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.

References 32

[1]	严芬, 程天印. 褐黄血蜱的形态学与分子生物学鉴定[J]. 中国兽医学报, 2015, 35(6):912-916.YAN F, CHENG T Y. Morphological and molecular identification of Haemaphysalis flava[J]. Chinese Journal of Veterinary Science, 2015, 35(6):912-916. (in Chinese)
[2]	JUNG M, KHO J W, LEE W G, et al. Seasonal occurrence of Haemaphysalis longicornis (Acari: Ixodidae) and Haemaphysalis flava, vectors of severe fever with thrombocytopenia syndrome (SFTS) in south Korea[J]. J Med Entomol, 2019, 56(4):1139-1144.
[3]	FANG L Z, LEI S C, YAN Z J, et al. Detection of multiple intracellular bacterial pathogens in Haemaphysalis flava ticks collected from hedgehogs in central China[J]. Pathogens, 2021, 10(2):115.
[4]	GUO J Y, SUN Y L, LUO X Y, et al. De novo transcriptome sequencing and comparative analysis of Haemaphysalis flava neumann, 1897 at larvae and nymph stages[J]. Infect Genet Evol, 2019, 75:104008.
[5]	EJIRI H, LIM C K, ISAWA H, et al. Isolation and characterization of Kabuto mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan[J]. Virus Res, 2018, 244:252-261.
[6]	苏学芳, 靳爱华, 宋红娥. 乐陵市2013-2020年发热伴血小板减少综合征的流行病学特点分析[J]. 临床医学, 2022, 42(3):1-3.SU X F, JIN A H, SONG H E. Epidemiological characteristics of severe fever with thrombocytopenia syndrome in Leling city from 2013 to 2020[J]. Clinical Medicine, 2022, 42(3):1-3. (in Chinese)
[7]	MAYER M P, BUKAU B. Hsp70 chaperones:cellular functions and molecular mechanism[J]. Cell Mol Life Sci, 2005, 62(6):670-684.
[8]	LIU L, CHENG T Y, YANG Y. Cloning and expression pattern of a heat shock cognate protein 70 gene in ticks (Haemaphysalis flava)[J]. Parasitol Res, 2017, 116(6):1695-1703.
[9]	HE X M, LIU L, CHENG T Y. HSC70 from Haemaphysalis flava (Acari:Ixodidae) exerts anticoagulation activity in vitro[J]. Ticks Tick-Borne Dis, 2019, 10(1):170-175.
[10]	廖芷卉. 褐黄血蜱中肠转录组分析和serpin 2表达、鉴定[D]. 长沙:湖南农业大学, 2016.LIAO Z H. Midgut transcriptome analysis of Haemaphysalis flava and expression and identification of serpin 2[D]. Changsha: Hunan Agricultural University, 2016. (in Chinese)
[11]	SKEIKY Y A, BENSON D R, GUDERIAN J A, et al. Immune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans[J]. Infect Immun, 1995, 63(10):4105-4114.
[12]	KUMAR J, CHAUDHURY A, BERA B C, et al. Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids[J]. Acta Parasitol, 2015, 60(4):727-734.
[13]	FANG L, SUN L, YANG J, et al. Heat shock protein 70 from Trichinella spiralis induces protective immunity in BALB/c mice by activating dendritic cells[J]. Vaccine, 2014, 32(35):4412-4419.
[14]	MULTHOFF G, PFISTER K, GEHRMANN M, et al. A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity[J]. Cell Stress Chaperones, 2001, 6(4):337-344.
[15]	和小明. 褐黄血蜱热休克蛋白70家族的克隆、表达及抗凝血功能研究[D]. 长沙:湖南农业大学, 2018.HE X M. The cloning, expression and anticoagulation activity studies of heat shock protein 70 family in Haemaphysalis flava[D]. Changsha: Hunan Agricultural University, 2018. (in Chinese)
[16]	MOGK A, BUKAU B. Molecular chaperones:structure of a protein disaggregase[J]. Curr Biol, 2004, 14(2):78-80.
[17]	RENSING S A, MAIER U G. Phylogenetic analysis of the stress-70 protein family[J]. J Mol Evol, 1994, 39(1):80-86.
[18]	BAUSERO M A, GASTPAR R, MULTHOFF G, et al. Alternative mechanism by which IFN-γ enhances tumor recognition:active release of heat shock protein 72[J]. J Immunol, 2005, 175(5):2900-2912.
[19]	DEMAND J, LUDERS J, HOHFELD J. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors[J]. Mol Cell Biol, 1998, 18(4):2023-2028.
[20]	杨亚. 褐黄血蜱HSP70基因克隆与表达分析及中肠微生物构成研究[D]. 长沙:湖南农业大学, 2015.YANG Y. Studies on gene clone and expression of Haemaphysalis flava HSP70 and identification of the microflora in the midgut[D]. Changsha: Hunan Agricultural University, 2015. (in Chinese)
[21]	RAN R Q, ZHOU G P, LU A G, et al. Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival[J]. Cell Stress Chaperones, 2004, 9(3):229-242.
[22]	FEDER M E, HOFMANN G E. Heat-shock proteins, molecular chaperones, and the stress response:evolutionary and ecological physiology[J]. Annu Rev Physiol, 1999, 61:243-282.
[23]	BROCCHIERI L, DE MACARIO E C, MACARIO A J L. Hsp70 genes in the human genome:conservation and differentiation patterns predict a wide array of overlapping and specialized functions[J]. BMC Evol Biol, 2008, 8:19.
[24]	ZHOU W S, TAHIR F, WANG J C Y, et al. Discovery of exosomes from tick saliva and salivary glands reveals therapeutic roles for CXCL12 and IL-8 in wound healing at the tick-human skin interface[J]. Front Cell Dev Biol, 2020, 8:554.
[25]	LI P C, YANG C C, HSU S P, et al. Repetitive progressive thermal preconditioning hinders thrombosis by reinforcing phosphatidylinositol 3-kinase/Akt-dependent heat-shock protein/endothelial nitric oxide synthase signaling[J]. J Vasc Surg, 2012, 56(1):159-170.
[26]	ALLENDE M, MOLINA E, GURUCEAGA E, et al. Hsp70 protects from stroke in atrial fibrillation patients by preventing thrombosis without increased bleeding risk[J]. Cardiovasc Res, 2016, 110(3):309-318.
[27]	SULTAN P, BUTWICK A. Platelet counts and coagulation tests prior to neuraxial anesthesia in patients with preeclampsia:a retrospective analysis[J]. Clin Appl Thromb Hemost, 2013, 19(5):529-534.
[28]	苏敏, 梁慧超, 孟江, 等. 姜炭对虚寒性出血证大鼠凝血功能, 血栓素B2和6-酮-前列腺素F1α的影响[J]. 中国实验方剂学杂志, 2013, 19(9):190-193.SU M, LIANG H C, MENG J, et al. Investigation of Zingiber officinale carbo on coagulation functionand TXB2, 6-keto-pgfIα in asthenia cold and blood deficiency syndrome rats[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2013, 19(9):190-193. (in Chinese)
[29]	RAND K N, MOORE T, SRISKANTHA A, et al. Cloning and expression of a protective antigen from the cattle tick Boophilus microplus[J]. Proc Natl Acad Sci U S A, 1989, 86(24):9657-9661.
[30]	SINGH-JASUJA H, HILF N, ARNOLD-SCHILD D, et al. The role of heat shock proteins and their receptors in the activation of the immune system[J]. Biol Chem, 2001, 382(4):629-636.
[31]	CHIARELLI F, CANFORA G, VERROTTI A, et al. Humoral and cellular immunity in children with active and quiescent atopic dermatitis[J]. Br J Dermatol, 1987, 116(5):651-660.
[32]	蔺焕然, 史泽风, 郭庆勇, 等. 犬吉氏巴贝斯虫南京分离株HSP70基因的原核表达及抗原性分析[J]. 中国兽医学报, 2020, 40(9):1754-1757, 1764.LIN H R, SHl Z F, GUO Q Y, et al. Prokaryotic expression of Babesia gibsoni HSP70 gene of Nanjing strain and antigenicity analysis of recombinant protein[J]. Chinese Journal of Veterinary Science, 2020, 40(9):1754-1757, 1764. (in Chinese)

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

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