Abstract: 【Objective】 The aim of this study was to study the mechanism of trypanosome invasion into host cells and to lay the foundation for the establishment of detection methods.【Method】 The isolated and preserved Trypanosoma evansi was used for culture propagation in Kunming White mice,and its paraflagellar fasciculus rod (PFR) gene was cloned and a phylogenetic tree was constructed.The physical and chemical properties,hydrophobicity,transmembrane region structure,secondary structure and tertiary structure of PFR protein were analyzed and predicted by bioinformatics methods.The prokaryotic expression vector pET28a-PFR was constructed,and the reactogenicity of PFR recombinant protein was detected by Western blotting.【Result】 The Yili strain of Trypanosoma evansi was successfully expanded in Kunming White mice,and the highest infection rate was reached on the 5th day.The PCR amplified fragment size of PFR gene was 834 bp,and the similarity with PFR gene of Trypanosoma brucei gambiense (XP_011775815.1) was 99.52%,and the phylogenetic tree based on the amino acid sequence of the PFR protein also showed that the strain had the closest genetic relationship with Trypanosoma brucei gambiense.The molecular formula of PFR protein was C₁₄₁₆H₂₂₈₆N₄₁₆O₄₄₂S₁₁,and the theoretical isoelectric point was 5.74.PFR protein was an alkaline,hydrophilic and unstable protein,without transmembrane region and signal peptide,and had 10 potential antigen epitopes,which were mainly located in cytoplasm.The secondary structure of PFR protein was mainly composed of alpha helix (92.34%),the tertiary structure was consistent with the secondary structure.The expression plasmid pET28a-PFR of PFR gene of Trypanosoma evansi was successfully constructed.After optimization of induction time,temperature and IPTG concentration,it was found that when IPTG concentration was 1 mmol/L and recombinant bacterial solution was induced at 37 ℃ for 6 hours,PFR protein expression was the highest and expressed in the form of inclusion body.Western blotting results showed that the recombinant protein reacted positively with Trypanosoma evansi positive serum.【Conclusion】 In this experiment, Trypanosoma evansi was successfully propagated in Kunming White mice,the PFR gene of Trypanosoma evansi was cloned,the prokaryotic expression vector of PFR was constructed,and the recombinant protein of PFR was induced to express.The protein had good reactivity,which provided a theoretical basis for the subsequent research on the function of the protein and the pathogenesis of Trypanosoma evansi.

Key words: Trypanosoma evansi; paraflagellar rod (PFR); prokaryotic expression; genetic evolution; biological characteristics