Zhao Hong, Xu Yongping, Li Xiaoyu, Li Gen, Zhao Haofei, Wang Lili
School of Bioengineering, Dalian University of Technology, Dalian, 116024, China.
Dalian SEM Bio-Engineering Technology Co. Ltd., Dalian, 116620, China.
Protein J. 2021 Apr;40(2):245-254. doi: 10.1007/s10930-021-09973-w. Epub 2021 Mar 15.
Infection by Enterotoxigenic Escherichia coli is a common cause of diarrhea in animals. The development of vaccines against enterotoxins can effectively control the infection. We have previously constructed a recombinant antigen SLS fused by STa, LTB and STb enterotoxin and it showed a high immunogenicity in mice. Herein, we evaluated the expression of SLS in three different E. coli cells with corresponding plasmids. SLS proteins expressed in E. coli BL21 (DE3) and Rosetta-gami B (DE3) were aggregated as inclusion bodies, and the proteins solubility were not obviously promoted in low temperature combined with adjustment of inducer concentration. In contrast, SLS protein with maltose-binding protein (MBP) yielded from TB1 (DE3) cells were partially soluble. After increasing the IPTG concentration in the medium up to 2 mM and incubating at 37 ℃ for 4 h, the soluble protein yield reached the highest level (4.533 mg/0.2 L culture), which was significantly higher than the expression of SLS protein in Rosetta-gami B (DE3) (P < 0.05). Therefore, the TB1-pMAL expression system can be used for mass extraction and purification of SLS antigen prior to measuring its immunogenicity in pregnant mammals.
产肠毒素大肠杆菌感染是动物腹泻的常见原因。开发抗肠毒素疫苗可有效控制感染。我们之前构建了一种由STa、LTB和STb肠毒素融合而成的重组抗原SLS，它在小鼠中表现出高免疫原性。在此，我们评估了SLS在三种携带相应质粒的不同大肠杆菌细胞中的表达情况。在大肠杆菌BL21(DE3)和Rosetta-gami B(DE3)中表达的SLS蛋白以包涵体形式聚集，低温结合调整诱导剂浓度并不能明显提高蛋白的溶解度。相比之下，在TB1(DE3)细胞中产生的带有麦芽糖结合蛋白(MBP)的SLS蛋白部分可溶。将培养基中的IPTG浓度提高到2 mM并在37℃孵育4小时后，可溶性蛋白产量达到最高水平(4.533 mg/0.2 L培养物)，这显著高于在Rosetta-gami B(DE3)中SLS蛋白的表达量(P<0.05)。因此，TB1-pMAL表达系统可用于在测量其在妊娠哺乳动物中的免疫原性之前大量提取和纯化SLS抗原。
