Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70(TKD) of Haemaphysalis flava.
Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70(TKD) of Haemaphysalis flava.
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黄血蜱HSC70与HSC70TKD抗凝活性及免疫原性对比分析
DOI：
10.1186/s13071-022-05521-2
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发表时间：
2022-11-05
影响因子：
3.2
通讯作者：
Duan DY
中科院分区：
文献类型：
--
作者：
Liu YK;Liu GH;Liu L;Wang AB;Cheng TY;Duan DY
黄血虫是一种嗜血的体外寄生虫，通过吸食血液和消化血粉来获取发育和繁殖所需的营养。在吸血和消化血粉的过程中，防止血液凝固对这种蜱来说很重要。已有研究表明，热休克同源蛋白70 （HSC70）具有一定的抗凝血活性，但其免疫原性尚不清楚。此外，通过重叠延伸法突变HSC70的tkd样肽的单个碱基是否会改变其抗凝血活性和免疫原性还有待研究。方法从h合成的互补DNA文库中克隆编码HSC70蛋白的基因。黄颜色。利用重叠延伸法将HSC70的TKD样肽编码基因突变为TKD肽编码基因HSC70TKD。构建大肠杆菌原核表达质粒，获得重组蛋白HSC70 （rHSC70）和HSC70TKD（rHSC70TKD）。采用四种体外凝血试验评估不同浓度的rHSC70和rhsc70tkd的抗凝活性。采用完全和不完全弗氏佐剂对重组蛋白进行皮下免疫。采用间接酶联免疫吸附法检测血清抗体滴度和血清干扰素γ (IFN-γ)和白细胞介素4 （IL-4）浓度，以评估rHSC70和rHSC70TKD的免疫原性。结果成功扩增出HSC70的开放阅读框，长度为1958 bp。对编码HSC70 tkd样肽的基因进行人工突变，将原序列的1373位腺嘌呤(A)突变为鸟嘌呤(G)， 1385位胞嘧啶(C)突变为G， 1386位G突变为C。利用表达质粒pET-28a-HSC70和pET-28a-HSC70TKD分别获得了与His-tag融合的rHSC70和rhsc70tkd。与阴性对照相比，rHSC70和rhsc70tkd可延长血浆凝血酶时间（TT），降低血浆纤维蛋白原（FIB）含量，但对凝血酶原时间（PT）和活化部分凝血活酶时间（APTT）无影响。有趣的是，rhsc70tkd延长TT和降低血浆中FIB含量的能力优于rHSC70。第三次免疫14天后，rHSC70和rHSC70TKDin大鼠血清特异性抗体滴度均达到1:12 . 4,000。rhsc70tkd组血清IFN-γ浓度高于rHSC70组。rHSC70组血清IL-4浓度最高，且rhsc70tkd组血清IL-4浓度高于阴性组。结论srhsc70和rhsc70tk通过延长体外TT和降低体外FIB含量表现出抗凝血活性。rhsc70tkd的抗凝血活性优于rHSC70。rHSC70和rhsc70tkd均具有良好的免疫原性和诱导的体液免疫和细胞免疫。
BackgroundHaemaphysalis 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.
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DOI：
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发表时间：
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影响因子：
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