华支睾吸虫钙调节蛋白的生物学特性及与肝纤维化的关系

发布时间：2018-11-07 07:59

【摘要】：目的对华支睾吸虫成虫(Clonorchis sinensis,Cs)钙调节蛋白(Ca M)进行生物学及功能分析,以确定其在肝纤维化中的作用。方法从Cs c DNA质粒文库中寻找Cs Ca M全长序列,以BLASTx搜索其同源序列并进行比对分析。以生物信息学进行同源比对、理化性质分析及功能域预测。以分子生物学方法进行原核克隆,大肠杆菌表达,亲和层析纯化,并将纯化蛋白免疫大鼠,产生多克隆抗体。ELISA检测Cs Ca M抗体滴度及产生曲线。免疫印迹实验分析Cs Ca M重组蛋白纯化及其抗体识别效果。免疫组化分析其组织定位;腹腔注射法建立Cs Ca M致大鼠肝纤维化模型。结果重组、表达及纯化了Cs Ca M,其编码150位氨基酸,理论相对分子质量23 400。结构域预测其具有EF手位模序。p ET-30a-Cs Ca M重组质粒其目的蛋白表达于宿主菌BL21 E.coli上清,相对分子质量约23 400。总Ig G抗体滴度于2~4周达较高峰,效价大于1∶51 200。免疫组织化学定位显示Cs Ca M在成虫睾丸表达丰富。Cs Ca M腹腔注射大鼠的肝脏均显示不同程度病变,HE染色可见炎症反应较严重,可见气球样变、门管区炎及碎片状坏死;网状纤维染色显示小胆管周围胶原增生,有轻到中度纤维化。结论 Cs Ca M促进大鼠肝脏炎症病变及纤维化的作用,提示其可能参与了华支睾吸虫病致肝纤维化的作用。
[Abstract]:Objective to study the role of calmodulin (Ca M) in hepatic fibrosis of adult Clonorchis sinensis (Clonorchis sinensis,Cs) by biological and functional analysis. Methods the full-length Cs Ca M sequence was found from the Cs c DNA plasmid library, and its homologous sequence was searched by BLASTx and analyzed by alignment. Bioinformatics was used for homology comparison, physicochemical properties analysis and functional domain prediction. Prokaryotic cloning, expression of Escherichia coli and purification by affinity chromatography were carried out by molecular biological methods. The purified protein was immunized to rats to produce polyclonal antibodies. ELISA was used to detect the titer and production curve of Cs Ca M antibody. The purification of Cs Ca M recombinant protein and the effect of antibody recognition were analyzed by Western blot. The liver fibrosis model of rats induced by Cs Ca M was established by intraperitoneal injection. Results Cs Ca M, was recombined, expressed and purified. It encodes 150 amino acids, and its theoretical molecular weight is 23 400. Its target protein was expressed in the supernatant of the host strain BL21 E.coli, and the relative molecular weight was about 23 400. The titer of total Ig G antibody reached its peak at 2 ~ 4 weeks, and its titer was more than 1:51. Immunohistochemical localization showed that Cs Ca M showed varying degrees of pathological changes in the liver of rats injected with. Cs Ca M, and HE staining showed severe inflammatory reaction, balloon degeneration, hilar inflammation and fragment necrosis. Reticular fiber staining showed collagen proliferation around the small bile duct with mild to moderate fibrosis. Conclusion Cs Ca M can promote hepatic inflammation and fibrosis in rats, suggesting that Cs Ca M may play a role in hepatic fibrosis induced by clonorchiasis sinensis.
【作者单位】：中山大学孙逸仙纪念医院检验科;中山大学中山医学院热带病防治重点实验室病原生物学教研室;中山大学药理学教研室;中山大学第三附属医院广东省肝脏疾病研究重点实验室;
【基金】：国家自然科学青年科学基金(81401689) 广东省医学科研基金(B2014137) 中央高校基本科研业务费专项资金资助(14ykpy23)~~
【分类号】：R575.2

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