关键词: 沙眼衣原体； 细菌噬菌体M13； DNA, 重组； 包涵体； IN5环 

Abstract: Lian Tingting, Wei Shijuan, Liu Yuanjun, Ren Jie, Wang Sheng, Guo Yuanli, Guo Rui, Liu Quanzhong, Shao Lili Department of Dermatology and Venereology, Tianjin Medical University General Hospital, Tianjin 300052, China Corresponding authors: Shao Lili, Email: lilishao_2007@163.com; Liu Quanzhong, Email: liuqztjykdxzyy@163.com 【Abstract】 Objective To construct active phages against Chlamydia trachomatis, and to evaluate its effect on Chlamydia trachomatis. Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1, and then the recombinant M13-IN5 phage was obtained. PCR amplification, enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully. The viability of the phage was evaluated by plaque formation assay. Cell counting kit-8（CCK8） assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units （PFU）/ml on the proliferation of Hela cells, and Hela cells uninfected with chlamydia served as the blank control group. Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage, M13 phage and Escherichia coli ER2738 at exponential growth phase. Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately, and chlamydia control group without the treatment with phages was set up. After 36-hour infection, confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage. Moreover, iodine staining was conducted to count inclusion bodies at 36, 48, 60 and 72 hours separately after infection. Statistical analysis was carried out by a two-sample t-test for comparisons between two groups, one-way analysis of variance （ANOVA） for intergroup comparison, and Bonferroni test for multiple comparisons. Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully, and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/pⅢ fusion protein with a high titer of 3.05 × 1011 PFU/ml. As CCK8 assay showed, there was no significant difference in proliferation of Hela cells among the blank control group, M13 phage group and recombinant M13-IN5 phage group （A450 values: 3.63 ± 0.01, 3.55 ± 0.02, 3.70 ± 0.01, respectively, F = 12.0, P > 0.05）. Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence. The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group （both P < 0.05） at 36 and 72 hours after chlamydial infection, and the number of inclusion bodies was significantly lower in the M13-IN5 phage group than in the M13 phage group （P > 0.05）. After 48, and 60 hours of chlamydial infection, the number of inclusion bodies did not differ among the M13 phage group, M13-IN5 phage group and control group （both P > 0.05）. Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein. In the in vitro experiments, the recombinant phage could enter into chlamydia inclusion bodies, and markedly inhibited the infection of Chlamydia trachomatis.

Key words: Chlamydia trachomatis, Bacteriophage M13, DNA, recombinant, Inclusion bodies, IN5 loop