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目的克隆、表达田鼠巴贝虫（Babesia microti）候选诊断抗原，评价其潜在的诊断价值。 方法目的基因来自本室前期构建的田鼠巴贝虫cDNA文库，分别是Bm2、Bm4、Bm6、Bm9和Bm15，用生物信息学分析软件对编码其ORF的氨基酸序列进行预测和分析。从阳性克隆的pBluscript重组质粒中PCR扩增目的基因。将扩增产物连接至pET28a质粒，构建重组质粒并转化至大肠埃希菌（Escherichia coli）BL21（DE3）感受态细胞中，用终浓度为1 mmol/L的异丙基-β-D-硫代半乳糖苷（IPTG）进行诱导表达，通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行蛋白表达和可溶性分析。对包涵体表达的蛋白，采用PAGE胶蛋白微量回收试剂盒对目的蛋白进行纯化。用田鼠巴贝虫感染小鼠血清和疟疾患者血清对纯化的重组蛋白进行ELISA分析，评价各重组抗原的敏感性和特异性。 结果 5个候选抗原Bm2、Bm4、Bm6、Bm9和Bm15基因经PCR扩增，大小分别约为480、300、750、200和700 bp，与理论值相符。构建的重组质粒经测序鉴定与cDNA文库筛选的阳性克隆的ORF序列一致。Bm4、Bm6和Bm15质粒经IPTG诱导，获得包涵体表达，相对分子质量（Mr）分别为13 000、30 000和30 000。但Bm2和Bm9未能表达成功。3个重组蛋白Bm4、Bm6和Bm15纯化后，经SDS-PAGE电泳分析，获得了单一条带的目的重组蛋白。使用田鼠巴贝虫感染小鼠血清和健康小鼠血清对Bm4、Bm6、Bm15和BmSA1（参照）重组蛋白的ELISA分析结果显示，重组蛋白Bm4、Bm6、Bm15及BmSA1的敏感性分别为15.0%、55.0%、80.0%、100.0％，特异性分别为100.0%、100.0%、90.0%、100.0%。Bm4、Bm6及Bm15与疟疾患者血清无交叉反应，BmSA1与疟疾患者血清有一定的交叉反应（假阳性率为13.3%）。 结论 表达了3个田鼠巴贝虫候选诊断抗原，初步评价发现Bm15抗原对检测田鼠巴贝虫具有良好的敏感性和特异性。
Objective To clone and express Babesia microti candidate diagnostic antigen and evaluate its potential diagnostic value. Methods The purpose of this study was to evaluate the amino acid sequence of Bombyx mori cDNA library of Bombyx mori, Bm2, Bm4, Bm6, Bm9 and Bm15. Bioinformatics software was used to predict and analyze the amino acid sequence of ORF. The target gene was PCR amplified from the pBluscript recombinant plasmid of positive clones. The amplified product was ligated into pET28a plasmid, and the recombinant plasmid was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. The recombinant plasmid was induced with isopropyl-β-D-sulfur Inducible expression of galactosyltransferase (IPTG) was detected by SDS-PAGE and protein expression and soluble analysis. The protein expressed in inclusion bodies was purified by PAGE gel protein recovery kit. The purified recombinant proteins were analyzed by ELISA using sera of mouse sera and malaria-infected mice, and the sensitivity and specificity of each recombinant antigen were evaluated. Results The five candidate antigens, Bm2, Bm4, Bm6, Bm9 and Bm15, were amplified by PCR and their sizes were about 480, 300, 750, 200 and 700 bp, respectively. The constructed recombinant plasmids were sequenced to identify the ORFs of the positive clones screened by cDNA library. The Bm4, Bm6 and Bm15 plasmids were induced by IPTG and expressed in inclusion bodies. The relative molecular weights (Mr) were 13 000, 30 000 and 30 000, respectively. However, Bm2 and Bm9 failed to express their success. Three recombinant proteins Bm4, Bm6 and Bm15 purified by SDS-PAGE electrophoresis analysis, a single band of the desired recombinant protein. ELISA analysis of Bm4, Bm6, Bm15 and BmSA1 (reference) recombinant proteins in mice sera infected with Babesia vovitae and healthy mice showed that the sensitivity of the recombinant proteins Bm4, Bm6, Bm15 and BmSA1 were 15.0% 55.0%, 80.0%, 100.0% and specificity of 100.0%, 100.0%, 90.0% and 100.0% respectively. Bm4, Bm6 and Bm15 did not cross-react with serum of malaria patients. BmSA1 had some cross-reaction with serum of malaria patients (the false-positive rate was 13.3%). Conclusion Three candidate diagnostic antigens of Babesia vaporariorum were expressed. The preliminary evaluation showed that the Bm15 antigen has good sensitivity and specificity for the detection of Babesia vivax.