大肠杆菌肠毒素ST_1-LT_B融合基因表达条件的优化

Optimization of Expression Condition of Fusion Gene Encoding Heat-stable Entero-Toxin Ⅰ and Heat-labile Enterotoxin B Subunit from Enterotoxigenic Escherichia coli

目的:对已构建的含有大肠杆菌肠毒素ST1-LTB融合基因的重组菌株进行鉴定和表达条件优化。方法:采用限制性核酸内切酶酶切鉴定含ST1-LTB融合基因的重组质粒,同时用SDS-PAGE检测不同条件下ST1-LTB融合基因的表达情况。结果:酶切鉴定证实重组质粒pXSL1含有ST1-LTB融合基因且阅读框架正确,以IPTG为诱导剂诱导ST1-LTB融合基因表达的优化条件是:培养基pH7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时ST1-LTB融合蛋白表达量达35.2%。结论:实现ST1-LTB融合基因的高效表达,为大肠杆菌肠毒素双价基因工程菌苗的生产工艺研究提供了可靠的实验数据。

Objective： To identify the recombinant strain containing ST1 - LTB fusion gene of Escherichia coli enterotoxin and optimive its expressional condition. Methods： To identify the recombinant plasmid pXSL1 with restriction endonucleases digestion. To detect expressional level of differently conditional ST1 - LTB fusion protein by SDS- PAGE. Results： The recombinant expression plasmid pXSL1 contained ST1 - LTB fusion gene having positive reading frame. The expression o ptimization result indicated that the ST1 - LTB fusion gene expression optimized condition with IPTG induction is culture medium pH 7.5,culture temperature 37℃, joining IPTG to final concentration 0.8mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the ST1 - LTB fusion proteins were about 34.8% of total cellular protein with IPTG induction by SDS - PAGE and gel system analysis. Conclusion： The ST1 - LTB fusion protein is expressed high by recombinant strain BL21 （DE3） （pXSL1）. This provided experimental data to research productive technique of bivalent genetically engineered vaccine.