Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70TKD of Haemaphysalis flava

Abstract

AbstractBackgroundHaemaphysalis flavais a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated.MethodsThe gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized fromH. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70TKD) using the overlap extension method.Escherichia coliprokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70TKD(rHSC70TKD). The purified rHSC70 and rHSC70TKDwere evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund’s adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70TKD.ResultsThe open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70TKDthat fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70TKD, respectively. rHSC70 and rHSC70TKDprolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70TKDto prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70TKDin rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70TKDgroup was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70TKDgroup was higher than that in the negative group.ConclusionsrHSC70 and rHSC70TKDexhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70TKDhad better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70TKDhad good immunogenicity and induced humoral and cellular immunity.