链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测

Expression, purification and characterization of bacteriophage lysin of Streptococcus in Escherichia coli

摘要:

噬菌体裂解酶是噬菌体产生的细胞壁水解酶, 通过水解宿主菌细胞壁使子代噬菌体释放, 在体外能高效且特异性地杀死细菌。本研究旨在克隆和表达链球菌噬菌体裂解酶PlyC, 并测定其生物学活性。利用PCR方法扩增PlyC的2条肽链PlyCA和PlyCB, 构建表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB, 分别转化至大肠杆菌BL21(DE3)中, 以0.7 mmol/L IPTG在30oC诱导7 h实现了高效表达, SDS-PAGE分析表明PlyCA和PlyCB表达量均可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法纯化目的蛋白, 其纯度大于95%。用透析复性方法得到目的产物重组链球菌噬菌体裂解酶PlyC, 以浊度法和平板计数法检测其体外抗菌效果, 扫描电子显微镜观察裂解酶作用前后细菌细胞形态变化。结果表明重组PlyC能特异性裂解化脓性链球菌(A组b-溶血性链球菌), 以4 mg/mL浓度作用于OD600为0.56的菌液60 min后杀菌率达99.6%, 扫描电镜观察结果显示该酶作用于菌体后, 链球菌细胞裂解, 呈碎片状态。本研究为开发一种新型、高效的链球菌感染疾病治疗药物打下了基础。

Abstract:

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 oC for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni2+-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A β-hemolytic streptococci). Under the incubation time of 60 min with 4 μg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.