Chen Weiqing, Wang Xiaofeng, Wang Pu, Zhang Deyong, Chen Hong, Ke Wei, Lu Yin, Zhang Jianfen
College of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Aug;25(8):1267-72.
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 degrees C 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 Ni(2+)-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 beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/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.
溶素是由噬菌体产生的胞壁质水解酶，作用于宿主细菌的细胞壁以释放子代噬菌体。研究表明，溶素在体外能有效且特异性地杀死细菌。为制备重组链球菌噬菌体溶素（PlyC）并分析其生物学活性，我们通过PCR扩增获得了PlyC的两个基因PlyCA和PlyCB，并将它们插入pET-32a(+)中，然后分别将重组表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB转化到大肠杆菌BL21(DE3)中。在30℃用0.7 mmol/L IPTG诱导7小时后，PlyCA和PlyCB成功表达，SDS-PAGE分析确定它们均占总细胞蛋白的30%以上。经过Ni(2+)-NTA亲和层析后，纯度超过95%。通过变性和蛋白质复性，我们获得了重组PlyC。为确定其生物学活性，我们采用比浊法和平板计数法。在用溶素处理前后，通过扫描电子显微镜（SEM）研究细胞形态。结果表明，重组PlyC能特异性裂解化脓性链球菌（A组β溶血性链球菌）。在OD600为0.56的化脓性链球菌稀释液中，用4μg/mL PlyC孵育60分钟，杀菌效果高达99.6%，而SEM观察显示细胞壁破裂并出现细胞碎片。这一发现为进一步研究和实现对链球菌感染的有效治疗奠定了基础。
