目的 表达田鼠巴贝虫重组肽基脯氨酸异构酶（BmPPIase）基因并分析其功能。 方法 利用生物信息学方法筛选分析BmPPIase基因信息。通过BmPPIase全基因合成获得目的片段BmPPIase，构建重组质粒pET28a-BmPPIase，转入大肠埃希菌BL21感受态细胞中进行原核表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析BmPPIase重组蛋白表达情况，镍柱亲和纯化重组蛋白，小牛血清蛋白（BSA）法测定重组蛋白浓度。取BmPPIase重组蛋白在新西兰白兔背部多点免疫4次（第1、15、29、43天，每次1.5 mg），首次免疫后第53天颈动脉采血，ELISA鉴定其特异性多克隆抗体效价。蛋白质免疫印迹（Western blotting）分析多克隆抗体的免疫原性。免疫荧光定位实验判断PPIase在田鼠巴贝虫虫体的位置。体外抑虫实验分别设置BmPPIase重组蛋白终浓度500、250、100、50、10 μg/ml组，以及重组剪接因子1组（无关蛋白对照组，500、250、100、50、10 μg/ml）、BSA组、空白对照组，各组加入小鼠感染红细胞孵育48 h，经溴化乙锭标记后，采用流式细胞仪检测感染红细胞的比例，分析重组蛋白在田鼠巴贝虫感染宿主红细胞过程中的作用。 结果 生物信息学分析结果显示，BmPPIase为亲环素类PPIases；在牛巴贝虫、泰勒虫、人芽囊原虫、疟原虫中均存在同源蛋白。重组质粒酶切结果显示，插入的基因大小为531 bp，与预期相符，测序结果正确。SDS-PAGE分析结果显示，BmPPIase重组蛋白为可溶性蛋白，相对分子质量为19 000，经纯化后获得蛋白浓度为1.5 mg/ml。ELISA检测结果显示，抗BmPPIase重组蛋白特异性多克隆抗体效价> 1 : 80 000。Western blotting分析结果显示，制备的抗BmPPIase多抗可特异识别重组蛋白。免疫荧光定位实验结果显示，BmPPIase分布在田鼠巴贝虫虫体表面，属于分泌蛋白。体外抑虫实验结果显示，重组BmPPIase对虫体入侵小鼠红细胞存在一定程度的抑制作用：BmPPIase重组蛋白浓度为500 μg/ml时，相对染虫率为（47.0 ± 1.2）%；随着重组蛋白浓度的降低，抑虫作用逐渐减弱，250、100、50 μg/ml时相对染虫率分别为（64.0 ± 0.3）%、（78.0 ± 1.9）%和（82.0 ± 0.25）%；至10 μg/ml时相对染虫率为（93.0 ± 0.22）%，已无明显抑虫作用。 结论 BmPPIase属于分泌蛋白，分布于虫体表面；可在体外明显抑制田鼠巴贝虫感染红细胞。

Objective To express the recombinant peptidyl-prolyl cis-trans isomerase gene of Babesia microti (BmPPIase) in prokaryotic cells and analyze its function. Methods BmPPIase information was screened and analyzed by bioinformatics. The target fragment BmPPIase was obtained via the total gene synthesis, to construct the recombinant plasmid pET28a-BmPPIase, which was then transfected into the competent cells of Escherichia coli BL21 for prokaryotic expression. The expressed recombinant protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was purified via nickel column affinity. The recombinant protein concentration was estimated based on the amount of bovine serum albumin (BSA) reference standard. New Zealand rabbits were immunized with the recombinant protein four times (1.5 mg dose each on the day 1, 15, 29, and 43) at multiple sites on their back to elicit antibodies. Carotid blood was collected on the day 53 after primary immunization, to examine specific polyclonal antibody titer using enzyme-linked immunosorbent assay (ELISA), and Western blotting was used to analyze the immunogenicity of the polyclonal antibody. Further, immunofluorescence assay was used to localize its distribution in B. microti. The suppressive effect of the protein was assayed by co-incubation with mice infected blood cells in vitro, and the experiment set recombinant BmPPIase groups (added the protein 500, 250, 100, 50, and 10 μg/ml at final concentration), splicing factor 1 group (unrelated protein control, added 500, 250, 100, 50, 10 μg/ml), BSA group (100 μg/ml), and blank control. After incubation for 48 h, the culture was stained with ethidium bromide, and the proportion of infected red blood cells was measured using flow cytometry to analyze the effect of the protein on the infection of B. microti to host red blood cells. Results Bioinformatics revealed that the BmPPIase was a cyclophilin type of PPIase and was homologous to the same protein in Babesia bovis, Theileria, Blastocystis hominis, and Plasmodium. As expected, the restriction enzyme digestion of the recombinant plasmid showed that the size of the inserted gene was 531 bp, which was validated using sequencing. SDS-PAGE showed that the recombinant protein BmPPIase was a soluble protein with a relative molecular weight of 19 000. After purification and elution, the protein concentration was 1.5 mg/ml. ELISA showed that the specific polyclonal antibody titer of the recombinant protein was > 1 : 80 000. In addition, Western blotting revealed that the polyclonal antibody could specifically recognize the recombinant protein, and the immunofluorescence localization assay showed that BmPPIase was distributed on the surface of the parasite as well as secreted. In vitro inhibition experiments showed that the rBmPPIase protein at different concentrations inhibited the infection of host cells to a certain level, the relative infection rate was (47.0 ± 1.2)% at 500 μg/ml. However, with the decrease of protein concentration, the inhibition effect of BmPPIase gradually weakened, the relative infection rate was (64.0 ± 0.3)% at 250 μg/ml, (78.0 ± 1.9)% at 100 μg/ml and (82.0 ± 0.25)% at 50 μg/ml respectively. The relative infection rate was (93.0 ± 0.22)% at 10 μg/ml with no significant effect. Conclusion The recombinant protein BmPPIase is a secretory protein distributed on the parasite surface, showing significant suppressive effect on B. microti infection of host cells in vitro.