Major of Biotechnology, Korea National University of Transportation, Jeungpyeong, 27909, Korea.
Major in IT·Biohealth Convergence, Department of IT·Energy Convergence, Graduate School, Korea National University of Transportation, Chungju, 27469, Korea.
Sci Rep. 2023 Sep 30;13(1):16430. doi: 10.1038/s41598-023-43559-z.
The bacteriophage CAP 10-3 forming plaques against Cutibacterium acnes which causes skin acne was previously isolated from human skin acne lesion. Incomplete whole genome sequence (WGS) of the bacteriophage CAP 10-3 was obtained and it had 29,643 bp long nucleotide with 53.86% GC content. The sequence was similar to C. acnes phage PAP 1-1 with a nucleotide sequence identity of 89.63% and the bacteriophage belonged to Pahexavirus. Bioinformatic analysis of the WGS predicted 147 ORFs and functions of 40 CDSs were identified. The predicted endolysin gene of bacteriophage CAP 10-3 was 858 bp long which was deduced as 285 amino acids (~ 31 kDa). The protein had the highest similarity with amino acid sequence of the endolysin from Propionibacterium phage PHL071N05 with 97.20% identity. The CAP 10-3 endolysin gene was amplified by PCR with primer pairs based on the gene sequence, cloned into an expression vector pET-15b and transformed into Escherichia coli BL21(DE3) strain. The predicted protein band (~ 33 kDa) for the recombinant endolysin was detected in an SDS-PAGE gel and western blot assay. The concentrated supernatant of cell lysate from E. coli BL21(DE3) (pET-15b_CAP10-3 end) and a partially purified recombinant CAP 10-3 endolysin showed antibacterial activity against C. acnes KCTC 3314 in a dose-dependent manner. In conclusion, the recombinant CAP 10-3 endolysin was successfully produced in E. coli strain and it can be considered as a therapeutic agent candidate for treatment of human skin acne.
先前从人类皮肤痤疮病变中分离出了一种针对痤疮丙酸杆菌（导致皮肤痤疮的细菌）的噬菌体能形成噬菌斑的 CAP 10-3 噬菌体。获得了该噬菌体的不完全全基因组序列（WGS），其核苷酸长度为 29643bp，GC 含量为 53.86%。该序列与痤疮丙酸杆菌噬菌体 PAP 1-1 的核苷酸序列同一性为 89.63%，噬菌体属于 Pahexavirus。WGS 的生物信息学分析预测了 147 个 ORF，鉴定了 40 个 CDS 的功能。噬菌体 CAP 10-3 的预测内切酶基因长 858bp，推断为 285 个氨基酸（31kDa）。该蛋白与丙酸杆菌噬菌体 PHL071N05 的氨基酸序列内切酶具有最高的相似性，同一性为 97.20%。根据基因序列设计引物对，通过 PCR 扩增 CAP 10-3 内切酶基因，将其克隆到表达载体 pET-15b 中，并转化到大肠杆菌 BL21(DE3)菌株中。在 SDS-PAGE 凝胶和 Western blot 分析中检测到重组内切酶的预测蛋白条带（33kDa）。来自大肠杆菌 BL21(DE3)（pET-15b_CAP10-3 end）的细胞裂解物的浓缩上清液和部分纯化的重组 CAP 10-3 内切酶在体外对 C. acnes KCTC 3314 表现出剂量依赖性的抗菌活性。总之，重组 CAP 10-3 内切酶在大肠杆菌菌株中成功表达，可以考虑作为治疗人类皮肤痤疮的候选治疗剂。
