人乳头瘤病毒16亚型L1基因在毕赤酵母GS115中的分泌表达
发布时间：2018-08-21 09:24
【摘要】：获得人乳头瘤病毒16亚型主要衣壳蛋白(L1)在Pichia Pastoris酵母分泌型表达系统中的高效表达。首先将人乳头瘤病毒16亚型主要衣壳蛋白L1基因重组到分泌型酵母表达载体(pPICZαB)形成整合质粒,电转酵母细胞GS115,经甲醇诱导表达,SDS-PAGE电泳、Western-Blot检测和PCR鉴定,筛选多拷贝阳性整合克隆,经过184个克隆筛选,获得了表达量较高的表达菌株。该菌株甲醇诱导后,发酵上清经SDS-PAGE电泳检测显示,上清中有特异蛋白条带,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,人乳头瘤病毒主要衣壳蛋白L1基因在毕赤酵母表达系统中获得了分泌表达。
[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 伪 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q786
本文编号：2195276
[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 伪 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q786
【参考文献】
相关期刊论文 前2条
1 仓尧卿,朱若英;人乳头瘤病毒及其疫苗的研究[J];微生物学免疫学进展;2000年04期
2 陈汶,刘彬,戎寿德,乔友林;人乳头状瘤病毒DNA检测进展[J];中华检验医学杂志;2005年05期
，本文编号：2195276
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