發(fā)布時間：2018-10-29 12:38

【摘要】： 優(yōu)化HPV16L1基因在Pichia Pastoris酵母中的表達。首先用酵母偏愛密碼子對HPV16型L1基因進行優(yōu)化并用引物延伸法合成該基因,將該基因構(gòu)建到分泌型酵母表達載體pPICZαB形成整合質(zhì)粒,電擊轉(zhuǎn)化GS115酵母細(xì)胞,篩選陽性重組子,經(jīng)SDS-PAGE電泳、Western-Blot檢測,選取表達量較高的菌株進行搖瓶表達,并摸索優(yōu)化表達條件,5L發(fā)酵罐表達蛋白,初步建立穩(wěn)定的發(fā)酵工藝。發(fā)酵上清經(jīng)SDS-PAGE電泳檢測顯示,其中有特異蛋白條帶,且在第四天表達量最高,表達產(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,HPV16L1基因經(jīng)過密碼子優(yōu)化和發(fā)酵條件的摸索,在Pichia Pastoris酵母中能夠穩(wěn)定地表達。
[Abstract]:To optimize the expression of HPV16L1 gene in Pichia Pastoris yeast. First, the HPV16 L1 gene was optimized by yeast preference codon and synthesized by primer extension method. The gene was constructed into secretory yeast expression vector pPICZ 偽 B to form an integrated plasmid, and transformed into GS115 yeast cells to screen positive recombinant. By SDS-PAGE electrophoresis and Western-Blot detection, the strains with high expression amount were selected for shake flask expression, and the expression conditions were optimized. The 5L fermenter expressed protein and established a stable fermentation process. The fermentation supernatant was detected by SDS-PAGE electrophoresis, and the specific protein band was found in the supernatant, and the highest expression level was found on the fourth day, and the molecular weight of the monomer 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 HPV16L1 gene could be expressed stably in Pichia Pastoris yeast by codon optimization and fermentation conditions.
【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】：2007
【分類號】：R346

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