌的發(fā)生有關(guān),與宮頸癌發(fā)生的關(guān)系尤為密切。宮頸癌是危及全球婦女健康的嚴(yán)重疾病,在世界范圍內(nèi)的發(fā)病率居女性惡性腫瘤的第二位�，F(xiàn)行的宮頸粘膜涂片篩查可實(shí)現(xiàn)宮頸癌的早期發(fā)現(xiàn),但是篩查的假陰性率高而且費(fèi)用大難以普及。同時(shí)對于中晚期宮頸癌的治療手術(shù)和化療后的效果并不理想,復(fù)發(fā)率高,花費(fèi)也大。因此在發(fā)展中國家研發(fā)一種HPV預(yù)防性疫苗是從根本上預(yù)防HPV感染,控制宮頸癌發(fā)病率的一種有效的方法。 HPV疫苗可分為預(yù)防性疫苗和治療性疫苗。目前研究成功的是針對HPV主要衣殼蛋白L1的VLPs預(yù)防性疫苗,已經(jīng)上市的HPV預(yù)防性疫苗有默克(Merk)公司的HPV預(yù)防性疫苗Gardasil,為HPV6、11、16和18型的VLP組成的四價(jià)疫苗和葛蘭素史克(GSK)公司的HPV預(yù)防性疫苗Cervarix,為HPV16和18型的VLP組成的兩價(jià)疫苗,在人群中的抗體陽轉(zhuǎn)率達(dá)到100%,4.5年保護(hù)率為100%,但由于存在價(jià)格昂貴,缺乏交叉保護(hù)作用等缺點(diǎn)限制了其在全球的廣泛使用。作為一種理想的HPV疫苗應(yīng)是價(jià)格低廉、具有較好的交叉保護(hù)活性,覆蓋盡可能多的HPV高危型別。上述內(nèi)容已成為第二代HPV預(yù)防性疫苗的研究目標(biāo)。L2是HPV的次要衣殼蛋白,對于衣殼的形成不是必要的,但已有的研究表明其N端可誘發(fā)產(chǎn)生具有交叉中和活性的抗體。本試驗(yàn)的主要研究目的就是以L2蛋白為靶抗原,探索研制一種低廉、廣譜的HPV預(yù)防性疫苗。 本論文的設(shè)計(jì)思路是：首先根據(jù)我國宮頸癌患者中HPV感染率的流行病學(xué)調(diào)查資料選擇HPV16、HPV18和HPV58為研究型別,以L2可誘發(fā)產(chǎn)生交叉中和抗體的N端為靶抗原,在原核表達(dá)系統(tǒng)中表達(dá)HPV16、18、58不同長度的L2蛋白,分別篩選不同型別HPV L2中誘發(fā)中和抗體滴度高的片段,然后將入選的個(gè)型別片段融合,進(jìn)行原核表達(dá)、純化,檢測其誘發(fā)的中和抗體及交叉中和抗體情況,為探討研制一種價(jià)格低廉、廣譜的HPV預(yù)防性疫苗提供實(shí)驗(yàn)依據(jù)。主要研究結(jié)果如下： 第一、構(gòu)建了分別表達(dá)HPV16、18和58型不同長度L2蛋白的十一種原核表達(dá)質(zhì)粒。上述十一個(gè)質(zhì)粒在IPTG誘導(dǎo)下均能表達(dá)相應(yīng)的目的蛋白,并對這十一種蛋白western-blot鑒定正確后進(jìn)行純化,得到十一種純化的蛋白,純度在90%。左右；純化蛋白免疫小鼠后血清ELISA結(jié)果顯示,三種HPV型別不同長度的L2蛋白均能誘發(fā)產(chǎn)生高滴度的特異性抗體,均在1：100000以上；用包裝的HPV16、18和58型假病毒顆粒,建立了HPV中和實(shí)驗(yàn)技術(shù)平臺(tái),用該方法檢測各純化蛋白免疫血清中和抗體,篩選出不同型別HPV最佳L2片段,它們分別是：HPV16L2(11-50aa), HPV18L2(11-200aa)和HPV58L2(11-150aa),針對同型假病毒的中和抗體滴度分別為1：640、1：1280和1：1280。 第二、將篩選出來的不同型別HPV L2蛋白片段進(jìn)行了兩種方式的融合,分別為18-16-58和5816-18,構(gòu)建了表達(dá)HPV16、18和58型L2融合蛋白的兩種pET-9a原核表達(dá)質(zhì)粒；上述兩個(gè)個(gè)質(zhì)粒在IPTG誘導(dǎo)下均能表達(dá)相應(yīng)的目的蛋白,對上述蛋白進(jìn)行western-blot鑒定正確并進(jìn)行純化,純度在90%左右；體外中和實(shí)驗(yàn)結(jié)果顯示,18-16-58L2融合蛋白誘發(fā)抗體對HPV16、18和58型假病毒的中和滴度分別為1：3200、1：3200和1：6400優(yōu)于58-16-18L2融合蛋白誘發(fā)的中和抗體水平(提示蛋白間不同的組合方式,可能對其表位的暴露有影響),而且能夠誘發(fā)產(chǎn)生針對HPV6、45和52型假病毒的交叉中和抗體,抗體滴度分別為1：640、1：1280和1：640；18-16-58L2融合蛋白與各型別單價(jià)蛋白相比,免疫原性加強(qiáng),對異型假病毒的中和抗體水平有了明顯的提高,具備廣譜性。 綜上所述,本研究分別篩選出HPV16、18和58型的能夠誘發(fā)較高中和活性的L2片段,并將篩選出的三種型別L2片段構(gòu)建融合蛋白,對其中和活性進(jìn)行了初步研究,對HPV廣譜中和抗體疫苗進(jìn)行了有意義的探索,為深入進(jìn)行這類疫苗的研究提供了有價(jià)值的實(shí)驗(yàn)資料。
[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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.1

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