一种新型的曲霉属洁净可逆性诱导表达系统  

Developing A Green and Reversiby Regulated Inducible Expression System in Fungus

作　　者：李宝意 周胜敏 LI Baoyi;ZHOU Shengmin(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200037

出　　处：《华东理工大学学报（自然科学版）》2023年第4期536-545,共10页Journal of East China University of Science and Technology

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2017YFE0129600)。

摘　　要：构建了一种以构巢曲霉为表达宿主、过氧化氢(H_(2)O_(2))抗性酶Peroxiredoxin(PRX)的编码基因AnPrxA的启动子P_(PRX)为表达用启动子、H_(2)O_(2)为表达诱导剂的新型、高效的曲霉表达体系。在双氧水诱导和非诱导条件下,评估P_(PRX)的上游调控区介导绿色荧光蛋白(GFP)表达的特征。利用基因工程构建了4个含不同长度启动子表达盒的克隆载体并转化至尿嘧啶生物合成缺陷型构巢曲霉宿主;分析了AnPrxA基因上游2033 bp序列中潜在的转录调控元件;利用荧光定量PCR测定表达盒拷贝数;考察与P_(PRX)融合的GFP表达情况;并且探讨了双氧水诱导浓度和作用时间与蛋白表达量的关系。结果表明,AnPrxA基因的5'-非编码区2033 bp序列中至少包含9种基因表达调控元件,大都呈多拷贝排列;虽然该2033 bp的非编码区域对gfp的转录均有贡献,但是非编码区缩短至500 bp也可以维持GFP蛋白的正常表达;在双氧水浓度为0.5~2.0 mmol/L的诱导条件下,P_(PRX)介导的GFP表达量提高3倍,与先前鉴定的2种曲霉属双氧水诱导型启动子的诱导率相似,但目标蛋白的绝对表达强度更高。研究还发现P_(PRX)介导gfp转录水平在H_(2)O_(2)诱导条件下提高30倍,显著高于其蛋白表达的诱导率(3倍),因此今后进一步提升蛋白翻译效率将有望大幅提高目标蛋白的合成水平。Aspergillus represents a potential host for expression of recombinant proteins after bacteria,yeast,plant and animal cells.Due to the late start of Aspergillus expression system research,the technology is still immature.And the number of alternative expression vectors and available promoters are limited,which limits the application of Aspergillus expression system.On our previous work,we found a peroxiredoxin from Aspergillus nidulans(AnPrxA)could be expressed at a high level and its expression level responded to H_(2)O_(2) in the medium.Based on the low price and self-decomposition properties of H_(2)O_(2),we explored a novel and efficient Aspergillus nidulans expression system using Aspergillus nidulans as the expression host,AnPrxA promoter as the expression promoter,and H_(2)O_(2) as the expression inducer.Four promoters of different lengths of the 5’untranslated regions(5’-UTR)of Aspergillus nidulans(P_(PRX)500,P_(PRX)1000,P_(PRX)1500 and P_(PRX)2033)were amplified and cloned in vectors,and the ability of the four promoters to drive GFP expression was evaluated.The results depicted that all four expression systems were sensitive to H_(2)O_(2),and P_(PRX)500 was sufficient for GFP expression,but the induction ratio of P_(PRX)2033 is higher.The optimal induction time of H_(2)O_(2) was determined to be 8 h.The optimal induction concentration range was 0.5—2.0 mmol/L.Comparison of the conditions with and without the inducer revealed that Pprx2033 mediated a 30-fold induction ratio of GFP at the transcriptional level and a 3-fold induction ratio at the protein expression level.In the induction phase of this expression system,the residual concentration could no longer be detected after 2 h of adding 2.0 mmol/L H_(2)O_(2) in the medium,and could no longer be detected after 1 h of adding 0.5 mmol/L H_(2)O_(2).

关 键 词：PRX 蛋白表达 构巢曲霉 过氧化氢 表达调控 

分 类 号：Q78[生物学—分子生物学]