發(fā)布時間：2018-03-01 10:27

  本文關(guān)鍵詞： 人類乳頭瘤病毒 L1重組蛋白 可溶性表達(dá) 純化 鑒定　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：宮頸癌是常見的婦科惡性腫瘤之一，，全球每年約有50萬新增病例，發(fā)病率在女性惡性腫瘤中居第二位，僅次于乳腺癌。人類乳頭瘤病毒HPV（Human Pa-pillomavirus，HPV）屬乳頭瘤病毒科乳頭瘤病毒屬，為無包膜DNA病毒，主要引起人類皮膚粘膜的增生性病變。大量分子流行病學(xué)和分子生物學(xué)研究已經(jīng)證明，高危型人類乳頭瘤病毒誘發(fā)宮頸癌，HPV58型在我國宮頸癌婦女中是繼HPV16和18之后的第三重要型別，由于HPV具有嚴(yán)格的種屬特異性且其增殖嚴(yán)格依賴宿主細(xì)胞的分化狀態(tài)，使得其難以在體外培養(yǎng)，使得傳統(tǒng)的滅活及減毒疫苗難以進(jìn)展，所以研究的重點走向了基因工程疫苗，HPV L1蛋白形成的病毒楊顆粒VLP（virus-like particle）是HPV疫苗的主要形式,在HPV VLP疫苗的研制中，如何以簡便高效的制備結(jié)構(gòu)完整、免疫原性強的VLP是所有問題的關(guān)鍵。 本研究利用重組表達(dá)質(zhì)粒pGex-6p-1-HPV58-L1轉(zhuǎn)入大腸桿菌E.coli BL21表達(dá)HPV58L1蛋白,通過對誘導(dǎo)劑IPTG的濃度、誘導(dǎo)時間以及誘導(dǎo)溫度等條件的優(yōu)化，篩選出最佳條件；由于HPV58L1蛋白的空間結(jié)構(gòu)復(fù)雜性等因素，使表達(dá)的目的蛋白易形成包涵體，我們通過包涵體復(fù)性的辦法，使目的蛋白的生物活性得以恢復(fù)，同時對目的蛋白的可溶性表達(dá)進(jìn)行了探索，轉(zhuǎn)入伴侶分子，同時通過不同溫度的誘導(dǎo)，得到的最佳條件是15℃低溫誘導(dǎo)表達(dá)的重組蛋白可溶性最好，并進(jìn)行過柱純化目的蛋白，western blotting鑒定，結(jié)果顯示表達(dá)的HPV58L1蛋白能HPV16亞型的單克隆抗體發(fā)生特異性反應(yīng)，進(jìn)行HPV58VLP的生物活性分析，為HPV新型疫苗的研制奠定一定得基礎(chǔ)。
[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

本文編號：1551523