Abstract: 【Objective】 This study aimed to express the invariant surface glycoprotein 65 (ISG 65) of Trypanosoma evansi (T.evansi),analyze its biological characteristics and immunogenicity,and prepare polyclonal antibody against T.evansi ISG 65 protein,so as to lay a foundation for the subsequent development of detection methods for this disease pathogen. 【Method】 ISG 65 gene of T.evansi Xinjiang strain was amplified by PCR.Similarity alignment of the measured sequences was performed and phylogenetic tree was constructed.Bioinformatics tools were used to predict and analyze the physicochemical properties,hydrophilicity and hydrophobicity,transmembrane region,signal peptide,phosphorylation sites,and subcellular localization of the ISG 65 protein.After homologous recombination,the ISG 65 gene fragment was transfected into E.coli BL21(DE3) competent cells,recombinant ISG 65 protein (rISG 65) was induced by IPTG and purified by nickel column affinity chromatography.The purification of the ISG 65 protein was verified by Western blotting and SDS-PAGE.The polyclonal antibodies were obtained by immunizing Kunming mice with the purified rISG 65 protein.The titer of the polyclonal antibodies and the reactivity of the recombinant protein were determined using indirect ELISA and Western blotting. 【Result】 The PCR amplification product of ISG 65 gene of T.evansi Xinjiang strain was 657 bp in size,and had the highest similarity with Trypanosoma brucei gambiense (accession No.:XM_011773430.1 and XM_011773437.1) at 99.06%.It was closely related to Trypanosoma brucei gambiense (accession No.:XM_011773430.1) and clustered into one branch.Bioinformatics analysis revealed that ISG 65 protein was a hydrophilic protein consisting of 436 amino acids with a theoretical isoelectric point of 8.02.ISG 65 protein was predicted to have 35 phosphorylation sites.The secondary structure comprised alpha helix (53.21%),random coil (33.03%),beta turn (2.98%),and extended strand (10.78%).ISG 65 protein had 14 B cell antigen epitopes,which showed good immunogenicity.pET-28a-ISG 65 recombinant plasmid was successfully constructed in this experiment.SDS-PAGE results showed that the recombinant pET-28a-ISG 65-BL21 was highly expressed in 1.0 mmol/L IPTG at 37 ℃ for 8 h,yielding a protein of approximately 24 ku after purification.The results of indirect ELISA detection showed that the titer of the obtained ISG 65 polyclonal antibody was 1∶1 638 400. 【Conclusion】 The recombinant ISG 65 protein expressed in prokaryotic cells exhibited good immunogenicity,could specifically bind to T.evansi positive serum,and possessed favorable biological characteristics.This study provided important reference data and theoretical support for future research on the biological characteristics of T.evansi ISG 65 protein and the establishment of ELISA diagnostic methods.

Key words: Trypanosoma evansi; ISG 65 protein; prokaryotic expression; polyclonal antibody