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目的 筛选细粒棘球蚴（Echinococcus granulosus, Eg）原头节抗原分子，并进行克隆、表达及免疫反应性分析。 方法 在对细粒棘球绦虫六钩蚴和原头节阶段表达的转录组测序数据进行差异分析的基础上，筛选出原头节阶段特异性表达的抗原分子。PCR扩增后克隆至pET28a载体，构建原核表达载体pET28a-Eg-08002，转化至大肠埃希菌（Escherichia coli）JM109，异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，抗His标签镍亲和层析柱纯化重组蛋白rEg-08002，快速免染聚丙烯酰胺电泳分析表达产物；蛋白质印迹（Western blotting）分析rEg-08002的免疫反应性。ELISA分析重组蛋白rEg-08002与细粒棘球蚴病患者血清和健康人血清的免疫反应性。 结果 筛选出了原头节中高表达的抗原分子Eg-08002，大小为612 bp，构建原核表达载体pET28a-Eg-08002。快速免染聚丙烯酰胺电泳和Western blotting分析结果显示重组蛋白rEg-08002在E. coli JM109中获得高效表达，在相对分子质量（Mr）约为23 000处可见重组蛋白rEg-08002条带，主要以包涵体形式存在，rEg-08002可被感染棘球蚴的小鼠血清识别。ELISA分析结果表明，12份细粒棘球蚴病患者血清和12份健康人血清的平均 吸光度（A450）值分别为1.245 ± 0.265和0.469 ± 0.006，差异具有统计学意义（P < 0.05）。 结论 筛选出了原头节中高表达的抗原分子Eg-08002，获得的重组蛋白rEg-08002具有较好的免疫反应性。
Objective To screen for protoscolei antigen molecules of Echinococcus granulosus (Eg) and to clone, express and immunoreactive them. Methods Based on the differential analysis of the transcriptome data of Echinococcus granulosus and protoscoid stage of Echinococcus granulosus, the antigenic molecules that were specifically expressed in the stage of protoscoleces were screened out. The PCR product was cloned into pET28a vector, and the prokaryotic expression vector pET28a-Eg-08002 was constructed. The recombinant plasmid was transformed into Escherichia coli JM109 and induced with IPTG. The recombinant protein rEg-08002 was purified by anti-His-tag nickel affinity chromatography and the expression product was analyzed by rapid polyacrylamide electrophoresis. The immunoreactivity of rEg-08002 was analyzed by Western blotting. ELISA analysis of the recombinant protein rEg-08002 and echinococcus granulosus patients serum and healthy human serum immunoreactivity. Results Eg-08002, a highly expressed prokaryotic expression antigen, was screened out and its size was 612 bp. The prokaryotic expression vector pET28a-Eg-08002 was constructed. The results of rapid polyacrylamide gel electrophoresis and Western blotting showed that the recombinant protein rEg-08002 was highly expressed in E. coli JM109, and the recombinant protein rEg-08002 was found at a relative molecular mass of about 23 000, mainly In the form of inclusion bodies, rEg-08002 can be recognized by mouse sera infected with hydatid cysts. ELISA analysis showed that the average absorbance (A450) values of serum of 12 Echinococcus granulosus patients and 12 healthy people were 1.245 ± 0.265 and 0.469 ± 0.006 respectively, with statistical significance (P <0.05). Conclusion Eg-08002, a highly expressed prokaryotic expression antigen, was screened out and the recombinant protein rEg-08002 obtained was highly immunoreactive.