论文部分内容阅读
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。
Objective: To optimize the expression of human papillomavirus type 16 major capsid protein L1 (HPV16L1) in Pichia pastoris and investigate the possible influencing factors. METHODS: Four different sequences of HPV 16 L1 genes M16, Y16, P16 and W16 (M16 and Y16 were optimized by yeast codon, P16 was mammalian codon optimized and W16 was wild-type) were cloned into Pichia Yeast expression plasmids p PinkTM-HC (high gene copy colony selection) and p PinkTM-LC (low gene copy colony selection) were performed and transformed into host strains with different protease deficiencies. After 24 hours of methanol induction, the samples of the bacterial cells were taken for Western blot to analyze the expression of L1 protein. Results: M16 showed the highest expression level, followed by Y16 and P16, while W16 showed almost no expression. The analysis of the codon usage of the gene sequences showed that the codon adaptation indices of the four genes were Y16, M16, W16 and P16 in descending order. The mRNA secondary structures of the four sequences were analyzed by free energy and GC content, Y16 was -409.40 kcal / mol and 43.85%, M16 was -451.50 kcal / mol and 47.83%, P16 was -606.50 kcal / mol and 64.10% W16 is -384.70 kcal / mol and 38.01%. The expression of protease-deficient strain L1 was higher than that of the wild-type strain, and there was no significant difference between p PinkTM-HC and p PinkTM-LC. Conclusion: Codon optimization can significantly improve the expression of HPV16L1 in Pichia pastoris, but its expression level does not correspond exactly with the codon usage, suggesting that codon optimization is only partly responsible for the changes in mRNA structure and stability . Protease deficient strains increased the stability of HPV16 L1 protein and significantly affected the expression level. Studies have shown that gene dose has no significant effect on the expression of HPV16L1.