牛樟芝免疫调节蛋白的过量表达及活性分析

Overexpression and activity analysis of Antrodia camphorata immunomodulatory protein

【目的】用大肠杆菌高效表达牛樟芝免疫调节蛋白(Antrodia camphorata immunomodulatory protein,ACA)。【方法】借助DNAworks3.2.4设计引物合成大肠杆菌密码子偏好性的ACA基因,构建于pET-32a和pGEX-4t-2,转化大肠杆菌,测序后分别命名为pET-32a-ACA/BL21和pGEX-4t-2-ACA/BL21;探索密码子偏好性、载体、培养基种类和诱导条件对ACA表达的影响并使用响应面法优化蛋白表达。纯化重组ACA(recombinant ACA,rACA)并干预小鼠巨噬细胞RAW264.7,通过测定rACA对巨噬细胞增殖能力、吞噬能力、细胞形态及产NO、肿瘤坏死因子α(tumor necrosis factor-α,TNFα)、白介素lβ(interleukin-1β,IL-1β)等细胞因子能力的影响来评估rACA的活性。【结果】pET-32a-ACA/BL21更适合ACA表达;在优化的SOB(酵母粉8.92 g/L)中培养pET-32a-ACA/BL21,添加0.42 mmol/L IPTG 25℃诱导7 h时rACA以可溶性表达为主,产量达187.122μg/mL,是目前大肠杆菌表达真菌免疫调节蛋白的最高报道。纯化的rACA能显著促进小鼠巨噬细胞增殖、提高细胞的吞噬活性和改变细胞形态,显著诱导巨噬细胞分泌NO、TNF-α和IL-1β。【结论】rACA的高效可溶性表达及类似天然ACA的活性使它可以替代天然ACA作为制药行业的食品添加剂或者免疫调节剂,甚至可以用于药物研究。

[Objective]The aim of our study is to express Antrodia camphorata immunomodulatory protein(ACA)in Escherichia coli efficiently.[Methods]We synthesized ACA gene with E.coli codon bias using DNAWorks 3.2.4 program to design and optimize primers.Then,we sequenced the PCR products,inserted the correct gene into expression vector pET-32a and pGEX-4t-2,transformed them into E.coli and named them pET-32a-ACA/BL21 and pGEX-4t-2-ACA/BL21 respectively after sequencing.We investigated the effects of codon bias,vectors,mediums and the induction conditions on the expression of recombinant ACA(rACA),and further optimized the protein expression using response surface methodology(RSM)subsequently.To evaluate the bioactivity of rACA,we determined the proliferation of RAW264.7 cells,analyzed the phagocytosis of macrophages,observed the cell morphology,detected the secretion of cytokine such as nitric oxide(NO),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)after treated RAW264.7 cells with purified rACA.[Results]pET-32a-ACA/BL21 was more suitable for the expression of ACA.The rACA was expressed mainly in soluble form with a yield of 187.122μg/mL,when the pET-32a-ACA/BL21 was cultured in optimized SOB medium(8.92 g/L yeast extract)and induced with 0.42 mmol/L IPTG at 25℃ for 7 h.This was the highest expression report for fungal immunomodulatory protein in E.coli so far.Moreover,the purified rACA promoted the proliferation of RAW264.7,enhanced the phagocytosis activity,changed the cell morphology as well as induced NO,TNF-α and IL-1β production within murine macrophages.[Conclusion]The highly soluble expression,and the very similar biological activities to native ACA make it a good candidate of native ACA for potential application as a food supplement or immunomodulatory agent in pharmaceuticals and even medical studies.