双价猪流行性腹泻病毒特异性双峰驼源单域抗体的重组表达和特征分析
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摘要
旨在构建抗猪流行性腹泻病毒(PEDV)膜蛋白(M)双峰驼源sdAb(sdAb-Mc19/29/30/37)的二价抗体,并比较研究单价和二价sdAb-Mc对温度的敏感性、pH耐受性、血清抗体竞争活性及抗原捕获活性等技术指标。研究结果证实,单价和二价sdAb-Mc在30～70℃和pH 2～10条件下,均可以结合M蛋白,当温度高于80℃和pH大于11时,其与M蛋白的结合活性显著下降。此外,研究发现,重组二价抗体的血清抗体竞争活性和抗原捕获活性强于单价的抗体。研究结果说明,重组表达的二价sdAb-Mc抗体对温度和pH耐受性没有改变,但其与靶抗原结合活性得到显著提高,为研制敏感和特异的PEDV病原学快速检测方法奠定基础。
Compared with conventional antibodies,the single-domain antibody(sdAb),characterized by small size, high solubility and higher stability, is caused extensive concern in medical and biotechnological applications. Herein, bivalent antibodies of bactrian camel-derived sdAb(sdAb-Mc19/29/30/37) against the membrane protein(M) of porcine epidemic diarrhea virus(PEDV) were constructed, and the stability in different temperature and pH conditions, competitive capacity with polyclonal serum antibodies against M protein, and capture capacity to M protein of the monovalent and bivalent sdAb-Mcwere compared. The results confirmed that both monovalent and bivalent sdAb-Mc have excellent binding activity to M protein under the condition of 30-70 ℃ and pH 2-10. However,the binding activity was decreased significantly when the temperature higher than 80 ℃ and pH greater than 11. Furthermore, the bivalent sdAb-Mc showing stronger competitive capacity to serum antibodies and capture capacity to M protein than monovalent sdAb-Mc.The results revealed that the properties of sdAb-Mc resistance to temperature and pH condition were not influenced by constructing bivalent sdAb, but its binding activity with target antigen was significantly improved.The bivalent sdAb-Mc obtained in this study can be a powerful tool for the development of sensitive and specific PEDV detection methods.
Compared with conventional antibodies,the single-domain antibody(sdAb),characterized by small size, high solubility and higher stability, is caused extensive concern in medical and biotechnological applications. Herein, bivalent antibodies of bactrian camel-derived sdAb(sdAb-Mc19/29/30/37) against the membrane protein(M) of porcine epidemic diarrhea virus(PEDV) were constructed, and the stability in different temperature and pH conditions, competitive capacity with polyclonal serum antibodies against M protein, and capture capacity to M protein of the monovalent and bivalent sdAb-Mcwere compared. The results confirmed that both monovalent and bivalent sdAb-Mc have excellent binding activity to M protein under the condition of 30-70 ℃ and pH 2-10. However,the binding activity was decreased significantly when the temperature higher than 80 ℃ and pH greater than 11. Furthermore, the bivalent sdAb-Mc showing stronger competitive capacity to serum antibodies and capture capacity to M protein than monovalent sdAb-Mc.The results revealed that the properties of sdAb-Mc resistance to temperature and pH condition were not influenced by constructing bivalent sdAb, but its binding activity with target antigen was significantly improved.The bivalent sdAb-Mc obtained in this study can be a powerful tool for the development of sensitive and specific PEDV detection methods.
引文
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[2] DORTMANS JCFM,LI W,VANDER WOLF P J,et al.Porcine epidemic diarrhea virus (PEDV) introduction into a naive Dutch pig population in 2014.[J].Vet Microbiol,2018,221:13-18.
[3] CHEN P,WANG K,HOU Y,et al.Genetic evolution analysis and pathogenicity assessment of porcine epidemic diarrhea virus strains circulating in part of China during 2011-2017[J].Infection,Genetics and Evolution:Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases,2019,69:153-165.
[4] 王婧,刁小龙,杨海峰,等.猪流行性腹泻病毒的鉴定及其S基因的序列分析[J].西北农业学报,2019,28(1):25-30.WANG J,DIAO X L,YANG H F,et al.Identification and sequece analysis of S gene from porcine epidemic diarrhea virus isolates[J].Acta Agriculturae Boreali-occidentalis Sinica,2019,28(1):25-30.
[5] 王艳丰,张丁华,李爱心,等.我国部分地区猪流行性腹泻、传染性胃肠炎及猪轮状病毒感染的流行现状及防控措施[J].动物医学进展,2018,39(6):121-125.WANG Y F,ZHANG D H,LI A X,et al.Epidemic status and control measure of porcine epidemic diarrhea,infectious gastroenteritis and porcine rotavirus infection in parts of China[J].Progress in Veterinary Medicine,2018,39(6):121-125.
[6] HAMERS-CASTERMAN C,ATARHOUCH T,MUYLDERMANS S,et al.Naturally occurring antibodies devoid of light chains[J].Nature,1993,363(6428):446-448.
[7] DUMOULIN M,CONRATH K,VAN MEIRHEAGHE A,et al.Single-domain antibody fragments with high conformational stability[J].Protein Science:a Publication of the Protein Society,2002,11(3):500-515.
[8] ANDERSON G P,LIU J H,ZABETAKIS D,et al.Thermal stabilization of anti-alpha-cobratoxin single domain antibodies[J].Toxicon:Official Journal of the International Society on Toxinology,2017,129:68-73.
[9] ARBAI-GHAHROUDI M.Camelid single-domain antibodies:historical perspective and future outlook[J].Frontiers in Immunology,2017,8:1589.
[10] BANNAS P,HAMBACH J,KOCH-NOLTE F.Nanobodies and nanobody-based human heavy Chain antibodies as antitumor therapeutics[J].Frontiers in Immunology,2017,8:1603.
[11] BEGHEIN E,GETTEMANS J.Nanobody technology:a versatile toolkit for microscopic imaging,protein-protein interaction analysis,and protein function exploration[J].Frontiers in Immunology,2017,8:771.
[12] GONZALEZ-SAPIENZA G,ROSSOTTI M A,TABARES-DA ROSA S.Single-domain antibodies as versatile affinity reagents for analytical and diagnostic applications[J].Frontiers in Immunology,2017,8:977.
[13] YANG S L,II L,YIN S H,et al.Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus[J].Applied Microbiology and Biotechnology,2018,102(20):8931-8942.
[14] DUHOO Y,ROCHE J,TRINH TTN,et al.Camelid nanobodies used as crystallization chaperones for different constructs of PorM,a component of the type IX secretion system from Porphyromonas gingivalis[J].Acta Crystallogr F,2017,73:286-293.
[15] YANG S,SHANG Y,YIN S,et al.A phage-displayed single domain antibody fused to alkaline phosphatase for detection of porcine circovirus type 2[J].Journal of Virological Methods,2015,213:84-92.
[16] BOND C J,MARSTERS J C,SIDHU S S.Contributions of CDR3 to VHH domain stability and the design of monobody scaffolds for naive antibody libraries[J].Journal of Molecular Biology,2003,332(3):643-655.
[17] HULTBERG A,TEMPERTON N J,ROSSEELS V,et al.Llama-derived single domain antibodies to build multivalent,superpotent and broadened neutralizing anti-viral molecules[J].PloS one,2011,6(4):e17665.
[18] VANDER LINDEN R H,FRENKEN L G,DE GEUS B,et al.Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies[J].Biochimica et Biophysica Acta,1999,1431(1):37-46.
[19] HARMSEN M M,VAN SOLT C B,VAN ZIJDERVELD-VAN BEMMEL A M,et al.Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy[J].Applied Microbiology and Biotechnology,2006,72(3):544-551.
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