M13-IN5重组噬菌体的构建与鉴定及其对沙眼衣原体的作用研究

Construction and identification of the recombinant M13-IN5phage and its effect on Chlamydia trachomatis

目的 构建针对沙眼衣原体的活性噬菌体,并评估其对沙眼衣原体的作用.方法 将衣原体噬菌体phiCPG1衣壳蛋白VP1的IN5序列与M13噬菌体重组,获得重组M13-IN5噬菌体.利用PCR扩增、酶切、测序重组噬菌体基因,验证目的片段是否成功插入;通过噬菌斑形成实验检测重组噬菌体的活性.利用CCK8法检测效价为1011噬斑形成单位(PFU)/ml的M13噬菌体和M13-IN5重组噬菌体对Hela细胞增殖的影响,同时设置空白对照组(未感染衣原体的Hela细胞).Western印迹检测重组噬菌体、M13噬菌体、对数期大肠杆菌ER2738中IN5环蛋白的表达.在沙眼衣原体E型标准株的培养过程中,分别加入效价为1011PFU/ml的M13噬菌体、M13-IN5重组噬菌体进行干预,并设置衣原体对照组.感染后36 h,共聚焦显微镜下观察M13噬菌体和M13-IN5重组噬菌体定位情况;在感染后36、48、60、72 h碘染色观察计数包涵体.两样本均数间比较用t检验;多组样本均数间比较采用方差分析,两两比较采用Bonferroni检验.结果 成功构建含有IN5环基因的具有生物活性的重组M13噬菌体,Western印迹证实重组噬菌体表达IN5环/pⅢ融合蛋白,重组噬菌体效价达3.05×1011 PFU/ml.CCK8法检测显示,空白对照组、M13噬菌体组、M13-IN5噬菌体组A450值分别为3.63±0.01、3.55±0.02、3.70±0.01,组间差异无统计学意义(F=12.0,P>0.05).共聚焦显微镜定位显示,噬菌体荧光与衣原体包涵体荧光存在重叠;衣原体感染后36、72h,M13-IN5噬菌体组和M13噬菌体组包涵体数目较对照组减少(均P< 0.05),且M13-IN5噬菌体组包涵体数目较M13噬菌体组有明显降低(P<0.05);衣原体感染后48、60h,M13噬菌体组、M13-IN5噬菌体组与对照组包涵体数目差异无统计学意义(P>0.05).结论 构建的M13-IN5重组噬菌体具有生物学活性,且能够成功表达IN5环蛋白;体外实验中,该噬菌体能进入衣原体包涵体内,且能明显抑制沙眼衣原体感染.

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/p Ⅲ 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,M 13 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 M 13-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.