肺炎链球菌中青霉素结合蛋白PBP3在大肠杆菌中的可溶性表达及活性鉴定

Soluble expression of pbp3 of Streptococcus pneumoniae in Escherichia coli and identification of its activity

摘要:

【目的】克隆肺炎链球菌R6的pbp3基因，构建原核表达载体并转化大肠杆菌表达，为PBP3的结构及应用研究创造条件。【方法】利用PCR法克隆肺炎链球菌R6中N端截短的pbp3基因(15?413 aa)，BamH Ⅰ和XhoⅠ酶切后插入pGEX-6p-1构建pGEX-6p-pbp3*表达质粒，在大肠杆菌BL21(DE3)中胞内表达GST-PBP3融合蛋白，Glutathione-Sepharose 4B column亲和纯化GST-PBP3融合蛋白，PreScission Protease切除GST标签，再次过谷胱甘肽亲和层析柱获得纯化的PBP3蛋白。利用PBP3对头孢喹诺的结合试验来鉴定表达蛋白是否有活性。【结果】经测序鉴定成功扩增出N端截短的pbp3基因，成功构建了pGEX-6p-pbp3*表达载体，并纯化出可溶性PBP3蛋白，而且有活性。【结论】肺炎链球菌pbp3基因原核表达系统的成功构建以及有活性重组蛋白的获得，为PBP3蛋白的结构及应用研究奠定了基础。

Abstract:

[Objective] To study the structure and application of penicillin-binding protein 3 (PBP3), the pbp3 gene of Streptococcus pneumoniae R6 was cloned and the recombinant protein of PBP3 was expressed in Escherichia coli with expression vector pGEX-6p-pbp3*. [Methods] The truncated pbp3 gene (15?413 aa) was cloned from S. pneumoniae R6 with PCR, and the pbp3* fragment digested by BamH Ⅰand Xho Ⅰwas inserted into pGEX-6p-1 to generate pGEX-6p-pbp3* expression plasmid. GST-PBP3* fusion protein was expressed in cytoplasm of E. coli BL21(DE3) and purified with Glutathione-Sepharose 4B column. The recombinant fusion protein, GST-PBP3, was firstly digested by PreScission Protease for cutting off the GST tag, and then pass through the Glutathione-Sepharose 4B column for getting the purified PBP3 protein. The PBP3 activity was identified by its binding activity to β-lactam antibiotics, such as cefquinome. [Results] The truncated pbp3 gene was verified by sequence analysis and the pGEX-6p-pbp3* expression plasmid was constructed successfully. Finally the soluble PBP3 protein with biologic activity was purified. [Conclusion] The active recombinant PBP3 was successfully obtained from prokaryotic expression system, and this laid a firm foundation for further study of the structure and application of PBP3.