关键词: 巴贝虫病, 田鼠巴贝虫, 肽基脯氨酸异构酶, 荧光定位, 体外抑虫实验

Abstract:

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.

Key words: Babesiosis, Babesia microti, Peptidyl-prolyl cis-trans isomerase, Immunofluorescence assay, Inhibition of invasion test