人乳头瘤病毒16型L1基因在毕赤酵母中表达的影响因素分析

发布时间：2018-08-22 07:10

【摘要】：目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密码子优化,P16为哺乳动物细胞密码子优化,而W16为野生型序列)分别克隆于毕赤酵母表达质粒p PinkTM-HC(高基因拷贝菌落筛选)和p PinkTM-LC(低基因拷贝菌落筛选),并转化不同蛋白酶缺陷的宿主菌。甲醇诱导24小时后,取菌体样品经Western blot分析L1蛋白的表达。结果:M16显示了最高的表达水平,其次是Y16与P16,而W16几乎无表达。基因序列密码子应用特征分析显示,4个基因的密码子适应指数从高到低依次为Y16、M16、W16和P16。通过自由能和GC含量分析4个序列的mRNA二级结构,Y16为-409.40 kcal/mol和43.85%;M16为-451.50 kcal/mol和47.83%;P 16为-606.50kcal/mol and 64.10%;W16为-384.70 kcal/mol and 38.01%。蛋白酶缺陷菌株L1表达高于野生型菌株,质粒p PinkTM-HC与p PinkTM-LC介导的表达无明显区别。结论:密码子优化操作显著改善了HPV16L1在毕赤酵母中的表达,但表达水平与密码子利用优劣并不完全对应,提示密码子优化仅是部分原因,而mRNA结构与稳定性变化值得探讨。蛋白酶缺陷菌株提高了HPV16L1蛋白的稳定性,显著影响了表达水平。研究证明基因剂量对HPV16L1的表达未产生明显影响。
[Abstract]:Objective: to optimize the expression of human papillomavirus 16 major capsid protein L1 (human papillomavirus type 16 major capsid protein L1 HPV16 L1 in Pichia pastoris and to investigate the possible influencing factors. Methods: four HPV16L1 genes, M16, Y16, P16 and W16, with different sequence characteristics, were optimized for mammalian cell codon optimization according to yeast codon. W16 was cloned into Pichia pastoris expression plasmids p PinkTM-HC (high gene copy colony screening) and p PinkTM-LC (low gene copy colony screening), respectively, and transformed into different protease deficient host bacteria. After methanol induction for 24 hours, the expression of L1 protein was analyzed by Western blot. Results: M16 showed the highest level of expression, followed by Y16 and P16, while W16 showed little expression. The codon adaptation index of the four genes was Y16M16W16 and P16s respectively from high to low. By free energy and GC analysis, the secondary structure of mRNA was -409.40 kcal/mol and 43.85m16 was -451.50 kcal/mol and 47.83 kcal/mol, respectively, and P16 was -606.50 kcal / mol and 64.10% W16 was -384.70 kcal/mol and 38.01. The expression of protease deficient strain L1 was higher than that of wild-type strain. There was no significant difference between plasmid p PinkTM-HC and p PinkTM-LC mediated expression. Conclusion: codon optimization can significantly improve the expression of HPV16L1 in Pichia pastoris, but the expression level is not exactly corresponding to the use of codon, suggesting that the codon optimization is only part of the reason, and the change of mRNA structure and stability is worth discussing. Protease deficient strains increased the stability of HPV16L1 protein and significantly affected the expression level. The results showed that the gene dose had no significant effect on the expression of HPV16L1.
【作者单位】：中国医学科学院北京协和医学院医学生物学研究所;云南省重大传染病疫苗研发重点实验室;云南省重大传染病疫苗工程技术研究中心;云南省重大传染病疫苗研发国家地方联合工程研究中心;
【基金】：云南省科技计划重点项目(2016FA049) 云南省应用基础研究面上项目(2010ZC232) 中央高校基本科研业务费(2012N08)资助项目
【分类号】：R373

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