本文關(guān)鍵詞：熱休克蛋白70與人乳頭瘤病毒E7融合DNA疫苗的免疫效應(yīng)研究　出處：《武漢大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

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【摘要】：宮頸癌是常見的婦科惡性腫瘤之一,已成為危害女性身體健康的最主要的殺手。世界衛(wèi)生組織統(tǒng)計資料表明,宮頸癌在全球婦女癌癥死亡率中位居第二,在一些發(fā)展中國家甚至位居首位,每年全球大約有50萬例新發(fā)宮頸癌病例,約20萬人死于宮頸癌。據(jù)不完全統(tǒng)計,我國現(xiàn)有宮頸癌病人約13.8萬,每年約有5萬人死于宮頸癌。大量研究資料證明,約80%的宮頸癌與4種高危型(16、18、31型和45型)的HPV感染相關(guān),其中約50%的宮頸癌與HPV16感染相關(guān),根據(jù)分子流行病學(xué)資料的結(jié)果,HPV16是導(dǎo)致我國婦女宮頸癌的主要型別。但是,目前臨床上尚無預(yù)防HPV16感染的措施,對中晚期宮頸癌的化療和手術(shù)治療效果也不理想。宮頸癌的復(fù)發(fā)率較高且治療費(fèi)用也高。因此,研制高效、安全和廉價的HPV DNA疫苗,采用特異性的免疫接種方式預(yù)防和治療HPV感染及其所引起的惡性病變,具有重要的現(xiàn)實(shí)意義。 大料文獻(xiàn)資料證實(shí),人乳頭瘤病毒16型E7基因是重要的轉(zhuǎn)化基因,在宮頸癌細(xì)胞中持續(xù)表達(dá)。大多數(shù)研究中靶基因主要集中于E7基因,然而由于E7基因本身具有轉(zhuǎn)化活性,直接應(yīng)用于人體有一定的危險性,去除E7的轉(zhuǎn)化活性基團(tuán),保留其抗原性成為基因疫苗研制的前提。E7蛋白C末端鋅指結(jié)構(gòu)“Cys-X-X-Cys”對其穩(wěn)定性有重要作用,鋅指結(jié)構(gòu)的突變可顯著降低E7蛋白的穩(wěn)定性和致癌性,提高DNA疫苗的安全性;改變E7C-末端鋅指結(jié)構(gòu)中的任何一個Cys(C58G、C61G、C91G、C94G),均能使E7蛋白完全失去永生化人上皮細(xì)胞的能力。 盡管DNA疫苗有很多優(yōu)點(diǎn),但是裸DNA疫苗并不能誘導(dǎo)強(qiáng)烈的、足以保護(hù)動物抵抗HPV感染的、高水平的免疫應(yīng)答,因此必須想方設(shè)法增強(qiáng)其免疫原性。近年來研究資料表明,熱休克蛋白(heat shock protein,HSP)與抗原基因融合具有增強(qiáng)DNA疫苗免疫效應(yīng)的潛能,HSP抗原肽復(fù)合物(來源于腫瘤或病毒感染細(xì)胞)能誘導(dǎo)較好的抗腫瘤和感染免疫應(yīng)答。HSP介導(dǎo)的抗原呈遞具有“cross-presentation”現(xiàn)象,即通過HSP介導(dǎo)進(jìn)入體內(nèi)的外源蛋白,可以誘導(dǎo)出抗原特異性的CTL。 基于以上原理,我們將突變(91位點(diǎn)C→G)鋅指結(jié)構(gòu)的E7基因、野生型E7基因、熱休克蛋白70基因分別克隆到真核表達(dá)載體pcDNA3.1~-質(zhì)粒中,構(gòu)建了pcd-mE7(突變)、pcd-wE7(野生)、pcd-HSP70三種重組質(zhì)粒,經(jīng)酶切和PCR鑒定構(gòu)建成功。與嵌合型 pcd-HPVmE7-HSP70 DNA 疫苗作比較,進(jìn)一步研究嵌合型pcd-HPVmE7-HSP70DNA疫苗的免疫增強(qiáng)效應(yīng)。我們用這四種重組質(zhì)粒、空載體及PBS
[Abstract]:Cervical cancer is one of the most common gynecological malignancies and has become the most important killer of women's health. The statistics of the World Health Organization show that cervical cancer is the second most common cancer death rate among women in the world. In some developing countries, there are about 500,000 new cervical cancer cases and 200,000 deaths from cervical cancer every year in the world. According to incomplete statistics, there are about 138,000 cervical cancer patients in China. About 50,000 people die of cervical cancer each year. A large number of studies have shown that about 80% of cervical cancer is associated with HPV infection in four high-risk types: type 161831 and type 45. About 50% of cervical cancer is associated with HPV16 infection. According to the results of molecular epidemiology, HPV16 is the main type of cervical cancer in Chinese women. At present, there is no clinical measures to prevent HPV16 infection, and the effect of chemotherapy and surgical treatment on advanced cervical cancer is not ideal. The recurrence rate of cervical cancer is high and the cost of treatment is also high. Therefore, the development of high efficiency. The safe and cheap HPV DNA vaccine has important practical significance to prevent and treat HPV infection and its malignant lesions by specific immunization. Large literature confirmed that human papillomavirus type 16 E7 gene is an important transformation gene, which is expressed in cervical cancer cells. The target gene is mainly focused on E7 gene in most studies. However, due to the transformation activity of E7 gene itself, it is dangerous to apply E7 gene directly to human body and remove the transformation active group of E7 gene. Preserving its antigenicity is a prerequisite for the development of gene vaccine. Zinc finger structure "Cys-X-X-Cys" at the C-terminal of E7 protein plays an important role in its stability. The mutation of zinc finger structure can significantly reduce the stability and carcinogenicity of E7 protein and improve the safety of DNA vaccine. Any of the CysC58GN C61GN C91GN C94 GG changes in the structure of E7C- terminal zinc finger can completely lose the ability of immortalized human epithelial cells. Despite the many advantages of the DNA vaccine, the naked DNA vaccine does not induce a high level of immune response that is strong enough to protect animals against HPV infection. Therefore, we must find ways to enhance its immunogenicity. Recent studies have shown that heat shock proteins (HSPs) are heat shock protein. The fusion of HSPand antigen gene has the potential to enhance the immune effect of DNA vaccine. HSP antigen peptide complex (derived from tumor or virus infected cells). HSP-mediated antigen presentation has the phenomenon of "cross-presentation". In other words, foreign proteins mediated by HSP can induce antigen-specific CTLs. On the basis of the above principle, we will change the mutation to locus C91. 鈫扵he E7 gene, wild type E7 gene and heat shock protein 70 gene of zinc finger structure were cloned into eukaryotic expression vector pcDNA3.1, respectively, and pcd-mE7 (mutagenesis) was constructed. Pcd-wE7 (wild pd-HSP70) recombinant plasmid. The results of restriction endonuclease digestion and PCR analysis were compared with the chimeric pcd-HPVmE7-HSP70 DNA vaccine. To further study the immune enhancement effect of chimeric pcd-HPVmE7-HSP70DNA vaccine. We use these four recombinant plasmids, empty vectors and PBS
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 熊金虎,伍欣星,譚云;不同地區(qū)HPV16 E7基因的克隆及序列差異分析[J];中國病毒學(xué);2002年03期

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本文編號：1392117