發(fā)布時間：2018-08-21 09:24

【摘要】：獲得人乳頭瘤病毒16亞型主要衣殼蛋白(L1)在Pichia Pastoris酵母分泌型表達(dá)系統(tǒng)中的高效表達(dá)。首先將人乳頭瘤病毒16亞型主要衣殼蛋白L1基因重組到分泌型酵母表達(dá)載體(pPICZαB)形成整合質(zhì)粒,電轉(zhuǎn)酵母細(xì)胞GS115,經(jīng)甲醇誘導(dǎo)表達(dá),SDS-PAGE電泳、Western-Blot檢測和PCR鑒定,篩選多拷貝陽性整合克隆,經(jīng)過184個克隆篩選,獲得了表達(dá)量較高的表達(dá)菌株。該菌株甲醇誘導(dǎo)后,發(fā)酵上清經(jīng)SDS-PAGE電泳檢測顯示,上清中有特異蛋白條帶,且在第四天表達(dá)量最高,表達(dá)產(chǎn)物單體分子量為55,000Da左右。發(fā)酵上清液經(jīng)純化,可獲得純度為90%以上的HPV16L1蛋白,電鏡觀察,所得HPV16L1蛋白可自組裝成病毒樣顆粒(VLPs),直徑約為55nm。結(jié)果表明,人乳頭瘤病毒主要衣殼蛋白L1基因在畢赤酵母表達(dá)系統(tǒng)中獲得了分泌表達(dá)。
[Abstract]:The main capsid protein (L1) of human papillomavirus 16 subtype was highly expressed in Pichia Pastoris yeast secretory expression system. Firstly, the main capsid protein L1 gene of human papillomavirus 16 subtype was recombined into secretory yeast expression vector (pPICZ 偽 B) to form an integrated plasmid. The recombinant plasmid was transformed into yeast cell GS115. The recombinant plasmid was expressed by methanol induced SDS-PAGE electrophoresis and identified by Western-Blot and PCR. After 184 clones were screened, a high expression strain was obtained. After methanol induction, the fermentation supernatants were detected by SDS-PAGE electrophoresis. The specific protein bands were found in the supernatants, and the highest expression level was found on the fourth day, and the molecular weight of the monomers was about 55000Da. The purified supernatant obtained HPV16L1 protein with purity of more than 90%. The obtained HPV16L1 protein was self-assembled into a virus like particle (VLPs), with a diameter of about 55 nm. The results showed that the main capsid protein L1 gene of human papillomavirus was secreted in Pichia pastoris.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392;Q786

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 倉堯卿,朱若英;人乳頭瘤病毒及其疫苗的研究[J];微生物學(xué)免疫學(xué)進展;2000年04期

2 陳汶,劉彬,戎壽德,喬友林;人乳頭狀瘤病毒DNA檢測進展[J];中華檢驗醫(yī)學(xué)雜志;2005年05期

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