目的 原核表达细粒棘球绦虫钙网蛋白，并对其功能进行初步探索。方法 以细粒棘球绦虫原头节cDNA为模板，PCR扩增目的基因，连接至pET-28a（+）载体，构建重组质粒pET28a-CRT，将测序正确的重组质粒转化至表达菌BL21诱导表达，经十二烷基硫酸钠聚丙烯酰胺（SDS-PAGE）电泳以及蛋白质免疫印迹（Western blotting）分析鉴定rEgCRT；应用在线生物信息学分析软件分析EgCRT表达蛋白理化性质及部分生物学功能；rEgCRT免疫小鼠制备多克隆抗体，并用间接ELISA和Western blotting检测其抗体效价及反应原性；应用间接免疫荧光分析CRT在原头节和成虫体内的分布以及rEgCRT与犬小肠上皮细胞的结合；应用RT-qPCR方法分析crt基因在虫体不同发育阶段转录水平及rEgCRT（实验组，PBS为对照组）对犬小肠上皮细胞凋亡、焦亡的影响。结果 CRT蛋白分子式为C₂₀₃₃H₃₀₅₁N₅₁₁O₆₅₀S₉，预测EgCRT蛋白α-螺旋占24.05%、无规则卷曲占60.00%，延伸链结构占15.95%。EgCRT与多房棘球绦虫和猪带绦虫的CRT在系统进化树上聚在一大分支。成功诱导表达纯化获得纯度较高且相对分子量约为50 000的rEgCRT；免疫学特性鉴定表明rEgCRT具有较好的抗原特异性；间接免疫荧光试验发现EgCRT可分别定位于原头节表皮层和成虫表皮层及部分实质组织。RT-qPCR分析结果显示，成虫阶段EgCRT的表达水平（24.83 ± 0）高于原头节阶段（27.05 ± 0.008）（t = 45.76，P < 0.01）。间接免疫荧光分析结果显示，rEgCRT共孵育的犬小肠上皮细胞有绿色荧光聚集。RT-qPCR结果显示，培养24 h后实验组和对照组Bax相对转录水平分别为31.63 ± 0.001、24.17 ± 0.027（t = 7.94，P < 0.01），表达上调；Bcl-2相对转录水平分别为34.55 ± 0.070、19.65 ± 0.042（t = 15.70，P < 0.01），表达下调；IL-18相对转录水平分别为25.62 ± 0.067、20.93 ± 0.014（t = 5.05，P < 0.01），表达下调；IL-1β相对转录水平分别为28.18 ± 0.001、28.71 ± 0.020（t = 0.26，P > 0.05），表达下调。结论 rEgCRT具有良好的抗原性，EgCRT在细粒棘球绦虫不同虫期均有表达，且其可能在细粒棘球绦虫固着、定植或生长发育中具有重要的作用；此外，rEgCRT与犬SIECs发生黏附并调节细胞的凋亡、焦亡，可能参与感染过程或宿主免疫应答，具有作为潜在疫苗候选分子、药物靶标的应用前景。

Objective To express Echinococcus granulosus (Eg) calreticulin (CRT) in prokaryotic cells and preliminarily explore the function of EgCRT. Methods The cDNA of Eg protoscoleces was used as a template to amplify the target gene using PCR assay, which was ligated into the pET-28a (+) vector to construct the recombinant plasmid pET28a-CRT. The recombinant plasmid was transformed into the Escherichia coli BL21 strain. The rEgCRT was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the physicochemical properties and some biological functions of the EgCRT protein were predicted using online bioinformatics tools. Mice were immunized with rEgCRT to prepare the polyclonal antibody, and the antibody titer and reactogenicity were detected using indirect ELISA and Western blotting, respectively. The distribution of CRT in protoscoleces and adults and the binding of rEgCRT to canine intestinal epithelial cells were analyzed using indirect immunofluorescence. In addition, the relative transcription level of the crt gene at different developmental stages and the effect of rEgCRT (experimental group and PBS as a control group) on apoptosis and pyroptosis of canine intestinal epithelial cells were examined using real-time quantitative reverse transcription PCR (RT-qPCR) assay. Results The molecular formula of the CRT protein was C₂₀₃₃H₃₀₅₁N₅₁₁O₆₅₀S₉, and the EgCRT protein was predicted to contain α-helix (24.05%), irregular coils (60.00%) and extended chain structures (15.95%). EgCRT, along with the CRTs of E. multilocularis and Taenia solium, were clustered into a clade on the phylogenetic tree. The rEgCRT protein was successfully expressed and highly purified, with a relative molecular weight of approximately 50 000. The rEgCRT protein showed a high antigenic specificity, and indirect immunofluorescence assay showed that EgCRT was localized in the epidermis of protoscoleces and adults and some parenchyma tissues. RT-qPCR assay showed higher expression of EgCRT in the adult stage (24.83 ± 0) than in the protoscoleces stage (27.05 ± 0.008) (t = 45.76, P < 0.01), and indirect immunofluorescence assay showed that the canine small intestinal epithelial cells co-cultured with rEgCRT presented green fluorescence aggregation. RT-qPCR assay showed that the relative transcription levels of the Bax gene were (31.63 ± 0.001) and (24.17 ± 0.027) in the experimental group and the control group 24 hours post-culture (t = 7.94, P < 0.01), indicating up-regulated expression, and the relative transcription levels of Bcl-2 were (34.55 ± 0.070) and (19.65 ± 0.042) (t = 15.70, P < 0.01), indicating downregulated expression, while the relative transcription levels of IL-18 were (25.62 ± 0.067) and (20.93 ± 0.014) (t = 5.05, P < 0.01), indicating downregulated expression, and the relative transcription levels of IL-1β were (28.18 ± 0.001) and (28.71 ± 0.020) (t = 0.26, P > 0.05), indicating downregulated expression. Conclusion The rEgCRT protein has a high antigenicity, and EgCRT is expressed in different developmental stages of E. granulosus, and may play an important role in the fixation, colonization, growth or development of E. granulosus. In addition, rEgCRT adheres to canine small intestinal epithelial cells and regulates cell apoptosis and pyroptosis, which may be involved in the process of E. granulosus infections or host immune responses, and has a promise as potential vaccine candidates and drug targets.