CAJ | 학술논문

目的：对华支睾吸虫成虫（Clonorchis sinensis, Cs）钙调节蛋白（CaM）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 cDNA质粒文库中寻找CsCaM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CsCaM抗体滴度及产生曲线。免疫印迹实验分析CsCaM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CsCaM致大鼠肝纤维化模型。结果重组、表达及纯化了CsCaM，其编码150位氨基酸，理论相对分子质量23400。结构域预测其具有EF手位模序。pET-30a-CsCaM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23400。总IgG抗体滴度于2~4周达较高峰，效价大于1∶51200。免疫组织化学定位显示CsCaM在成虫睾丸表达丰富。CsCaM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 CsCaM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。
목적：대화지고흡충성충（Clonorchis sinensis, Cs）개조절단백（CaM）진행생물학급공능분석，이학정기재간섬유화중적작용。방법종Cs cDNA질립문고중심조CsCaM전장서렬，이BLASTx수색기동원서렬병진행비대분석。이생물신식학진행동원비대、이화성질분석급공능역예측。이분자생물학방법진행원핵극륭，대장간균표체，친화층석순화，병장순화단백면역대서，산생다극륭항체。ELISA검측CsCaM항체적도급산생곡선。면역인적실험분석CsCaM중조단백순화급기항체식별효과。면역조화분석기조직정위；복강주사법건립CsCaM치대서간섬유화모형。결과중조、표체급순화료CsCaM，기편마150위안기산，이론상대분자질량23400。결구역예측기구유EF수위모서。pET-30a-CsCaM중조질립기목적단백표체우숙주균BL21 E. coli상청，상대분자질량약23400。총IgG항체적도우2~4주체교고봉，효개대우1∶51200。면역조직화학정위현시CsCaM재성충고환표체봉부。CsCaM복강주사대서적간장균현시불동정도병변，HE염색가견염증반응교엄중，가견기구양변、문관구염급쇄편상배사；망상섬유염색현시소담관주위효원증생，유경도중도섬유화。결론 CsCaM촉진대서간장염증병변급섬유화적작용，제시기가능삼여료화지고흡충병치간섬유화적작용。
Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1:51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis. Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis-associated hepatic fibrosis.