含不同个数基序乳酸乳球菌肽聚糖锚钩蛋白结合活性比较

Comparison of the binding activity of Lactococcus lactis peptidoglycan protein anchor with different number of motifs

摘要:

摘要:【目的】比较两种含不同个数基序乳酸乳球菌肽聚糖锚钩蛋白(protein anchor，PA)的结合活性。【方法】首先应用PCR技术分别扩增得到含有2个或3个自溶素基序(Lysin Motif，LysM)基因片段的PA2与PA3;然后应用pET-32a(+)质粒构建原核表达载体，将其转化大肠杆菌BL-21(DE3)，进行诱导表达，获得目的蛋白;最后将经复性后的PA2、PA3融合蛋白与GEM(Gram-positive Enhancer Matrix，GEM)颗粒结合，经Western blot、透射电镜与SDS-PAGE进行结合鉴定与结合活性比较分析。【结果】融合蛋白PA2、PA3复性后都能与GEM结合，PA3与GEM颗粒的结合活性明显好于PA2。【结论】含有3个LysMs的PA对GEM的结合活性明显优于含有2个LysMs的PA。本研究可为进一步完善乳球菌外壳-蛋白锚钩展示系统提供理论基础。

Abstract:

Abstract:［Objective］The aim of the present study was to compare the binding activity of Lactococcus lactis peptidoglycan protein anchor (PA) with different number of motifs.［Methods］L．lactis PA gene sequences with 2 and 3 lysin motifs (LysM) were obtained by PCR amplification．Then，the recombinant plasmid of pET-32a(+) containing PA gene was constructed and transformed into E．coli BL21 (DE3)，and induced to express the fusion protein PA2 and PA3．The purified and refolded PA2 and PA3 were incubated with gram positive enhancer matrix (GEM)．The binding activities of PA2 and PA3 were identified and compared by Western blot，TEM and SDS-PAGE.［Results］The PA2 and PA3 could bind to GEM．The anchoring activity of PA3 was obviously superior to the PA2.［Conclusion］The data indicated that PA with 3 LysMs had a better binding capacity compared with 2 LysMs．The results provided foundations to further improve the design of GEM-PA display system．