人乳头瘤病毒16亚型L1基因在毕赤酵母GS115中的优化表达
发布时间：2018-10-29 12:38
【摘要】： 优化HPV16L1基因在Pichia Pastoris酵母中的表达。首先用酵母偏爱密码子对HPV16型L1基因进行优化并用引物延伸法合成该基因,将该基因构建到分泌型酵母表达载体pPICZαB形成整合质粒,电击转化GS115酵母细胞,筛选阳性重组子,经SDS-PAGE电泳、Western-Blot检测,选取表达量较高的菌株进行摇瓶表达,并摸索优化表达条件,5L发酵罐表达蛋白,初步建立稳定的发酵工艺。发酵上清经SDS-PAGE电泳检测显示,其中有特异蛋白条带,且在第四天表达量最高,表达产物单体分子量为55,000Da左右。发酵上清液经纯化,可获得纯度为90%以上的HPV16L1蛋白,电镜观察,所得HPV16L1蛋白可自组装成病毒样颗粒(VLPs),直径约为55nm。结果表明,HPV16L1基因经过密码子优化和发酵条件的摸索,在Pichia Pastoris酵母中能够稳定地表达。
[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 伪 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
[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 伪 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
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