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

摘要/Abstract

摘要： 褐黄血蜱(Haemaphysalis flava, H.flava)热休克蛋白70-b2(heat shock protein 70-b2, HSP70-b2)属于HSP70家族。为评估褐黄血蜱HSP70-b2的抗凝血活性、免疫原性及其类14-Mer肽的作用,本研究通过RACE技术扩增褐黄血蜱HSP70-b2基因全长;定点突变其编码类14-Mer肽段基因序列获得HSP70-b2^M基因全长;分别构建原核表达质粒,经原核表达、镍柱层析纯化获得可溶性重组蛋白,体外凝血四项试验评估rHSP70-b2与rHSP70-b2^M的抗凝血活性;将重组蛋白与弗氏(不)完全佐剂按推荐剂量乳化,皮下免疫Sprague Dawley(SD)大鼠,间接ELISA检测血清抗体效价,探究rHSP70-b2与rHSP70-b2^M的免疫原性。结果显示:HSP70-b2基因全长2 413 bp,开放阅读框长1 905 bp,编码蛋白中具有HSP70蛋白家族标签。成功将HSP70-b2中编码类14-Mer肽段的核酸序列突变为编码14-Mer肽段的核酸序列。与对照组相比,rHSP70-b2和rHSP70-b2^M对凝血酶原时间(prothrombin time, PT)和活化部分凝血酶时间(activated partial thromboplastin time, APTT)无显著作用(P＞0.05),但均可延长凝血酶时间(thrombin time, TT)和纤维蛋白原(FIB)凝血时间(P＜0.05),且rHSP70-b2^M与rHSP70-b2的抗血凝效果相近(P＞0.05)。在3次免疫后,大鼠血清中抗体效价随免疫次数的增加而提升,且rHSP70-b2诱导产生的抗体水平极显著高于rHSP70-b2^M(P＜0.01)。综上表明:rHSP70-b2和rHSP70-b2^M在体外可通过延长凝血酶时间和降低纤维蛋白原含量发挥抗凝血作用;rHSP70-b2和rHSP70-b2^M均具有良好的免疫原性,可诱发体液免疫。

关键词: 褐黄血蜱, HSP70-b2, 定点突变, 原核表达, 抗凝活性, 免疫原性

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

中图分类号:

S852.746

程天印, 吴聪颖, 刘雨珂, 段德勇. 褐黄血蜱HSP70-b2及其类14-Mer肽的作用研究[J]. 畜牧兽医学报, 2023, 54(6): 2570-2580.

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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