y集學(xué)位答辯mu1納米抗體 高通量篩

1、1 碩博生：穆碩博生：穆 彬彬?qū)?dǎo) 師：閻錫蘊(yùn)師：閻錫蘊(yùn) 研究員研究員23Nobel Prize in Physiology or Medicine in 1984 Georges J.F. Khler Csar MilsteinNiels K. Jerne Discovery of the hybridoma technique for the production of monoclonal antibodiesmyeloma cellmyeloma celldie4IgG, IgD, IgEIgAIgMHcAbNARCamelShark5Classical AbHeavy-chain A

2、b6FR2FR3FR4CDR1CDR2CDR3FR1CDR1FR2CDR2CDR3FR3FR4VHVHH (nanobody)disulfide linkage V37F37G44E44L45R45W47G47FR1Classical AbHeavy-chain AbVHHydrophobic amino acidHydrophilic amino acidFR2: hydrophilic amino acid substitutionCDR1 & CDR3: longerExtra sulfide linkage78DiseasePathogen Target antigenVHH

3、valencyAdditional fusion partnerSleeping sicknessTrypanosomesVSG oligomannnoseMonovalentApolipoprotein L-1Infant diarrheaRotavirusUnknownMonovalentNoneInfant diarrheaRotavirusUnknownMonovalentLactobacillus cell-surface anchorPiglet SiarrheaE. coliF4 fimbriaeMonovalentNoneAIDSHIVgp120MonovalentNoneCa

4、riesS. mutansI/II adhesionMonovalentNoneFMDFMD virusVP1MonovalentPEGSepsisN. meningitidesLPSMonovalentNoneCancerCEAMonovalentb b-LactamaseCancerEGF receptorBivalentAnti-albumin VHHRheumatoid arthritisTNF-a aBivalentAnti-albumin VHHBrain disordersa a(2,3)- SialoglycoproteinMonovalentNoneNeurodegenera

5、tive diseasesBaxMonovalentNone910flu1112 lothers13Heavy-chain AbCooperated Shanxi Alpaca Co. Ltd14leukocyte Immunized with Inactivated H5N110ml peripheral bloodRNA extractionSize：2106l Diversity： sequencingl Positive rate15Classical AbHeavy-chain AbFirst PCRSecond PCR16M13KO7Influenza viral particle

6、sEnrichment of specific clones1718Binding specificitySDS-PAGEWB: anti-His19ompA1VT1B2sdAb3HmycLow affinity202122Pentabody dissociated from influenza virus more slowly2324Viral protein concentration030ag3fg3pg3ng020004000600080001000012000140002500000OD390nmhydroquinonequinone25Viral particlepVHH3B f

7、or capture, mAb 3C8 for detectionmAb 3C8 for capture, mAb 3C8 for detection26Viral protein concentration030ag3fg3pg3ng020004000600080001000012000140002500000OD390nmhydroquinonequinone27MNP biosensorDAS-ELISA28 New structural antibody- Pantabody New detection system- MNPs based biosensor New reagent

8、for readout the optical density of quinone at 390nm Less-time consumingBin Mu, et.al., Influenza virus detection with pentabody-functionalized nanoparticles. J Virol Method (under review)29(Phage ELISA)nanobodies 3031Purification of nanobodiesNanobody characterizing B12 binderPhage ELISAB12 binderB1

9、2 binderB12 binderB12 binderB12 tagSecreted diabody in culture mediumExpressionBSAB12 binderStability SPRAffinity anti-BSA nanobody32Amplification of genes of nanobodies from phagespFASEBAB12B12 Tag: anti-BSA nanobodynanobodyLB12LHissecretionB12 binderB12 binderB12 binderB12 binderB12 binderRandomly

10、 select clonesIgG immunized nanobody library2 rounds panning33Culture mediumELISA on target antigenlStability of immobilized BSAlAccuracy lReproducibility4 ChannelsRegenerated for analysis of other nanobodies SPR analysis34BSA on the chip is stable Most nanobodies-B12 bind to immobilized BSA very we

11、ll chipB12B12nanobody35Sequence: 12 different clones36lDoes B12 affect the binding capacity of nanobodies?lCan nanobody-B12 in culture medium represent purified nanobody?Different?B12nanobodynanobody37FC7-B12FC12-B12FC75-B12Novel binderNovel binderNovel binder38Dissociation phase to FcCorrelation be

12、tween amounts of Fc17-B12 captured by BSA and amounts of Fc bound to Fc178 x 10-3 1/s SD 6.7%Kd :27 independent transformants of Fc17-B1239Culture mediumELISA on BSABiophysical properties of a fusion protein with a stable and less-stable partner will be mainly dependent on the features of the latter

13、B12 is very soluble and with high expression levelHypothesis: fusion protein amount in culture medium reflect the expression of different cloneBSAB12 binder40Different expression level in culture medium. Amino acid sequencesBSAB12 binderCDR1CDR310 clones41 Does the nanobody-B12 amount in culture med

14、ium reflect the expression level and the solubility of nanobody? 42WB: Cell lysate anti-His43 Whether B12 affect the expression of nanobodies?44S: supernatantP: pelletWestern Blot: Cell lysateanti-HisELISA:Culture mediumB12nanobodynanobodyWestern Blotanti-HisheatingfiltrationELISA on BSAExclude prot

15、eins prone to aggregation BSAB12 binder46Protein ELISA with heated or non-heated culture medium47 Does B12 affect the heat stability of nanobodies? 48SEC of purified human VH 49Purification of nanobodiesNanobody characterizing B12 binderPhage ELISAB12 binderB12 binderB12 binderB12 binderB12 tagSecre

16、ted diabody in culture mediumExpressionBSAB12 binderStability SPRAffinity 50 Select clones with good expression level, solubility, stability and affinity High-throughput Less time-consuming No need for protein purification Automatic analysis the BSA coated chips could be reused This new method is no

17、t only suitable for nanobodies isolation but also for the evaluation of other engineered proteinsBin Mu, et al., Fast screening for expression levels, biophysical properties and affinities of large number of proteins. Nucleic Acids Res (under review)511. Bin Mu, Xinglu Huang, Pengcheng Bu, Jie Zhuan

18、g, Jing Feng, Dongling Yang, Changsheng Dong, Jianbing Zhang, Xiyun Yan. Influenza virus detection with pentabody-functionalized nanoparticles. J Virol Method (under review)2. Bin Mu, Tomoko Hirama, Shenghua Li, Toya Nath Baral, Yiling Chen, Xinyun Yan, Jianbing Zhang. Fast screening for expression levels, biophysical properties and affinities of large number of proteins. Nucleic Acids Research (under review)3. Bin Mu, Fei Wang, Yufei Long, Tao Tu, Xiyun Yan. A panel of neutralization single domain antibodies again HIV. (In preparation)4. Bin Mu