沙眼衣原体主要外膜蛋白affibody的筛选及特性研究

The characteristics of persistent Chlamydia trachomatis (Ct) infection are immune evasion and antibiotic resistance, therefore, persistent infection and the high risk of recurrence result in ectopic pregnancy and tubal infertility. Targeted therapy based on monoclonal antibodies (mAb) is the most promising way for tumor and infectious diseases. However, applications of mAb in medicine are limited because of its weakness tissue penetration and adverse side effects. Affibody molecules bind specifically to target protein with high affinity. In addition, affibody molecules have more advantages over mAb, such as higher tissue penetration, more robust stability against heat and enzymes, and relative low production costs. Granzyme B is a serine protease produced by cytotoxic lymphotytes and capable of inducing rapid target cell death by apoptosis, so, GrB is widely used in tumor targeted therapy bases on mAb. In the present study, we intend to screen and identify Ct MOMP specific affibody by genetic engineering and phage display technology. Then, granzyme B (GrB) will be used as the toxin, MOMP specific affibody-GrB recombinant protein will be prepared. The specific binding of affibody-GrB to Ct and clearance of Ct will be investigated further in cell culture model of persistent Ct infection. Combined with animal experiment of Ct tract infection mouse model in vivo, the efficacy and safety of MOMP specific affibody-GrB in target therapy for Ct will be evaluated. This study will provide a new way for the target therapy based on affibody for infectious diseases.

沙眼衣原体(Ct)持续感染时既逃避免疫又具耐药性，故易复发致异位妊娠、不孕等后果。基于单抗的靶向治疗是目前肿瘤和感染性疾病治疗中具前景的新手段，但其组织渗透性弱及副作用大等问题需解决。亲和体(affibody)分子能以高亲和力与靶蛋白特异结合，且穿透性强、稳定性好和易制备等诸多优点，在靶向治疗方面比单抗更具优势。颗粒酶B可作为单抗负载毒素通过诱导靶细胞凋亡而被用于肿瘤靶向治疗。本研究拟通过基因工程和噬菌体展示技术，筛选Ct主要外膜蛋白(MOMP)特异affibody分子，并以其为载体携带颗粒酶B，通过原核表达系统制备重组毒素蛋白，在体外细胞水平检测MOMP特异性重组毒素蛋白对Ct的靶向结合和对持续性Ct感染的清除作用，结合小鼠Ct生殖道感染模型，评价MOMP特异性重组毒素蛋白对Ct靶向治疗的有效性和安全性。本研究为发展基于MOMP亲和体的Ct持续感染特异性靶向治疗提供新途径。

女性生殖道感染沙眼衣原体（Ct）后可引起慢性盆腔炎、异位妊娠及输卵管性不孕等疾病。目前尚无疫苗特异性防治Ct感染，虽可采用抗生素治疗，但治疗后易复发引起慢性持续性感染。基于单抗的分子靶向治疗为肿瘤和持续性感染性疾病带来了希望。但仍有一些问题尚待解决，如毒副作用大、成本高等。本研究针对上述问题开展研究，通过分子生物学和噬菌体展示技术筛选了Ct MOMP特异性结合亲和体ZMOMP:461 affibody，并以其为基础携带细菌毒素PE38KDEL，制备了重组融合蛋白ZMOMP:461 affitoxin （Z461X），进一步通过蛋白水平、细胞水平和小鼠生殖道Ct感染模型对筛选的affibody 及其affitoxin分子的分子靶向结合和Ct的清除能力进行了验证。结果如下：（1）构建了MOMP affibody的随机组合噬菌体展示文库；（2）获得了MOMP affibody 45个，选择了其中的1个ZMOMP:461进行了原核表达，并制备了纯化蛋白；（3）SPR技术证实筛选的affibody分子与MOMP有极高的亲和力；（4）免疫荧光共定位技术验证了筛选的affibody分子特异性靶向结合Ct感染HeLa229细胞内的天然MOMP蛋白；（5）利用原核表达系统制备并纯化了Z461X，验证了affitoxin在蛋白水平、细胞水平对MOMP的靶向特异性，证实PE38KDEL毒素的加入并未影响affibody对靶蛋白的特异性亲和力；（6）对affitoxin的IC50进行了研究；（7）Z461X对小鼠Ct生殖道感染模型的治疗作用：1）成功建立了Ct小鼠生殖道感染模型；2）生殖道给予Z461X后，PCR及HE染色等多种方法验证了其对Ct感染有显著的清除作用。本研究为感染性疾病Ct感染的分子靶向治疗带来全新的途径。研究成果申请发明专利1项；发表论著3篇（SCI论文2篇），1篇在总结中；培养硕士研究生1人。