应用正交实验法优化大肠杆菌表达CTB-PSMA624-632蛋白的条件

Conditions for Optimizing Expression of CTB-PSMA624-632 Protein in E. coli by Orthogonal Experiment

通过基因工程方法,将含霍乱毒素B亚单位(CTB)和前列腺特异性膜抗原(PSMA)表位肽的重组质粒pET28a-CTB-PSMA624-632,转化表达宿主菌BL21 (DE3)。CTB蛋白的表达有利于开发粘膜佐剂和霍乱弧菌基因工程疫苗,同时抗原肽疫苗也是肿瘤免疫治疗的理想候选。本研究采用正交实验法,优化条件以增加大肠杆菌中CTB-PSMA624-632蛋白的表达量。选取了5个单因素作为考察因子,分别为:预诱导时间、诱导时间、诱导温度、IPTG浓度、诱导转速。先做了4水平的实验L16(45)。根据极差分析的数据对影响较大的3个因素又做了细调型的3水平实验L9(33)。结果表明,在早期指数生长期的诱导能得到相对高水平的包涵体形式的CTB-PSMA624-632蛋白,并且此蛋白的表达随着诱导温度升高到37℃而逐渐增加;相比于使用正常浓度的IPTG诱导得到的蛋白表达量,用100%μmol/L诱导足以诱导蛋白的表达,这比通常使用的IPTG浓度小10倍;此外,诱导时间5 h与7 h对蛋白的表达量差距甚微,因此选定较短时间的5 h,240 r/min的诱导转速也被认为是能诱导目的蛋白CTB-PSMA624-632具有高表达量的最优条件。

The recombinant plasmid p ET28 a-CTB-PSMA624-632, containing cholera toxin B subunit(CTB) and prostate specific membrane antigen(PSMA) epitope antigen, was transformed and expressed in host strain BL21(DE3) by genetic engineering method. The expression of CTB protein was beneficial to the development of mucosal adjuvant and Vibrio cholera gene engineering vaccine, while antigen peptide vaccine was also an ideal candidate for tumor immunotherapy. Herein, orthogonal experiments were used in this study to optimize the conditions to increase the expression level of CTB-PSMA624-632 protein in Escherichia coli. There were five selected single influencing factors which were post-induction time, induction time, induction temperature, IPTG concentration, and induction speed, respectively. We did a four-level experiment L16(45) at first. According to the range analysis, the three factors that attested most were further studied in the finely adjusting three-level experiment L9(33). The results showed that the relatively high-level CTB-PSMA624-632 protein in the form of inclusion bodies was obtained at the early exponential growth phase, and the expression of this protein increased gradually with the increase of induction temperature to 37℃. Inducting with 100 μmol/L IPTG was sufficient to induce the expression of CTB-PSMA624-632 which was 10 times less than normally used IPTG concentration. In addition, the induction time between 5 h and 7 h showed a little difference on protein expression, accordingly we selected the shorter one.Furthermore, 240 r/min of induction speed was also considered to be optimal to induce the target protein CTBPSMA624-632 with higher expression.