中东呼吸综合征冠状病毒特异性纳米抗体噬菌体展示库的构建和鉴定
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摘要
目的构建中东呼吸综合征冠状病毒(MERS-CoV)特异性纳米抗体噬菌体展示库,并应用于纳米中和抗体筛选。方法使用MERS-CoV的受体结合区(RBD)重组蛋白免疫羊驼后分离外周血单个核细胞(PBMC)并提取总RNA。使用简并引物通过PCR扩增重链抗体的重链可变区(VHH)基因构建重组噬菌粒并转化TG1大肠杆菌克隆菌株,构建特异性纳米抗体噬菌体展示库,利用噬菌体展示技术进行纳米中和抗体筛选和功能鉴定。结果构建的纳米抗体噬菌体展示库库容为1.31×10~8个,丰度为5.65×10~(10)个/mL,VHH片段插入率达到96%,且具有较好的多样性,并利用构建的纳米抗体噬菌体展示文库成功筛选并表达出具有中和活性的纳米抗体。结论成功构建了能有效应用于MERS-CoV中和纳米抗体筛选的噬菌体库。
Objective To construct a phage display library of specific nano-antibodies against the Middle East respiratory syndrome coronavirus( MERS-CoV) and apply it to the screening of neutralizing nano-antibodies. Methods MERS-CoV receptor-binding domain( RBD) recombinant protein was used to immunize alpaca. After the last immunization,peripheral blood mononuclear cells( PBMCs) were isolated from the whole blood and total RNA was extracted. The VHH gene was amplified by PCR and used to construct recombinant phages. TG1 Escherichia coli was transformed by these recombinant phages. A phage display library of specific nano-antibodies against the MERS-CoV were obtained and used to screen and characterize the nano-antibodies. Results The volume of this library of nano-antibodies was 1. 31 × 108 and its abundance rate was 5. 65 × 1010/mL. The ratio of VHH insertion in the constructed library reached 96%. There was a rich diversity of nano-antibodies in this library. Nano-antibodies with neutralizing activity were identified and expressed from this library.Conclusion We successfully constructed a library of phages which could be effectively applied to the screening of nano-antibodies against MERS-CoV virus.
Objective To construct a phage display library of specific nano-antibodies against the Middle East respiratory syndrome coronavirus( MERS-CoV) and apply it to the screening of neutralizing nano-antibodies. Methods MERS-CoV receptor-binding domain( RBD) recombinant protein was used to immunize alpaca. After the last immunization,peripheral blood mononuclear cells( PBMCs) were isolated from the whole blood and total RNA was extracted. The VHH gene was amplified by PCR and used to construct recombinant phages. TG1 Escherichia coli was transformed by these recombinant phages. A phage display library of specific nano-antibodies against the MERS-CoV were obtained and used to screen and characterize the nano-antibodies. Results The volume of this library of nano-antibodies was 1. 31 × 108 and its abundance rate was 5. 65 × 1010/mL. The ratio of VHH insertion in the constructed library reached 96%. There was a rich diversity of nano-antibodies in this library. Nano-antibodies with neutralizing activity were identified and expressed from this library.Conclusion We successfully constructed a library of phages which could be effectively applied to the screening of nano-antibodies against MERS-CoV virus.
引文
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[25]Wang Y,Tai W,Yang J,et al.Receptor-binding domain of MERS-Co Vwith optimal immunogen dosage and immunization interval protects human transgenic mice from MERS-Co V infection[J].Hum Vaccin Immunother,2017,13(7):1615-1624.
[26]Tang J,Zhang N,Tao X,et al.Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERScoronavirus[J].Hum Vaccin Immunother,2015,11(5):1244-1250.
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[2]Tai W,Zhao G,Sun S,et al.A recombinant receptor-binding domain of MERS-Co V in trimeric form protects human dipeptidyl peptidase 4(h DPP4)transgenic mice from MERS-Co V infection[J].Virology,2016,499:375-382.
[3]Rabaan A A,Alahmed S H,Bazzi A M,et al.A review of candidate therapies for Middle East respiratory syndrome from a molecular perspective[J].J Med Microbiol,2017,66(9):1261-1274.
[4]Qiu H,Sun S,Xiao H,et al.Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome(MERS)-coronavirus infection[J].Antiviral Res,2016,132:141-148.
[5]Du L,Zhao G,Yang Y,et al.A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein[J].J Virol,2014,88(12):7045-7053.
[6]Jiang L,Wang N,Zuo T,et al.Potent neutralization of MERS-Co Vby human neutralizing monoclonal antibodies to the viral spike glycoprotein[J/OL].Sci Transl Med,2014,6(234):234ra59.doi:10.1126/scitranslmed.3008140.Epub 2014 Apr 28.
[7]Zhang N,Jiang S,Du L.Current advancements and potential strategies in the development of MERS-Co V vaccines[J].Expert Rev Vaccines,2014,13(6):761-774.
[8]Hamers-Casterman C,Atarhouch T,Muyldermans S,et al.Naturally occurring antibodies devoid of light chains[J].Nature,1993,363(6428):446-448.
[9]Alibakhshi A,Abarghooi Kahaki F,Ahangarzadeh S,et al.Targeted cancer therapy through antibody fragments-decorated nanomedicines[J].J Control Release,2017,268:323-334.
[10]Dmitriev O Y,Lutsenko S,Muyldermans S.Nanobodies as probes for protein dynamics in vitro and in cells[J].J Biol Chem,2016,291(8):3767-3775.
[11]Nuttall S D,Krishnan U V,Hattarki M,et al.Isolation of the new antigen receptor from wobbegong sharks,and use as a scaffold for the display of protein loop libraries[J].Mol Immunol,2001,38(4):313-326.
[12]Wang Y,Fan Z,Shao L,et al.Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications[J].Int J Nanomedicine,2016,11:3287-3303.
[13]Tai W,Wang Y,Fett C A,et al.Recombinant receptor-binding domains of multiple Middle East respiratory syndrome coronaviruses(MERS-Co Vs)induce cross-neutralizing antibodies against divergen human and camel MERS-Co Vs and antibody escape mutants[J/OL].J Virol,2016,91(1):e01651-16.doi:10.1128/JVI.01651-16.Print 2017 Jan 1.
[14]Maass D R,Sepulveda J,Pernthaner A,et al.Alpaca(Lama pacos)as a convenient source of recombinant camelid heavy chain antibodies(VHHs)[J].J Immunol Methods,2007,324(1/2):13-25.
[15]Kastelic D,Frkovic-Grazio S,Baty D,et al.Pompon D.A single-step procedure of recombinant library construction for the selection o efficiently produced llama VH binders directed against cance markers[J].J Immunol Methods,2009,350(1/2):54-62.
[16]Wen Y,Ouyang Z,Schoonooghe S,et al.Structural evaluation of a nanobody targeting complement receptor Vsig4 and its cross reactivity[J].Immunobiology,2017,222(6):807-813.
[17]Veugelen S,Dewilde M,De Strooper B,et al.Screening and characterization strategies for nanobodies targeting membrane proteins[J].Methods Enzymol,2017,584:59-97.
[18]Fu X,Gao X,He S,et al.Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2(PCV2)[J/OL].PLo S One,2013,8(3):e56222.doi:10.1371/journal.pone.0056222.Epub 2013Mar 1.
[19]Yan J,Wang P,Zhu M,et al.Characterization and applications of nanobodies against human procalcitonin selected from a novel naive nanobody phage display library[J/OL].J Nanobiotechnology,2015,13:33.doi:10.1186/s12951-015-0091-7.
[20]Liu X,Tang Z,Duan Z,et al.Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference[J].Talanta,2017,164:154-158.
[21]Lu G,Wang Q,Gao G F.Bat-to-human:spike features determining‘host jump’of coronaviruses SARS-Co V,MERS-Co V,and beyond[J].Trends Microbiol,2015,23(8):468-478.
[22]Ma C,Wang L,Tao X,et al.Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments--the importance of immunofocusing in subunit vaccine design[J].Vaccine,2014,32(46):6170-6176.
[23]Raj V S,Mou H,Smits S L,et al.Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC[J].Nature,2013,495(7440):251-254.
[24]Du L,Tai W,Yang Y,et al.Introduction of neutralizing immunogenicity index to the rational design of MERS coronavirus subunit vaccines[J/OL].Nat Commun,2016,7:13473.doi:10.1038/ncomms13473.
[25]Wang Y,Tai W,Yang J,et al.Receptor-binding domain of MERS-Co Vwith optimal immunogen dosage and immunization interval protects human transgenic mice from MERS-Co V infection[J].Hum Vaccin Immunother,2017,13(7):1615-1624.
[26]Tang J,Zhang N,Tao X,et al.Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERScoronavirus[J].Hum Vaccin Immunother,2015,11(5):1244-1250.
[27]Arbabi-Ghahroudi M.Camelid single-domain antibodies:Historical perspective and future outlook[J/OL].Front Immunol,2017,8:1589.doi:10.3389/fimmu.2017.01589.e Collection 2017.