利用翻译延伸因子1-α启动子在毕赤酵母中表达人乳头瘤病毒16L1蛋白

Expression of human papilloma virus 16 L1 protein in Pichia pastoris using translation elongation factor 1-α promoter

目的利用翻译延伸因子1-α(translation elongation factor 1-α,TEF1-α)启动子在毕赤酵母中表达人乳头瘤病毒(human papilloma virus,HPV)16 L1蛋白。方法从毕赤酵母GS115中扩增组成型TEF1-α启动子(PTEF1-α)基因,通过基因重组替换商业化表达质粒pPink-HC和pPink-LC的启动子PAOX1;在PTEF1-α下游分别克隆绿色荧光蛋白(green fluorescent protein,GFP)基因和HPV 16 L1基因,构建重组质粒pTEF-GFP和pTEF-16 L1;将重组质粒电转化感受态毕赤酵母PichiaPink strain 1,培养不同时间后,荧光显微镜下观察GFP的表达;分别在YPD、YPG、YPM、YPSor培养基中培养pTEF-16 L1转化菌,检测菌体在不同培养基中的增殖情况;Western blot检测HPV 16 L1蛋白的表达水平。结果重组质粒pTEF-GFP和pTEF-16 L1经双酶切鉴定和序列分析表明构建正确;荧光显微镜检测显示,在不同培养时间PTEF1-α指导了GFP的成功表达,且表达量随培养时间的延长而降低;重组毕赤酵母在YPD和YPG培养基中生长迅速;4种培养基中均有HPV 16 L1蛋白的特异性表达,且在YPG和YPSor培养基中获得了HPV 16 L1蛋白较持续的表达。结论成功实现了组成型启动子PTEF1-α控制的HPV16 L1蛋白的表达,为HPV疫苗的低成本及方便生产提供了一个可供选择的有效方案。

Objective To express human papilloma virus （HPV） 16 L1 protein in Pichiapastoris using translation elongation factor 1-αpromoter. Methods The oligonucleotide fragment of constitutive translation elongation factor 1-α promoter （PFEF1-α） was amplified from Pichia pastoris GS115 by PCR. Recombinant plasmids pTEF-GFP and pTEF-16 L1 were constructed by replacing the alcohol oxidase 1 promoter （PAOX1） of commercial expression vector PichiaPink-HC and PichiaPink-LC with PTEF1-α and cloning the genes of green fluorescence protein （GFP） and HPV16 L1 protein downstream to the promoter respectively. The recombinant plasmids were transformed to competent P. pastoris strain 1 by electrotransformation, cultured for various hours, and observed for expression of GFP by fluorescent microscopy. The P. pastoris strain 1 transformed with pTEF-16 L1 was cultured in YPD, YPG, YPM, and YPSor media respectively, and determined for proliferation level. The expression level of HPV 16 L1 protein was determined by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmids pTEF-GFP and pTEF-16 L1 were constructed correctly. Fluorescent microscopy showed that GFP was expressed successfully under the control of constitutive PTEF1-α, and the expression level decreased with the increasing time for culture. The recombinant P. pastoris grew rapidly in YPD and YPG media. Westenl blot showed specific expression of HPV16 L1 protein in four media. However, persistent expression of HPV 16 L1 protein was observed in YPG and YPSor media. Conclusion HPV16 L1 protein were constitutively expressed under the control of PTEF1-α, which provided an alternative protocol for cost-valuable and convenient production of HPV vaccine.