褐黄血蜱HSP70-b2及其类14-Mer肽的作用研究

doi:10.11843/j.issn.0366-6964.2023.06.034

Abstract

Abstract: Heat shock protein 70-b2 (HSP70-b2) of Haemaphysalis flava is a member of the HSP70 family. The research was to evaluate the anticoagulant activity and immunogenicity of HSP70-b2, and to investigate the role of 14-Mer-like peptide of HSP70-b2 from H. flava. The gene of H. flava HSP70-b2 was amplified in vitro by RACE technology. The coding sequence of 14-Mer-like peptide of HSP70-b2 was site-directed mutated to amplify the HSP70-b2^M gene. Prokaryotic expression plasmids were constructed. The soluble recombinant proteins were expressed by prokaryotic expression system and were purified by nickel column chromatography. The anticoagulant activities of rHSP70-b2 and rHSP70-b2^M were evaluated using four clotting assays in vitro. Emulsifying recombinant proteins with complete and incomplete Freund’s adjuvants were subcutaneously immunized in Sprague Dawley (SD) rats. The serum antibody titers were detected by indirect enzyme linked immunosorbent assay (ELISA) to assess the immunogenicity of rHSP70-b2 and rHSP70-b2^M. Results revealed that the length of HSP70-b2 was 2 413 bp, the open reading frame (ORF) of HSP70-b2 was 1 905 bp, and the encoded protein had typical HSP70 protein family tags. The HSP70-b2 gene sequence encoding the 14-Mer-like peptide was successfully mutated into 14-Mer gene sequence encoding the 14-Mer peptide. Compared with the control group, rHSP70-b2 and rHSP70-b2^M had no significant effect on the prothrombin time (PT) and activated partial thromboplastin time (APTT) (P>0.05). However, rHSP70-b2 and rHSP70-b2^M prolonged the thrombin time (TT) and fibrinogen (FIB) coagulation time (P<0.05), which had similar anticoagulant activities (P>0.05). The antibody titers in rat serum increased with the increase of immunization times, and the antibody titers induced rHSP70-b2 was significantly higher than that of rHSP70-b2^M (P<0.01). The results suggested that rHSP70-b2 and rHSP70-b2^M exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. Both rHSP70-b2 and rHSP70-b2^M had nice immunogenicity and could induce humoral immunity.

Key words: Haemaphysalis flava, HSP70-b2, site directed mutagenesis, prokaryotic expression, anticoagulation, immunogenicity

CLC Number:

S852.746

CHENG Tianyin, WU Congying, LIU Yuke, DUAN Deyong. Studies on the Role of HSP70-b2 and Its 14-Mer-like Peptide from Haemaphysalis flava[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2570-2580.

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Studies on the Role of HSP70-b2 and Its 14-Mer-like Peptide from Haemaphysalis flava

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