基于增强型绿色荧光蛋白研究中性粒细胞对大肠杆菌的抑制作用

Inhibitory effect of neutrophils on EGFP-expressing Escherichia coli

目的基于增强型绿色荧光蛋白(EGFP)研究中性粒细胞(PMN)对大肠杆菌的抑制作用。方法以p EGFP-N1为模板,扩增出750 bp的EGFP基因片段,克隆到p ET-20b(+)载体上,构建p ET-20b(+)-EGFP并转入宿主BL21,PCR鉴定、IPTG诱导表达后的EGFP-E.coli与中性粒细胞共同培养后,测定指定时间的荧光强度,并线性拟合中性粒细胞抗菌的动力学方程。结果成功构建重组质粒p ET-20b(+)-EGFP和BL21表达增强型绿色荧光蛋白。中性粒细胞对大肠杆菌的作用时间与ln(F/F0)呈正相关(R2=0.823 8)。结论基于EGFP改进传统的PMN抑菌方法。

[Objective] To investigate the inhibitory effect of neutrophils on enhanced green fluorescent protein （EGFP） -expressing E. coll. [Methods] Using pEGFP-N1 as a template, a 750 bp segment of EGFP gene was amplified and cloned into the vector of pET-20b （＋） to build pET-20b （＋） -EGFP which was transferred to the host BL21. EGFP-E. coli by IPTG induction and neutrophils were co-cultured, fluorescence intensity was determined at the given time and linear fitting of dynamic equation of neutrophil antimicrobial effect was performed. [Results] The recombinant plasmid pET-20b （＋） -EGFP was successfully constructed and BL21 expressed enhanced green fluorescent protein. The effect time of neutrophils on E. coli was positively correlated with in （F/Fc, RE= 0.8238）. [Conclusion] Based on the EGFP, the traditional study of polymorphonuclear neutrophils （PMN） in bacteriostasis has been improved.