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Cov ntshav-hlwb barrier thiab cov ntshav-hlwb barrier tiv thaiv biotherapeutic cov neeg ua hauj lwm mus txog lawv lub hom phiaj nyob rau hauv lub hauv paus paj hlwb, yog li cuam tshuam txoj kev kho mob ntawm neurological kab mob.Txhawm rau nrhiav pom tshiab lub hlwb thauj khoom hauv vivo, peb tau qhia txog T7 phage peptide lub tsev qiv ntawv thiab sau cov ntshav thiab cerebrospinal kua (CSF) siv lub tshuab ua kom nco qab lub pas dej loj ntawm cov nas.Tshwj xeeb phage clones tau enriched nyob rau hauv CSF tom qab plaub rounds ntawm xaiv.Kev ntsuam xyuas ntawm tus neeg sib tw peptides qhia ntau tshaj 1000-fold enrichment hauv CSF.Lub bioactivity ntawm peptide-mediated xa mus rau lub hlwb tau lees paub los ntawm 40% txo qis ntawm amyloid-β nyob rau hauv cov kua cerebrospinal siv BACE1 peptide inhibitor txuas mus rau cov ntaub ntawv tshiab hloov pauv peptide.Cov txiaj ntsig no qhia tias peptides txheeb xyuas los ntawm vivo phage xaiv txoj hauv kev yuav yog lub tsheb muaj txiaj ntsig rau kev xa tawm ntawm macromolecules rau lub hlwb nrog kev kho mob.
Central nervous system (CNS) lub hom phiaj kev tshawb fawb tau tsom mus rau kev txheeb xyuas cov tshuaj zoo thiab cov neeg sawv cev uas nthuav tawm CNS-targeting zog, nrog rau kev siv zog tsawg dua rau kev tshawb pom cov txheej txheem uas ua rau muaj kev xa tshuaj mus rau lub hlwb.Qhov no yog pib hloov tam sim no raws li kev xa tshuaj, tshwj xeeb tshaj yog cov molecules loj, yog ib feem tseem ceeb ntawm kev txhim kho tshuaj neuroscience niaj hnub no.Ib puag ncig ntawm lub hauv nruab nrab lub paj hlwb muaj kev tiv thaiv zoo los ntawm lub paj hlwb barrier system, muaj cov ntshav-hlwb barrier (BBB) ​​​​thiab cov ntshav-hlwb barrier (BCBB) 1, ua rau nws nyuaj rau xa tshuaj mus rau lub hlwb 1,2.Nws kwv yees tias yuav luag tag nrho cov tshuaj molecule loj thiab ntau dua 98% ntawm cov tshuaj molecule me raug tshem tawm ntawm lub hlwb 3.Tias yog vim li cas nws yog ib qho tseem ceeb heev los txheeb xyuas lub hlwb tshiab thauj cov tshuab uas muab cov tshuaj kho tau zoo thiab tshwj xeeb xa mus rau CNS 4,5.Txawm li cas los xij, BBB thiab BCSFB kuj tseem nthuav tawm lub sijhawm zoo rau kev xa tshuaj thaum lawv nkag mus thiab nkag mus rau txhua yam ntawm lub hlwb los ntawm nws cov vasculature.Yog li, kev siv zog tam sim no los siv cov txheej txheem tsis muaj kev cuam tshuam ntawm kev xa mus rau lub hlwb feem ntau ua raws li cov txheej txheem ntawm receptor-mediated transport (PMT) siv cov endogenous BBB6 receptor.Txawm hais tias tsis ntev los no tseem ceeb kev nce qib uas siv cov hloov pauv receptor pathway7,8, kev txhim kho ntxiv ntawm kev xa khoom tshiab nrog cov khoom txhim kho yog xav tau.Txog qhov kawg no, peb lub hom phiaj yog txhawm rau txheeb xyuas cov peptides uas muaj peev xwm los kho CSF ​​kev thauj mus los, raws li lawv tuaj yeem siv los xa cov macromolecules rau CNS lossis qhib txoj hauv kev tshiab receptor.Tshwj xeeb, cov receptors tshwj xeeb thiab cov neeg thauj khoom ntawm lub paj hlwb (BBB thiab BSCFB) tuaj yeem ua lub hom phiaj muaj peev xwm rau kev ua haujlwm thiab tshwj xeeb ntawm cov tshuaj biotherapeutic.Cerebrospinal kua (CSF) yog ib yam khoom secretory ntawm choroid plexus (CS) thiab yog nyob rau hauv kev sib cuag nrog cov kua interstitial ntawm lub hlwb los ntawm lub subarachnoid qhov chaw thiab ventricular chaw4.Tsis ntev los no nws tau pom tias subarachnoid cerebrospinal kua diffuses ntau dhau mus rau hauv interstitium ntawm lub hlwb 9.Peb cia siab tias yuav nkag mus rau qhov chaw parenchymal siv qhov subarachnoid inflow ib sab lossis ncaj qha los ntawm BBB.Txhawm rau ua tiav qhov no, peb tau siv lub zog zoo hauv vivo phage xaiv lub tswv yim uas zoo txheeb xyuas cov peptides thauj los ntawm ob qho ntawm ob txoj hauv kev no.
Peb tam sim no piav qhia txog ib ntus hauv vivo phage kev tshuaj xyuas txoj hauv kev nrog CSF sampling ua ke nrog kev sib tw siab dhau los (HTS) los saib xyuas cov kev xaiv thawj zaug nrog ntau lub tsev qiv ntawv ntau haiv neeg.Kev tshuaj ntsuam xyuas tau ua rau cov nas nco qab nrog kev cog qoob loo loj cisterna (CM) cannula kom tsis txhob muaj ntshav paug.Qhov tseem ceeb, txoj hauv kev no xaiv ob lub hlwb-lub hom phiaj thiab peptides nrog kev thauj mus los thoob plaws cerebrovascular barrier.Peb siv T7 phages vim lawv qhov me me (~ 60 nm) 10 thiab qhia tias lawv tsim nyog rau kev thauj cov vesicles uas tso cai rau kev hla ntawm lub endothelial thiab / lossis epithelial-medulla barrier.Tom qab plaub lub voj voog ntawm panning, cov pej xeem phage raug cais tawm uas qhia tau hais tias muaj zog hauv vivo CSF ​​​​enrichment thiab cerebral microvessel koom haum.Qhov tseem ceeb, peb tuaj yeem lees paub peb qhov kev tshawb pom los ntawm kev ua kom pom tias qhov nyiam thiab tshuaj lom neeg zoo tshaj plaws cov neeg sib tw peptides tuaj yeem thauj cov khoom thauj protein mus rau hauv cov kua cerebrospinal.Ua ntej, cov tshuaj pharmacodynamic ntawm CNS tau tsim los ntawm kev sib txuas ua ke ntawm kev hloov pauv peptide nrog ib qho inhibitor ntawm BACE1 peptide.Ntxiv rau qhov ua kom pom tias hauv vivo cov tswv yim kev tshuaj xyuas kev ua haujlwm tuaj yeem txheeb xyuas lub hlwb tshiab thauj peptides ua cov neeg nqa khoom muaj txiaj ntsig zoo, peb cia siab tias cov kev xaiv ua haujlwm zoo sib xws kuj tseem yog qhov tseem ceeb hauv kev txheeb xyuas lub hlwb tshiab txoj hauv kev.
Raws li cov plaque-forming units (PFU), tom qab phage ntim cov kauj ruam, lub tsev qiv ntawv ntawm random 12-mer linear T7 phage peptides nrog ntau haiv neeg ntawm kwv yees li 109 tau tsim thiab tsim (saib Cov Khoom Siv thiab Cov Txheej Txheem).Nws yog ib qho tseem ceeb kom nco ntsoov tias peb ua tib zoo txheeb xyuas lub tsev qiv ntawv no ua ntej hauv vivo panning.PCR amplification ntawm phage tsev qiv ntawv cov qauv siv hloov kho primers generated amplicons uas ncaj qha siv tau rau HTS (Ntxiv daim duab 1a).Vim a) HTS11 sequencing yuam kev, b) cuam tshuam rau qhov zoo ntawm primers (NNK) 1-12, thiab c) muaj cov qus-hom (wt) phage (skeleton inserts) nyob rau hauv lub standby tsev qiv ntawv, ib tug sequence filtering txheej txheem yog siv los rho tawm cov ntaub ntawv pov thawj ib ntus xwb (Ntxiv daim duab 1b).Cov kauj ruam lim dej no siv rau txhua lub tsev qiv ntawv HTS.Rau cov tsev qiv ntawv tus qauv, tag nrho ntawm 233,868 tau nyeem, ntawm 39% dhau los ntawm cov txheej txheem lim dej thiab tau siv rau kev txheeb xyuas cov tsev qiv ntawv thiab xaiv rau cov kev sib txuas ntxiv tom ntej (Cov duab ntxiv 1c–e).Cov ntawv nyeem feem ntau yog qhov sib npaug ntawm 3 lub hauv paus khub ntev nrog lub ncov ntawm 36 nucleotides (Supplementary Fig. 1c), lees paub lub tsev qiv ntawv tsim (NNK) 1-12.Qhov tseem ceeb, kwv yees li 11% ntawm cov tswv cuab hauv tsev qiv ntawv muaj 12-dimensional wild-type (wt) backbone PAGISRELVDKL insert, thiab yuav luag ib nrab ntawm cov sequences (49%) muaj cov ntxig lossis tshem tawm.Lub HTS ntawm lub tsev qiv ntawv lub tsev qiv ntawv tau lees paub qhov muaj ntau haiv neeg ntawm peptides hauv lub tsev qiv ntawv: ntau dua 81% ntawm peptide sequences tau pom ib zaug thiab tsuas yog 1.5% tshwm sim hauv ≥4 daim ntawv (Cov Lus Ntxiv 2a).Cov zaus ntawm cov amino acids (aa) ntawm tag nrho 12 txoj hauj lwm nyob rau hauv repertoire correlated zoo nrog cov zaus xav tau rau tus naj npawb ntawm codons generated los ntawm degenerate NKK repertoire (Suab ntxiv Fig. 2b).Qhov pom zaus ntawm aa residues encoded los ntawm cov inserts no correlated zoo nrog cov zauv zaus (r = 0.893) (Suab ntxiv Fig. 2c).Kev npaj cov tsev qiv ntawv phage rau kev txhaj tshuaj suav nrog cov kauj ruam ntawm kev nthuav dav thiab tshem tawm endotoxin.Qhov no yav dhau los tau pom tias muaj peev xwm txo tau qhov sib txawv ntawm cov tsev qiv ntawv phage12,13.Yog li ntawd, peb tau ua raws cov tsev qiv ntawv phaj-amplified phage uas tau raug tshem tawm cov tshuaj endotoxin thiab muab piv nrog cov tsev qiv ntawv qub los kwv yees qhov zaus ntawm AA.Ib qho kev sib raug zoo (r = 0.995) tau pom nyob nruab nrab ntawm lub pas dej qub thiab lub pas dej ua kom muaj zog thiab purified (Supplementary Fig. 2d), qhia tias kev sib tw ntawm clones amplified ntawm daim hlau siv T7 phage tsis ua rau muaj kev tsis ncaj ncees loj.Qhov kev sib piv no yog nyob ntawm qhov zaus ntawm tripeptide motifs nyob rau hauv txhua lub tsev qiv ntawv, vim muaj ntau haiv neeg ntawm cov tsev qiv ntawv (~109) tsis tuaj yeem ntes tau tag nrho txawm tias HTS.Kev soj ntsuam ntau zaus ntawm aa ntawm txhua txoj haujlwm tau qhia txog txoj haujlwm me me-nyob ntawm kev tsis ncaj ncees nyob rau hauv peb txoj haujlwm kawg ntawm kev nkag mus rau repertoire (Ntxiv daim duab 2e).Hauv kev xaus, peb tau txiav txim siab tias qhov zoo thiab ntau haiv neeg ntawm lub tsev qiv ntawv tau txais thiab tsuas yog kev hloov pauv me me ntawm ntau haiv neeg tau pom vim yog kev nthuav dav thiab kev npaj cov tsev qiv ntawv phage ntawm ntau qhov kev xaiv.
Serial cerebrospinal kua sampling tuaj yeem ua tau los ntawm kev phais implanting cannula rau hauv CM ntawm cov nas nco qab los pab qhia txog T7 phage txhaj tshuaj intravenously (iv) ntawm BBB thiab / lossis BCSFB (Fig. 1a-b).Peb siv ob lub caj npab xaiv ywj siab (caj npab A thiab B) hauv thawj peb qhov kev xaiv hauv vivo (Fig. 1c).Peb maj mam nce qhov nruj ntawm kev xaiv los ntawm kev txo qis tag nrho cov phage qhia hauv thawj peb qhov kev xaiv.Rau qhov thib plaub ntawm panning, peb muab cov qauv ntawm cov ceg A thiab B thiab ua peb qhov kev xaiv ywj pheej ntxiv.Txhawm rau kawm txog cov khoom hauv vivo ntawm T7 phage hais hauv cov qauv no, cov tsiaj qus-hom phage (PAGISRELVDKL tus tswv ntxig) tau txhaj rau hauv nas ntawm tus Tsov tus tw leeg.Rov qab cov phages los ntawm cov kua cerebrospinal thiab ntshav ntawm lub sijhawm sib txawv ntawm cov ntsiab lus tau pom tias T7 icosahedral phages me me muaj qhov pib tshem tawm sai sai los ntawm cov ntshav ntim (Ntxiv daim duab 3).Raws li cov titers tswj hwm thiab cov ntshav ntim ntawm nas, peb suav tias tsuas yog kwv yees li 1% wt.phage los ntawm cov koob tshuaj tau kuaj pom hauv cov ntshav 10 feeb tom qab txhaj tshuaj.Tom qab qhov pib sai sai no, qhov kev ncua qeeb qeeb tau ntsuas nrog ib nrab-lub neej ntawm 27.7 feeb.Qhov tseem ceeb, tsuas yog ob peb phages tau rov qab los ntawm CSF compartment, qhia txog keeb kwm qis rau cov tsiaj qus phage tsiv teb tsaws rau hauv CSF compartment (Ntxiv daim duab 3).Qhov nruab nrab, tsuas yog kwv yees li 1 x 10-3% titers ntawm T7 phage hauv cov ntshav thiab 4 x 10-8% ntawm cov phages thawj zaug tau kuaj pom hauv cov kua dej cerebrospinal dhau tag nrho lub sijhawm kuaj (0-250 min).Qhov tseem ceeb, ib nrab-lub neej (25.7 min) ntawm cov tsiaj qus phage hauv cov kua cerebrospinal zoo ib yam li cov ntshav pom.Cov ntaub ntawv no qhia tau hais tias qhov teeb meem sib cais CSF compartment los ntawm cov ntshav yog tsis zoo nyob rau hauv CM-cannulated nas, tso cai nyob rau hauv vivo xaiv phage tsev qiv ntawv los txheeb xyuas cov clones uas nkag tau los ntawm cov ntshav mus rau hauv CSF compartment.
(a) Teeb tsa ib txoj hauv kev rau rov kuaj cov kua dej cerebrospinal (CSF) los ntawm lub pas dej loj.(b) Daim duab qhia txog lub cellular qhov chaw ntawm lub hauv nruab nrab lub paj hlwb barrier (CNS) barrier thiab xaiv lub tswv yim siv los txheeb xyuas peptides uas hla cov ntshav-hlwb barrier (BBB) ​​​​thiab cov ntshav-hlwb barrier.(c) Nyob rau hauv vivo phage zaub tshuaj ntsuam flowchart.Hauv txhua qhov kev xaiv, phages (tus cim tsiaj hauv cov xub) tau txhaj tshuaj intravenously.Ob txoj kev ywj pheej xaiv (A, B) raug cais kom txog thaum 4th puag ncig ntawm kev xaiv.Rau kev xaiv rounds 3 thiab 4, txhua phage clone muab rho tawm los ntawm CSF tau manually sequenced.(d) Kinetics ntawm phage cais tawm ntawm cov ntshav (lub voj voog liab) thiab cov kua dej cerebrospinal (daim duab peb sab ntsuab) thaum thawj zaug ntawm kev xaiv hauv ob lub cannulated nas tom qab txhaj tshuaj ntawm lub tsev qiv ntawv T7 peptide (2 x 1012 phages / tsiaj).Xiav squares qhia qhov nruab nrab qhov pib concentration ntawm phage hauv cov ntshav, xam los ntawm tus nqi ntawm cov tshuaj phage, coj mus rau hauv tus account tag nrho cov ntshav ntim.Cov squares dub qhia lub ntsiab lus ntawm kev sib tshuam ntawm y kab extrapolated los ntawm cov ntshav phage concentrations.(e,f) Qhia qhov txheeb ze zaus thiab kev faib tawm ntawm txhua qhov sib tshooj tripeptide motifs pom hauv peptide.Tus naj npawb ntawm motifs pom nyob rau hauv 1000 nyeem yog qhia.Qhov tseem ceeb (p <0.001) enriched motifs yog cim nrog liab dots.(e) Kev sib txheeb scatterplot piv cov txheeb ze zaus ntawm tripeptide motif ntawm lub tsev qiv ntawv txhaj tshuaj nrog cov ntshav los phage los ntawm cov tsiaj #1.1 thiab #1.2.(f) Kev sib txheeb scatterplot piv cov txheeb ze zaus ntawm tsiaj phage tripeptide motifs #1.1 thiab #1.2 cais nyob rau hauv cov ntshav thiab cerebrospinal kua.(g, h) Sequence ID sawv cev ntawm phage enriched nyob rau hauv cov ntshav (g) tiv thaiv txhaj cov tsev qiv ntawv thiab phage enriched nyob rau hauv CSF (h) piv rau ntshav tom qab ib puag ncig ntawm nyob rau hauv vivo xaiv nyob rau hauv ob qho tib si tsiaj.Qhov loj ntawm ib tsab ntawv code qhia ntau npaum li cas cov amino acid tshwm sim ntawm txoj hauj lwm ntawd.Ntsuab = polar, ntshav = nruab nrab, xiav = yooj yim, liab = acidic thiab dub = hydrophobic amino acids.Daim duab 1a, b tau tsim thiab tsim los ntawm Eduard Urich.
Peb txhaj ib lub tsev qiv ntawv phage peptide rau hauv ob lub CM ntsuas nas (clades A thiab B) thiab cais phage los ntawm cov kua cerebrospinal thiab ntshav (Daim duab 1d).Qhov pib nrawm nrawm ntawm lub tsev qiv ntawv tau hais tsawg dua piv rau cov tsiaj qus-hom phage.Qhov nruab nrab ntawm lub neej ntawm cov tsev qiv ntawv txhaj tshuaj hauv ob qho tib si tsiaj yog 24.8 feeb hauv cov ntshav, zoo ib yam li cov tsiaj qus phage, thiab 38.5 feeb hauv CSF.Ntshav thiab cerebrospinal kua phage kuaj los ntawm txhua tus tsiaj tau raug HTS thiab tag nrho cov peptides tau txheeb xyuas rau qhov muaj qhov luv tripeptide motif.Tripeptide motifs raug xaiv vim lawv muab lub hauv paus me me rau kev tsim qauv thiab kev sib cuam tshuam ntawm peptide-protein14,15.Peb pom muaj kev sib raug zoo nyob rau hauv kev faib cov motifs ntawm cov tshuaj phage tsev qiv ntawv thiab clones muab rho tawm los ntawm cov ntshav ntawm ob leeg tsiaj (Fig. 1e).Cov ntaub ntawv qhia tau hais tias muaj pes tsawg leeg ntawm lub tsev qiv ntawv tsuas yog marginally enriched nyob rau hauv cov ntshav compartment.Amino acid frequencies thiab kev pom zoo ua ntu zus tau txheeb xyuas ntxiv ntawm txhua txoj haujlwm siv kev hloov kho ntawm Weblogo16 software.Interestingly, peb pom muaj zog enrichment nyob rau hauv cov ntshav glycine residues (Fig. 1g).Thaum cov ntshav tau muab piv nrog cov clones uas tau xaiv los ntawm CSF, kev xaiv muaj zog thiab qee qhov kev xaiv ntawm motifs tau pom (Daim duab 1f), thiab qee cov amino acids tau nyiam nyob rau ntawm txoj haujlwm predetermined hauv 12-tus tswv cuab (Fig. 1h).Qhov tshwj xeeb tshaj yog, ib tus tsiaj txawv qhov sib txawv ntawm cov kua cerebrospinal, thaum cov ntshav glycine enrichment tau pom nyob rau hauv ob qho tib si tsiaj (Ntxiv Fig. 4a–j).Tom qab kev lim dej nruj ntawm cov ntaub ntawv ua ntu zus hauv cov kua cerebrospinal ntawm cov tsiaj #1.1 thiab #1.2, tag nrho ntawm 964 thiab 420 qhov tshwj xeeb 12-mer peptides tau txais (Cov Lus Ntxiv 1d–e).Cov phage clones cais tau nthuav dav thiab raug rau qhov thib ob ntawm kev xaiv vivo.Phage muab rho tawm los ntawm qhov thib ob ntawm kev xaiv tau raug HTS hauv txhua tus tsiaj thiab tag nrho cov peptides uas tau txheeb xyuas tau siv los ua cov tswv yim rau ib qho kev paub txog motif los soj ntsuam qhov tshwm sim ntawm tripeptide motifs (Fig. 2a, b, ef).Piv rau thawj lub voj voog ntawm phage rov qab los ntawm CSF, peb tau soj ntsuam ntxiv kev xaiv thiab deselection ntawm ntau motifs hauv CSF hauv ceg A thiab B (Fig. 2).Kev txheeb xyuas lub network algorithm tau siv los txiav txim seb lawv sawv cev cov qauv sib txawv ntawm qhov sib xws.Ib qho zoo sib xws tau pom ntawm 12-dimensional sequences rov qab los ntawm CSF hauv lwm qhov clade A (Fig. 2c, d) thiab clade B (Fig. 2g, h).Cov kev soj ntsuam sib koom ua ke hauv txhua ceg tau nthuav tawm cov kev xaiv sib txawv rau 12-mer peptides (Ntxiv daim duab 5c, d) thiab kev nce hauv CSF / ntshav titer piv rau lub sij hawm rau pooled clones tom qab qhov kev xaiv thib ob piv rau thawj qhov kev xaiv (Ntxiv daim duab 5e).).
Ua kom muaj txiaj ntsig ntawm motifs thiab peptides hauv cerebrospinal kua los ntawm ob qhov kev sib tw ua tiav ntawm hauv vivo functional phage zaub xaiv.
Tag nrho cov cerebrospinal kua phages rov qab los ntawm thawj puag ncig ntawm txhua tus tsiaj (tsiaj # 1.1 thiab # 1.2) tau sib sau ua ke, nthuav dav, HT-sequenced thiab reinjected ua ke (2 x 1010 phages / tsiaj txhu) 2 SM cannulated nas (#1.1 → #).2.1 thiab 2.2, 1.2 → 2.3 thiab 2.4).(a,b,e,f) Kev sib txheeb scatterplots piv cov txheeb ze zaus ntawm tripeptide motifs ntawm tag nrho cov CSF-derived phages nyob rau hauv thawj thiab thib ob xaiv rounds.txheeb ze zaus thiab kev faib tawm ntawm motifs sawv cev rau tag nrho cov overlapping tripeptides pom nyob rau hauv peptides nyob rau hauv ob qho tib si orientations.Tus naj npawb ntawm motifs pom nyob rau hauv 1000 nyeem yog qhia.Motifs uas tau loj heev (p < 0.001) xaiv los yog tsis suav nrog hauv ib qho ntawm cov tsev qiv ntawv sib piv yog qhov tseem ceeb nrog cov dots liab.(c, d, g, h) Sequence logo sawv cev ntawm tag nrho CSF-nplua nuj 12 amino acid ntev sequences raws li nyob rau hauv rounds 2 thiab 1 ntawm nyob rau hauv vivo xaiv.Qhov loj ntawm ib tsab ntawv code qhia ntau npaum li cas cov amino acid tshwm sim ntawm txoj hauj lwm ntawd.Txhawm rau sawv cev rau lub logo, qhov zaus ntawm CSF ib ntus muab rho tawm los ntawm ib tus tsiaj nruab nrab ntawm ob qhov kev xaiv rounds yog muab piv thiab cov khoom sib txuas ntxiv hauv qhov thib ob yog qhia: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 thiab (h) #1.2–#2.4.Feem ntau enriched amino acids ntawm ib tug muab txoj hauj lwm nyob rau hauv (c, d) tsiaj no.2.1 thiab no.2.2 los yog (g, h) nyob rau hauv cov tsiaj no.2.3 i nr.2.4 muaj nyob rau hauv cov xim.Ntsuab = polar, ntshav = nruab nrab, xiav = yooj yim, liab = acidic thiab dub = hydrophobic amino acids.
Tom qab qhov kev xaiv thib peb, peb tau txheeb xyuas 124 qhov tshwj xeeb peptide ib ntus (#3.1 thiab #3.2) los ntawm 332 CSF-reconstituted phage clones cais los ntawm ob tug tsiaj (Ntxiv daim duab 6a).Qhov sib lawv liag LGSVS (18.7%) muaj cov txheeb ze tshaj plaws, ua raws li cov tsiaj qus PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), thiab SARGSWREIVSLS (2.2%).Hauv qhov thib plaub thib plaub, peb tau sau ob ceg ntawm nws tus kheej xaiv los ntawm peb tus tsiaj cais (Fig. 1c).Ntawm 925 sequenced phage clones rov qab los ntawm CSF, nyob rau hauv lub thib plaub puag ncig peb pom 64 cim peptide sequences (Supplementary Fig. 6b), uas cov txheeb ze ntawm cov tsiaj qus-hom phage poob mus rau 0.8%.Qhov feem ntau CSF clones nyob rau hauv plaub puag ncig yog LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) thiab RLSSVDSDLSGC (3, RLSSVDSDLSGC).%)).Qhov ntev ntawm cov peptides xaiv yog vim nucleotide insertions / deletions lossis ntxov ntxov nres codons hauv tsev qiv ntawv primers thaum siv degenerate codons rau NNK tsev qiv ntawv tsim.Tsis ntev los no nres codons tsim cov peptides luv thiab raug xaiv vim tias lawv muaj qhov zoo siab aa motif.Ntev peptides yuav tshwm sim los ntawm kev ntxig / tshem tawm hauv primers ntawm cov tsev qiv ntawv hluavtaws.Qhov no tso cai rau tus tsim nres codon sab nraum tus ncej thiab nyeem nws kom txog thaum tus tshiab nres codon tshwm hauv qab.Feem ntau, peb suav cov txiaj ntsig zoo rau tag nrho plaub qhov kev xaiv los ntawm kev sib piv cov ntaub ntawv nkag nrog cov ntaub ntawv tso tawm cov qauv.Rau thawj qhov kev tshuaj ntsuam xyuas, peb siv cov tsiaj qus-hom phage titers uas tsis yog siv keeb kwm yav dhau los.Interestingly, tsis zoo phage xaiv muaj zog heev nyob rau hauv thawj CSF lub voj voog, tab sis tsis nyob rau hauv cov ntshav (Fig. 3a), uas tej zaum yuav yog vim tsis tshua muaj tshwm sim ntawm passive diffusion ntawm feem ntau cov tswv cuab ntawm lub tsev qiv ntawv peptide mus rau hauv lub CSF compartment los yog cov txheeb ze phages yuav zoo dua khaws cia los yog tshem tawm ntawm cov hlab ntsha tshaj bacteriophages.Txawm li cas los xij, nyob rau hauv qhov thib ob ntawm panning, kev xaiv muaj zog ntawm phages hauv CSF tau pom nyob rau hauv ob qho tib si clades, qhia tias qhov kev sib tw dhau los tau ua kom muaj txiaj ntsig zoo hauv phages qhia peptides uas txhawb nqa CSF uptake (Fig. 3a).Ib zaug ntxiv, tsis muaj ntshav ntxiv.Tsis tas li ntawd nyob rau hauv peb thiab plaub puag ncig, phage clones tau ua kom muaj txiaj ntsig zoo hauv CSF.Sib piv cov txheeb ze zaus ntawm txhua qhov tshwj xeeb peptide sib lawv liag ntawm ob qhov kev xaiv dhau los, peb pom tias cov kev sib tw tseem muaj ntau dua nyob rau hauv plaub qhov kev xaiv (Fig. 3b).Tag nrho ntawm 931 tripeptide motifs tau muab rho tawm los ntawm tag nrho 64 qhov sib txawv peptide uas siv ob qho tib si peptide orientations.Cov motifs enriched tshaj plaws nyob rau hauv lub thib plaub puag ncig tau soj ntsuam zoo dua rau lawv enrichment profiles nyob rau hauv tag nrho cov rounds piv rau txhaj tshuaj (txiav-off: 10% enrichment) (Suab ntxiv Fig. 6c).Cov qauv kev xaiv dav dav pom tau tias feem ntau ntawm cov kev xav tau kawm tau ua kom muaj txiaj ntsig hauv txhua qhov kev sib tw dhau los ntawm ob ceg xaiv.Txawm li cas los xij, qee qhov motifs (piv txwv li SGL, VSG, LGS GSV) feem ntau yog los ntawm lwm cov clade A, thaum lwm tus (xws li FGW, RTN, WGF, NTR) tau ntxiv rau lwm cov clade B.
Kev lees paub ntawm CSF thauj ntawm CSF-enriched phage-displayed peptides thiab biotinylated thawj coj peptides conjugated rau streptavidin payloads.
(a) Enrichment ratios xam nyob rau hauv tag nrho plaub rounds (R1-R4) raws li txhaj (input = I) phage (PFU) titers thiab txiav txim CSF phage titers (tso tawm = O).Cov yam tseem ceeb ntxiv rau peb qhov kawg (R2-R4) raug xam los ntawm kev sib piv nrog rau qhov kev sib tw dhau los thiab thawj puag ncig (R1) nrog cov ntaub ntawv hnyav.Qhib bar yog cerebrospinal kua, ntxoov ntxoo bar yog plasma.(***p<0.001, raws li Tub Ntxhais Kawm T-test).(b) Daim ntawv teev cov phage peptides ntau tshaj, nyob rau hauv raws li lawv cov txheeb ze sib piv rau tag nrho cov phages sau nyob rau hauv CSF tom qab round 4 ntawm xaiv.Lub rau feem ntau phage clones yog highlighted nyob rau hauv cov xim, naj npawb thiab lawv enrichment yam ntawm rounds 3 thiab 4 ntawm xaiv (insets).(c,d) Lub rau feem ntau enriched phage clones, khoob phage thiab niam txiv phage peptide tsev qiv ntawv los ntawm puag ncig 4 tau soj ntsuam ib tus zuj zus nyob rau hauv ib tug CSF sampling qauv.CSF thiab cov ntshav kuaj tau raug sau los ntawm lub sijhawm teev tseg.(c) Qhov sib npaug ntawm 6 tus neeg sib tw phage clones (2 x 1010 phages / tsiaj txhu), phages khoob (#1779) (2 x 1010 phages / tsiaj txhu) thiab cov tsev qiv ntawv phage peptide (2 x 1012 phages / tsiaj txhu) Txhaj tsawg kawg 3 CM yog muab rau cov tsiaj sib cais ntawm cov cannulated.CSF pharmacokinetics ntawm txhua qhov kev txhaj tshuaj phage clone thiab phage peptide tsev qiv ntawv nyob rau lub sijhawm tau qhia.(d) qhia qhov nruab nrab CSF / ntshav piv rau txhua qhov rov qab phages / mL nyob rau lub sijhawm kuaj.(e) Plaub tus thawj coj hluavtaws peptides thiab ib qho scrambled tswj tau txuas nrog biotin rau streptavidin los ntawm lawv cov N-terminus (tetramer zaub) tom qab txhaj tshuaj (tail vein iv, 10 mg streptavidin/kg).Yam tsawg kawg peb tus nas intubated (N = 3).).CSF cov qauv raug sau los ntawm lub sijhawm qhia cov ntsiab lus thiab streptavidin concentrations tau ntsuas los ntawm CSF anti-streptavidin ELISA (nd = tsis kuaj).(*p<0.05, **p<0.01, ***p<0.001, raws li kev xeem ANOVA).(f) Kev sib piv ntawm cov amino acid sib lawv liag ntawm feem ntau enriched phage peptide clone #2002 (ntsuab) nrog rau lwm yam phage peptide clones los ntawm 4th puag ncig ntawm kev xaiv.Ib yam thiab zoo sib xws cov amino acid fragments yog xim-coded.
Ntawm tag nrho cov phages enriched nyob rau hauv plaub puag ncig (Fig. 3b), rau tus neeg sib tw clones raug xaiv rau ntxiv ib tug neeg soj ntsuam nyob rau hauv lub CSF sampling qauv.Qhov sib npaug ntawm rau tus neeg sib tw phage, khoob phage (tsis muaj qhov ntxig) thiab cov tsev qiv ntawv prophage peptide tau txhaj rau hauv peb lub cannulated CM tsiaj, thiab cov tshuaj pharmacokinetics tau txiav txim siab hauv CSF (Fig. 3c) thiab ntshav (Ntxiv daim duab 7) kev soj ntsuam.Tag nrho cov phage clones kuaj tau tsom rau CSF compartment ntawm qib 10-1000 lub sij hawm siab dua li ntawm qhov khoob tswj phage (#1779).Piv txwv li, clones # 2020 thiab # 2077 muaj txog 1000 lub sij hawm siab dua CSF titers dua li tswj phage.Pharmacokinetic profile ntawm txhua tus peptide xaiv txawv, tab sis txhua tus ntawm lawv muaj peev xwm CSF homing siab.Peb tau pom qhov txo qis mus tas li rau lub sijhawm rau clones #1903 thiab #2011, thaum rau clones #2077, #2002 thiab #2009 nce ntxiv thaum thawj 10 feeb yuav qhia tau tias muaj kev thauj mus los tab sis yuav tsum tau kuaj xyuas.Clones #2020, #2002, thiab #2077 ruaj khov nyob rau theem siab, thaum lub CSF concentration ntawm clone #2009 maj mam txo qis tom qab thawj zaug nce.Peb mam li muab piv cov txheeb ze zaus ntawm txhua tus neeg sib tw CSF nrog nws cov ntshav concentration (Fig. 3d).Kev sib raug zoo ntawm qhov nruab nrab titer ntawm txhua tus neeg sib tw CSF nrog nws cov ntshav titer ntawm txhua lub sijhawm kuaj pom tau tias peb ntawm rau tus neeg sib tw tau muaj txiaj ntsig zoo hauv cov ntshav CSF.Interestingly, clone #2077 pom cov ntshav ruaj khov dua (Cov duab ntxiv 7).Txhawm rau kom paub meej tias cov peptides lawv tus kheej muaj peev xwm thauj cov khoom thauj uas tsis yog phage particles rau hauv CSF compartment, peb tau tsim plaub tus thawj coj peptides derivatized nrog biotin ntawm N-terminus qhov twg peptides txuas rau phage particle.Biotinylated peptides (nos. 2002, 2009, 2020 thiab 2077) tau sib txuas nrog streptavidin (SA) kom tau txais cov ntaub ntawv multimeric me ntsis mimicking phage geometry.Hom ntawv no kuj tau tso cai rau peb ntsuas SA raug rau hauv cov ntshav thiab cov kua cerebrospinal li cargo-transporting protein peptides.Qhov tseem ceeb, cov ntaub ntawv phage feem ntau tuaj yeem rov ua dua thaum cov khoom siv peptides tau tswj hwm hauv hom SA-conjugated (Daim duab 3e).Cov scrambled peptides tau pib raug tsawg dua thiab sai dua CSF tshem tawm nrog cov qib tsis paub meej hauv 48 teev.Txhawm rau kom nkag siab txog txoj hauv kev xa khoom ntawm cov peptide phage clones mus rau hauv qhov chaw CSF, peb tau txheeb xyuas qhov chaw ntawm ib tus neeg phage peptide hits siv immunohistochemistry (IHC) txhawm rau txheeb xyuas cov phage ncaj qha 1 teev tom qab txhaj tshuaj hauv vivo.Qhov tseem ceeb, clones # 2002, # 2077, thiab # 2009 tuaj yeem kuaj pom los ntawm kev muaj zog staining hauv hlwb capillaries, thaum tswj phage (#1779) thiab clone # 2020 tsis tau kuaj pom (Cov duab ntxiv 8).Qhov no qhia tau hais tias cov peptides no ua rau muaj kev cuam tshuam rau lub paj hlwb los ntawm kev hla BBB.Kev soj ntsuam ntxaws ntxiv yog yuav tsum tau kuaj xyuas qhov kev xav no, vim tias txoj hauv kev BSCFB kuj tseem tuaj yeem koom nrog.Thaum muab piv cov amino acid ib theem zuj zus ntawm feem ntau enriched clone (#2002) nrog rau lwm yam peptides xaiv, nws tau muab sau tseg tias ib co ntawm lawv muaj zoo xws li cov amino acid extensions, uas tej zaum yuav qhia ib tug zoo xws li kev thauj mechanism (Fig. 3f).
Vim nws qhov tshwj xeeb plasma profile thiab qhov tseem ceeb nce hauv CSF lub sijhawm dhau los, phage zaub clone #2077 tau tshawb nrhiav ntxiv rau lub sijhawm ntev 48-teev thiab muaj peev xwm rov tsim dua qhov nce sai hauv CSF pom nyob rau hauv kev koom tes nrog SA qib (Fig. 4a).Hais txog lwm yam phage clones, #2077 stained rau lub paj hlwb capillaries thiab pom colocalization tseem ceeb nrog capillary marker lectin thaum saib ntawm qhov kev daws teeb meem siab dua thiab tej zaum qee qhov staining hauv qhov chaw parenchymal (Daim duab 4b).Txhawm rau tshawb xyuas seb peptide-mediated pharmacological teebmeem puas tuaj yeem tau txais hauv CNS, peb tau ua qhov kev sim uas biotinylated versions ntawm i) # 2077 transit peptide thiab ii) BACE1 inhibitor peptide tau tov nrog SA ntawm ob qhov sib txawv.Rau ib qho kev sib xyaw ua ke peb tsuas yog siv BACE1 peptide inhibitor thiab rau lwm qhov peb siv 1: 3 piv ntawm BACE1 peptide inhibitor rau # 2077 peptide.Ob qho piv txwv tau muab tso rau hauv cov hlab ntsha thiab cov ntshav thiab cerebrospinal kua qib ntawm beta-amyloid peptide 40 (Abeta40) tau ntsuas lub sijhawm.Abeta40 tau ntsuas hauv CSF raws li nws qhia txog BACE1 inhibition hauv lub hlwb parenchyma.Raws li xav tau, ob qho tib si complexes txo cov ntshav ntawm Abeta40 (Fig. 4c, d).Txawm li cas los xij, tsuas yog cov qauv uas muaj qhov sib xyaw ntawm peptide no.2077 thiab ib qho inhibitor ntawm BACE1 peptide conjugated rau SA ua rau muaj kev txo qis hauv Abeta40 hauv cov kua cerebrospinal (Fig. 4c).Cov ntaub ntawv qhia tias peptide no.2077 muaj peev xwm thauj tau 60 kDa SA protein rau hauv CNS thiab tseem ua rau cov tshuaj muaj txiaj ntsig nrog SA-conjugated inhibitors ntawm BACE1 peptide.
(a) Kev txhaj tshuaj Clonal (2 × 10 phages / tsiaj txhu) ntawm T7 phage uas qhia txog cov tshuaj pharmacokinetic ntev ntawm CSF peptide #2077 (RLSSVDSDLSGC) thiab cov tshuaj tswj tsis tau tshuaj phage (#1779) tsawg kawg peb CM-intubated nas.(b) Confocal microscopic duab ntawm tus neeg sawv cev cortical microvessels nyob rau hauv phage-injected nas (2 × 10 10 phages / tsiaj) uas qhia counterstaining ntawm peptide #2077 thiab cov hlab ntsha (lectin).Cov phage clones tau muab rau 3 nas thiab tso cai rau 1 teev ua ntej perfusion.Lub hlwb raug muab faib thiab stained nrog polyclonal FITC-labeled antibodies tiv thaiv T7 phage capsid.Kaum feeb ua ntej perfusion thiab tom qab kho, DyLight594-labeled lectin tau muab tso rau hauv cov hlab ntsha.Fluorescent dluab qhia lectin staining (liab) ntawm luminal sab ntawm microvessels thiab phages (ntsuab) nyob rau hauv lub lumen ntawm capillaries thiab perivascular paj hlwb.Qhov ntsuas bar sib haum mus rau 10 µm.(c, d) Biotinylated BACE1 inhibitory peptide ib leeg los yog ua ke nrog biotinylated transit peptide #2077 tau ua ke rau streptavidin tom qab txhaj tshuaj txhaj tshuaj tsawg kawg peb lub cannulated CM nas (10 mg streptavidin / kg).BACE1 peptide inhibitor-mediated txo nyob rau hauv Aβ40 tau ntsuas los ntawm Aβ1-40 ELISA hauv cov ntshav (liab) thiab kua cerebrospinal (txiv kab ntxwv) ntawm lub sijhawm qhia.Txhawm rau kom pom tseeb zoo dua, kab dotted yog kos rau ntawm daim duab ntawm qhov ntsuas ntawm 100%.(c) Kev txo qis hauv Aβ40 hauv cov ntshav (daim duab peb sab liab) thiab cerebrospinal kua (txiv kab ntxwv daim duab peb sab) hauv nas kho nrog streptavidin conjugated rau transit peptide #2077 thiab BACE1 inhibitory peptide hauv 3: 1 piv.(d) Kev txo qis hauv cov ntshav Aβ40 (lub voj voog liab) thiab cerebrospinal kua (lub voj voog txiv kab ntxwv) ntawm cov nas kho nrog streptavidin ua ke rau BACE1 inhibitory peptide nkaus xwb.Aβ concentration hauv kev tswj yog 420 pg / ml (tus qauv sib txawv = 101 pg / ml).
Phage zaub tau ua tiav hauv ntau qhov chaw ntawm kev tshawb fawb biomedical17.Txoj kev no tau siv rau hauv vivo vascular diversity kev tshawb fawb 18,19 nrog rau cov kev tshawb fawb tsom rau cov hlab ntsha hlwb 20,21,22,23,24,25,26.Hauv txoj kev tshawb no, peb tau txuas ntxiv rau daim ntawv thov ntawm txoj kev xaiv no tsis yog rau kev txheeb xyuas ncaj qha ntawm peptides tsom rau cov hlab ntsha hauv hlwb, tab sis kuj mus rau kev tshawb pom ntawm cov neeg sib tw nrog cov khoom siv thauj mus los hla cov hlab ntsha hauv hlwb.Tam sim no peb piav qhia txog kev txhim kho ntawm cov txheej txheem xaiv vivo hauv CM intubated nas thiab ua kom pom nws lub peev xwm los txheeb xyuas peptides nrog CSF homing zog.Siv T7 phage tso saib lub tsev qiv ntawv ntawm 12-mer random peptides, peb muaj peev xwm ua kom pom tau tias T7 phage me txaus (kwv yees li 60 nm inch) 10 kom yoog rau cov ntshav-hlwb barrier, yog li ncaj qha hla cov ntshav-hlwb barrier lossis choroid plexus.Peb pom tias CSF sau los ntawm cannulated CM nas yog ib qho kev tswj xyuas zoo nyob rau hauv vivo functional screening method, thiab hais tias cov extracted phage tsis tsuas yog khi rau lub vasculature tab sis kuj ua hauj lwm raws li ib tug transporter hla cov ntshav-hlwb barrier.Tsis tas li ntawd, los ntawm kev sau cov ntshav ib txhij thiab thov HTS rau CSF ​​thiab cov ntshav tau txais cov phages, peb tau lees paub tias peb txoj kev xaiv CSF tsis cuam tshuam los ntawm cov ntshav ntxiv lossis kev nyab xeeb rau kev nthuav dav ntawm cov kev xaiv.Txawm li cas los xij, cov ntshav ntim yog ib feem ntawm cov txheej txheem xaiv, txij li cov phages muaj peev xwm ncav cuag CSF compartment yuav tsum muaj sia nyob thiab ncig hauv cov hlab ntsha ntev txaus los txhawb lawv lub hlwb.Txhawm rau kom rho tawm cov ntaub ntawv txhim khu kev qha los ntawm cov ntaub ntawv HTS nyoos, peb tau siv cov lim dej hloov mus rau cov txheej txheem tshwj xeeb hauv kev ua haujlwm tsis raug hauv kev tsom xam.Los ntawm kev sib koom ua ke ntawm cov kev ntsuas kinetic rau hauv txoj kev tshuaj ntsuam, peb tau lees paub cov tshuaj sai sai ntawm cov tsiaj qus T7 phages (t½ ~ 28 min) hauv cov ntshav 24, 27, 28 thiab kuj tau txiav txim siab lawv ib nrab-lub neej hauv cov kua cerebrospinal (t½ ~ 26 min) ib feeb).Txawm hais tias muaj cov tshuaj pharmacokinetic zoo sib xws hauv cov ntshav thiab CSF, tsuas yog 0.001% ntawm cov ntshav concentration ntawm phage tuaj yeem kuaj pom hauv CSF, qhia tias tsis tshua muaj kev txav mus los ntawm cov tsiaj qus-hom T7 phage hla cov ntshav-hlwb barrier.Qhov kev ua haujlwm no qhia txog qhov tseem ceeb ntawm thawj qhov kev xaiv thaum siv hauv vivo panning cov tswv yim, tshwj xeeb tshaj yog rau phage systems uas tau tshem tawm sai sai ntawm kev ncig, raws li ob peb clones tuaj yeem ncav cuag CNS compartment.Yog li, nyob rau hauv thawj puag ncig, qhov txo qis hauv cov tsev qiv ntawv ntau haiv neeg yog qhov loj heev, tsuas yog qee tus lej ntawm cov clones thaum kawg tau sau rau hauv tus qauv CSF nruj heev.Qhov no hauv vivo panning lub tswv yim suav nrog ntau yam kev xaiv xws li kev ua kom nquag plias hauv CSF compartment, clone ciaj sia nyob hauv cov ntshav, thiab tshem tawm sai ntawm T7 phage clones los ntawm cov ntshav hauv thawj 10 feeb (Fig. 1d thiab Supplementary Fig. 4M).).Yog li, tom qab thawj puag ncig, qhov sib txawv phage clones tau txheeb xyuas hauv CSF, txawm hais tias tib lub pas dej tau siv rau ib tus tsiaj.Qhov no qhia tau hais tias ntau yam kev xaiv nruj rau cov tsev qiv ntawv uas muaj coob tus tswv tsev qiv ntawv ua rau muaj kev txo qis hauv ntau haiv neeg.Yog li ntawd, cov xwm txheej random yuav dhau los ua ib feem tseem ceeb ntawm kev xaiv thawj zaug, cuam tshuam rau qhov tshwm sim.Nws zoo li ntau tus clones hauv thawj lub tsev qiv ntawv muaj qhov zoo sib xws CSF enrichment propensity.Txawm li cas los xij, txawm tias nyob rau hauv tib txoj kev sim, cov txiaj ntsig kev xaiv yuav txawv vim muaj tsawg tsawg ntawm txhua qhov tshwj xeeb clone hauv thawj pas dej.
Cov motifs enriched hauv CSF txawv ntawm cov ntshav.Interestingly, peb tau sau tseg thawj zaug hloov mus rau glycine-nplua nuj peptides nyob rau hauv cov ntshav ntawm ib tug neeg tsiaj.(Fig. 1g, Supplementary Fig. 4e, 4f).Phage uas muaj glycine peptides tuaj yeem ruaj khov thiab tsis tshua muaj peev xwm coj tawm ntawm kev ncig.Txawm li cas los xij, cov glycine-nplua nuj peptides no tsis tau kuaj pom hauv cov kua cerebrospinal, qhia tias cov tsev qiv ntawv curated tau dhau los ntawm ob txoj kev xaiv sib txawv: ib qho hauv cov ntshav thiab lwm qhov tso cai rau hauv cov kua cerebrospinal.CSF-enriched clones uas tshwm sim los ntawm plaub puag ncig ntawm kev xaiv tau raug kuaj ntau.Yuav luag tag nrho cov clones kuaj ib tus zuj zus tau lees paub tias muaj txiaj ntsig zoo hauv CSF piv rau kev tswj hwm phage.Ib qho peptide ntaus (#2077) tau tshuaj xyuas ntau yam ntxiv.Nws pom lub plasma ntev ib nrab-lub neej piv rau lwm yam hits (Daim duab 3d thiab ntxiv daim duab 7), thiab nthuav, cov peptide no muaj cov cysteine ​​​​residue ntawm C-terminus.Nws tsis ntev los no tau pom tias qhov sib ntxiv ntawm cysteine ​​​​rau peptides tuaj yeem txhim kho lawv cov tshuaj pharmacokinetic los ntawm kev khi rau albumin 29 .Qhov no tam sim no tsis paub txog peptide #2077 thiab yuav tsum tau kawm ntxiv.Qee cov peptides tau pom qhov valence-dependency nyob rau hauv CSF enrichment (cov ntaub ntawv tsis qhia), uas tej zaum yuav muaj feem xyuam rau cov displayed nto geometry ntawm T7 capsid.Lub T7 system peb siv tau pom 5-15 daim ntawv luam ntawm txhua peptide ib phage particle.IHC tau ua rau cov neeg sib tw ua cov tshuaj phage clones txhaj rau hauv lub hlwb cortex ntawm nas (Ntxiv Fig. 8).Cov ntaub ntawv qhia tias tsawg kawg peb lub clones (No. 2002, No. 2009 thiab No. 2077) cuam tshuam nrog BBB.Nws tseem yuav tau txiav txim siab seb qhov kev sib cuam tshuam BBB no puas ua rau muaj kev cuam tshuam ntawm CSF lossis kev txav ntawm cov clones ncaj qha mus rau BCSFB.Qhov tseem ceeb, peb qhia tau tias cov peptides xaiv khaws lawv lub peev xwm thauj khoom CSF thaum ua ke thiab khi rau cov khoom thauj khoom protein.Kev khi ntawm N-terminal biotinylated peptides rau SA tseem ceeb rov ua dua cov txiaj ntsig tau nrog lawv cov phage clones hauv cov ntshav thiab cerebrospinal kua (Fig. 3e).Thaum kawg, peb qhia tau tias cov hmoov lead peptide #2077 muaj peev xwm txhawb lub hlwb ua haujlwm ntawm biotinylated peptide inhibitor ntawm BACE1 conjugated rau SA, ua rau muaj kev cuam tshuam pharmacodynamic hauv CNS los ntawm txo qis Abeta40 qib hauv CSF (Fig. 4).Peb tsis tuaj yeem txheeb xyuas cov homologues hauv cov ntaub ntawv los ntawm kev ua cov peptide ib ntus homology tshawb nrhiav ntawm txhua qhov hits.Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias qhov loj ntawm T7 lub tsev qiv ntawv yog kwv yees li 109, thaum lub tsev qiv ntawv theoretical loj rau 12-mers yog 4 x 1015. Yog li ntawd, peb tsuas yog xaiv ib feem me me ntawm qhov chaw sib txawv ntawm 12-mer peptide tsev qiv ntawv, uas tej zaum yuav txhais tau tias ntau qhov zoo tshaj peptides no tuaj yeem txheeb xyuas los ntawm kev ntsuam xyuas qhov sib txawv ntawm qhov chaw sib txawv.Hypothetically, ib qho ntawm cov laj thawj yog vim li cas peb tsis pom ib yam dab tsi homologues ntawm cov peptides no tej zaum yuav deselection thaum lub sij hawm evolution los mus tiv thaiv lub uncontrolled nkag ntawm tej yam peptide motifs mus rau hauv lub hlwb.
Ua ke, peb cov txiaj ntsig tau muab lub hauv paus rau kev ua haujlwm yav tom ntej txhawm rau txheeb xyuas thiab ua tus yam ntxwv ntawm kev thauj mus los ntawm cov kab mob cerebrovascular barrier hauv vivo kom ntxaws ntxiv.Kev teeb tsa yooj yim ntawm txoj kev no yog ua raws li kev xaiv ua haujlwm uas tsis yog tsuas yog txheeb xyuas cov clones nrog lub paj hlwb vascular binding zog, tab sis kuj suav nrog cov kauj ruam tseem ceeb uas ua tiav cov clones muaj kev ua haujlwm zoo los hla cov kab mob lom hauv vivo rau hauv CNS compartment.yog txhawm rau txheeb xyuas cov txheej txheem ntawm kev thauj mus los ntawm cov peptides thiab lawv nyiam rau kev khi rau microvasculature tshwj xeeb rau thaj tsam ntawm lub hlwb.Qhov no tuaj yeem ua rau pom txoj hauv kev tshiab rau kev thauj mus los ntawm BBB thiab receptors.Peb cia siab tias cov peptides uas tau txheeb xyuas tuaj yeem khi ncaj qha rau cerebrovascular receptors lossis mus ncig ligands thauj los ntawm BBB lossis BCSFB.Cov peptide vectors nrog CSF thauj kev ua haujlwm nrhiav pom hauv txoj haujlwm no yuav raug tshawb xyuas ntxiv.Tam sim no peb tab tom tshawb xyuas lub hlwb tshwj xeeb ntawm cov peptides rau lawv lub peev xwm hla BBB thiab / lossis BCSFB.Cov peptides tshiab no yuav yog cov cuab yeej muaj txiaj ntsig zoo rau kev tshawb pom muaj peev xwm ntawm cov receptors tshiab lossis txoj hauv kev thiab rau kev txhim kho cov platforms tshiab uas muaj txiaj ntsig zoo rau kev xa cov macromolecules, xws li biologics, mus rau lub hlwb.
Cannulate lub cisterna loj (CM) siv kev hloov kho ntawm txoj kev piav qhia yav dhau los.Anesthetized Wistar nas (200-350 g) tau muab tso rau ntawm lub stereotaxic apparatus thiab ib tug nruab nrab incision yog ua nyob rau hauv lub shaved thiab aseptically npaj tawv taub hau kom nthuav tawm lub pob txha taub hau.Laum ob lub qhov nyob rau hauv cheeb tsam ntawm lub sab sauv sash thiab fasten lub fixing screws nyob rau hauv lub qhov.Ib lub qhov ntxiv tau drilled nyob rau sab occipital crest rau stereotactic kev taw qhia ntawm stainless hlau cannula rau hauv CM.Siv cov cement nyob ib ncig ntawm lub cannula thiab ruaj ntseg nrog screws.Tom qab cov duab kho thiab cement hardening, daim tawv nqaij raug kaw nrog 4/0 supramid suture.Kev tso cai ntawm cannula tau lees paub los ntawm qhov tshwm sim ntawm cov kua dej cerebrospinal (CSF).Tshem cov nas tawm ntawm lub tshuab stereotaxic, tau txais kev kho mob tom qab tsim nyog thiab kev tswj xyuas qhov mob, thiab cia nws rov zoo li tsawg kawg ib lub lis piam kom txog thaum pom cov ntshav hauv cov kua cerebrospinal.Wistar nas (Crl:WI/Han) tau los ntawm Charles River (Fabkis).Txhua tus nas tau khaws cia nyob rau hauv cov xwm txheej tshwj xeeb uas tsis muaj kab mob.Txhua qhov kev sim tsiaj tau pom zoo los ntawm Veterinary Office of the City of Basel, Switzerland, thiab tau ua raws li Tsiaj Daim Ntawv Tso Cai No. 2474 (Kev soj ntsuam ntawm Lub Ncauj Tsheb Thauj Mus Los los ntawm Kev Ntsuas Cov Qib Kev Kho Mob hauv Cerebrospinal Fluid thiab Brain of Rat).
Maj mam ua kom tus nas nco qab nrog CM cannula hauv tes.Tshem tawm Datura los ntawm cannula thiab sau 10 µl ntawm spontaneously ntws cerebrospinal kua.Txij li thaum lub patency ntawm cannula tau raug cuam tshuam thaum kawg, tsuas yog kuaj cov kua dej cerebrospinal uas tsis muaj pov thawj ntawm cov ntshav paug los yog tsis muaj xim muaj nyob rau hauv txoj kev tshawb no.Nyob rau tib lub sijhawm, kwv yees li 10-20 μl ntawm cov ntshav tau coj los ntawm qhov me me ntawm qhov kawg ntawm tus Tsov tus tw rau hauv cov hlab nrog heparin (Sigma-Aldrich).CSF thiab ntshav tau sau los ntawm ntau lub sijhawm cov ntsiab lus tom qab txhaj tshuaj T7 phage.Kwv yees li 5-10 μl ntawm cov kua raug muab pov tseg ua ntej txhua tus qauv CSF tau sau, uas sib haum rau qhov ntim ntawm lub catheter tuag.
Cov tsev qiv ntawv tau tsim los siv T7Select 10-3b vector raws li tau piav qhia hauv T7Select system phau ntawv (Novagen, Rosenberg li al., InNovations 6, 1-6, 1996).Luv luv, ib qho random 12-mer DNA ntxig tau ua ke hauv cov qauv hauv qab no:
NNK codon tau siv los tiv thaiv ob chav nres codons thiab amino acid overexpression hauv qhov ntxig.N yog qhov sib xyaw ua ke ntawm tus kheej ntawm txhua tus nucleotide, thiab K yog qhov sib xyaw ua ke ntawm adenine thiab cytosine nucleotides.Ib cheeb tsam uas tau tso tseg tau hloov pauv mus rau ob npaug ntawm DNA los ntawm kev sib txuas ntxiv nrog dNTP (Novagen) thiab Klenow enzyme (New England Biolabs) hauv Klenow buffer (New England Biolabs) rau 3 teev ntawm 37 ° C.Tom qab cov tshuaj tiv thaiv, ob-stranded DNA tau zoo los ntawm EtOH nag lossis daus.Cov txiaj ntsig DNA tau zom nrog kev txwv enzymes EcoRI thiab HindIII (ob leeg los ntawm Roche).Cov cleaved thiab purified (QIAquick, Qiagen) ntxig (T4 ligase, New England Biolabs) ces ligated in-frame rau hauv pre-cleaved T7 vector tom qab amino acid 348 ntawm 10B capsid noob.Ligation cov tshuaj tiv thaiv tau incubated ntawm 16 ° C. rau 18 teev ua ntej ntim hauv vitro.Phage ntim hauv vitro tau ua raws li cov lus qhia muab nrog T7Select 10-3b cloning kit (Novagen) thiab cov tshuaj ntim tau nthuav dav ib zaug rau lysis siv Escherichia coli (BLT5615, Novagen).Cov lysates tau centrifuged, titrated thiab khov ntawm -80 ° C. raws li cov khoom lag luam ntawm glycerol.
Direct PCR amplification ntawm phage variable regions amplified nyob rau hauv broth los yog phaj siv proprietary 454 / Roche-amplicon fusion primers.Lub pem hauv ntej fusion primer muaj ib ntus flanking thaj tsam sib txawv (NNK) 12 (template-specific), GS FLX Titanium Adapter A, thiab plaub lub hauv paus tsev qiv ntawv tseem ceeb ib ntus (TCAG) (Cov duab ntxiv 1a):
Qhov rov qab fusion primer kuj muaj biotin txuas rau kev ntes cov hlaws thiab GS FLX Titanium Adapter B xav tau rau clonal amplification thaum lub sij hawm emulsion PCR:
Cov amplicons tau raug rau 454 / Roche pyrosequencing raws li 454 GS-FLX Titanium raws tu qauv.Rau phau ntawv Sanger sequencing (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA yog amplified los ntawm PCR thiab sequenced nrog cov nram qab no primer khub:
Ntxig los ntawm ib tus neeg cov plaques raug rau PCR amplification siv Roche Fast Start DNA Polymerase Kit (raws li cov chaw tsim khoom cov lus qhia).Ua qhov pib kub (10 min ntawm 95 ° C) thiab 35 boost cycles (50 s ntawm 95 ° C, 1 min ntawm 50 ° C, thiab 1 min ntawm 72 ° C).
Phage los ntawm cov tsev qiv ntawv, phage tsiaj qus, phage cawm los ntawm CSF thiab ntshav, los yog ib tug neeg clones tau amplified nyob rau hauv Escherichia coli BL5615 nyob rau hauv TB broth (Sigma Aldrich) los yog nyob rau hauv 500 cm2 tais diav (Thermo Scientific) rau 4 h ntawm 37 ° C.Phage tau muab rho tawm los ntawm daim hlau los ntawm kev yaug cov phaj nrog Tris-EDTA tsis muaj (Fluka Analytical) los yog los ntawm kev sau cov plaques nrog cov lus qhia tsis muaj menyuam.Phage raug cais tawm ntawm kab lis kev cai supernatant lossis extraction tsis nrog ib puag ncig ntawm polyethylene glycol (PEG 8000) nag lossis daus (Promega) thiab rov ua dua hauv Tris-EDTA tsis nyob.
Lub amplified phage tau raug rau 2-3 rounds ntawm endotoxin tshem tawm siv endotoxin tshem tawm hlaws dai (Miltenyi Biotec) ua ntej intravenous (IV) txhaj tshuaj (500 μl / tsiaj).Hauv thawj puag ncig, 2 × 1012 phages tau qhia;hauv ob, 2 × 1010 phages;nyob rau hauv lub thib peb thiab plaub xaiv rounds, 2 × 109 phages rau ib tug tsiaj.Phage cov ntsiab lus nyob rau hauv CSF thiab cov ntshav kuaj sau ntawm lub sij hawm cov ntsiab lus tau txiav txim los ntawm cov quav hniav suav raws li cov chaw tsim tshuaj paus cov lus qhia (T7Select system phau ntawv).Kev xaiv phage tau ua los ntawm kev txhaj tshuaj ntawm cov tsev qiv ntawv purified rau hauv cov hlab ntsha ntawm tus Tsov tus tw los yog rov txhaj tshuaj phage rho tawm los ntawm CSF los ntawm kev xaiv yav dhau los, thiab tom qab sau qoob loo tau ua nyob rau ntawm 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, thiab 240 min.Tag nrho ntawm plaub puag ncig ntawm vivo panning tau ua nyob rau hauv uas ob ceg xaiv tau cais cais thiab tshuaj xyuas thaum thawj peb qhov kev xaiv.Tag nrho cov phage inserts muab rho tawm los ntawm CSF los ntawm thawj ob qhov kev xaiv tau raug rau 454 / Roche pyrosequencing, thaum tag nrho cov clones muab rho tawm los ntawm CSF los ntawm ob qhov kawg ntawm kev xaiv tau manually sequenced.Tag nrho cov ntshav phages los ntawm thawj puag ncig ntawm kev xaiv kuj raug rau 454 / Roche pyrosequencing.Rau kev txhaj tshuaj phage clones, xaiv phages tau nthuav dav hauv E. coli (BL5615) ntawm 500 cm2 daim hlau ntawm 37 ° C rau 4 teev.Ib tus kheej xaiv thiab ua raws li tus kheej cov clones tau nthuav tawm hauv nruab nrab TB.Tom qab phage extraction, purification thiab tshem tawm ntawm endotoxin (raws li tau piav qhia saum toj no), 2 × 1010 phages / tsiaj nyob rau hauv 300 μl tau txhaj tshuaj intravenously rau hauv ib tus Tsov tus tw leeg.
Preprocessing thiab qualitative filtering ntawm sequence cov ntaub ntawv.Raw 454/Roche cov ntaub ntawv tau hloov dua siab tshiab los ntawm binary standard kwj daim ntawv qhia hom ntawv (sff) mus rau Pearson human readable format (fasta) siv cov neeg muag khoom software.Kev ua tiav ntxiv ntawm nucleotide ib ntus tau ua tiav siv cov kev pabcuam C thiab cov ntawv sau (tsis tau tso tawm software pob) raws li tau piav qhia hauv qab no.Kev tsom xam ntawm cov ntaub ntawv tseem ceeb suav nrog cov txheej txheem lim dej ntau theem nruj.Txhawm rau lim tawm cov ntawv nyeem uas tsis muaj qhov siv tau 12mer ntxig DNA ib ntus, cov ntawv nyeem tau sib txuas ua ke kom pib daim ntawv lo (GTGATGTCGGGGATCCGAATTCT), nres daim ntawv lo (TAAGCTTGCGGCCGCACTCGAGTA) thiab keeb kwm yav dhau (CCCTGCAGGATATCCCGGGAAGCTCGTCGAC.man-Wuned test.alignment cia mus txog 2 inconsistencies ib alignment31.Yog li ntawd, nyeem yam tsis tau pib thiab tso tseg cov cim npe thiab nyeem uas muaj cov ntaub ntawv keeb kwm yav dhau los, piv txwv li, cov kev sib raug zoo uas ntau tshaj qhov tso cai tsis sib haum, raug tshem tawm ntawm lub tsev qiv ntawv.Raws li cov ntawv nyeem ntxiv, N-mer DNA ntu txuas ntxiv los ntawm lub cim pib thiab xaus ua ntej lub cim nres tau raug tshem tawm los ntawm cov ntawv nyeem thawj zaug thiab ua tiav ntxiv (tom qab no hu ua "insert").Tom qab kev txhais lus ntawm qhov ntxig, qhov feem tom qab thawj nres codon ntawm 5' kawg ntawm cov primer raug tshem tawm ntawm qhov ntxig.Tsis tas li ntawd, nucleotides ua rau tsis tiav codons ntawm 3′ kawg ntawm primer kuj raug tshem tawm.Txhawm rau tshem tawm cov ntawv ntxig uas tsuas muaj keeb kwm yav dhau los, cov ntawv txhais lus pib nrog tus qauv amino acid "PAG" kuj raug tshem tawm.Peptides nrog qhov ntev tom qab kev txhais lus tsawg dua 3 amino acids raug tshem tawm ntawm lub tsev qiv ntawv.Thaum kawg, tshem tawm redundancy nyob rau hauv lub insert pas dej ua ke thiab txiav txim seb qhov zaus ntawm txhua qhov tshwj xeeb insert.Cov txiaj ntsig ntawm qhov kev tshuaj ntsuam no suav nrog cov npe ntawm nucleotide sequences (inserts) thiab lawv (nyeem) zaus (Cov duab ntxiv 1c thiab 2).
Pab pawg N-mer DNA inserts los ntawm qhov sib npaug zoo sib xws: Txhawm rau tshem tawm 454 / Roche-specific sequencing yuam kev (xws li teeb meem nrog sequencing homopolymer extensions) thiab tshem tawm cov tsis tseem ceeb redundancies, yav tas los lim N-mer DNA sequence inserts (inserts) yog txheeb los ntawm qhov zoo sib xws.insertions (txog li 2 lub hauv paus tsis sib piv tau tso cai) siv cov txheej txheem rov ua dua uas tau teev tseg raws li hauv qab no: cov ntawv ntxig raug txheeb xyuas ua ntej los ntawm lawv qhov zaus (siab tshaj plaws rau qis tshaj), thiab yog tias lawv zoo ib yam, los ntawm lawv qhov thib ob los ntawm qhov ntev (ntev mus rau luv tshaj).Yog li, feem ntau thiab qhov ntev tshaj plaws insertions txhais thawj "pab pawg".Pawg zaus yog teem rau qhov tseem ceeb zaus.Tom qab ntawd, txhua qhov kev ntxig ntxiv nyob rau hauv daim ntawv teev npe tau sim ntxiv rau hauv pab pawg los ntawm kev sib koom ua ke ntawm Needleman-Wunsch.Yog tias tus naj npawb ntawm qhov tsis sib haum, kev ntxig, lossis kev tshem tawm hauv qhov kev sib dhos tsis tshaj qhov pib ntawm 2, ib qho kev ntxig ntxiv rau hauv pab pawg, thiab tag nrho pawg ntau zaus tau nce los ntawm ntau npaum li cas qhov kev ntxig ntxiv.Ntxig ntxiv rau ib pab pawg raug cim tias siv thiab tsis suav nrog kev ua haujlwm ntxiv.Yog tias cov txheej txheem ntxig tsis tuaj yeem muab ntxiv rau ib pawg uas twb muaj lawm, cov txheej txheem ntxig yog siv los tsim ib pawg tshiab nrog qhov tsim nyog ntxig zaus thiab cim tias siv.Qhov kev rov ua dua yuav xaus thaum txhua qhov kev sib txuas ua ke tau siv los tsim ib pab pawg tshiab lossis tuaj yeem suav nrog hauv ib pab pawg uas twb muaj lawm.Tom qab tag nrho, pawg inserts uas muaj nucleotides nws thiaj li muab txhais ua peptide sequences (peptide qiv).Qhov tshwm sim ntawm qhov kev tshuaj ntsuam no yog ib txheej ntawm cov ntawv ntxig thiab lawv cov zaus sib thooj uas ua rau cov ntawv nyeem sib law liag (Ntxiv Fig. 2).
Motif Generation: Raws li cov npe ntawm cov peptides tshwj xeeb, lub tsev qiv ntawv tau tsim muaj tag nrho cov amino acid qauv (aa) raws li qhia hauv qab no.Txhua tus qauv ua tau ntev 3 tau muab rho tawm los ntawm peptide thiab nws cov qauv hloov pauv tau ntxiv nrog rau cov tsev qiv ntawv motif uas muaj tag nrho cov qauv (tripeptides).Cov tsev qiv ntawv ntawm cov motifs repetitive heev tau sequenced thiab redundancy tshem tawm.Tom qab ntawd, rau txhua tripeptide hauv lub tsev qiv ntawv motif, peb tau kuaj xyuas nws qhov muaj nyob hauv lub tsev qiv ntawv siv cov cuab yeej suav.Hauv qhov no, qhov zaus ntawm peptide uas muaj qhov pom motif tripeptide tau ntxiv thiab muab tso rau hauv motif hauv lub tsev qiv ntawv motif ("number of motifs").Qhov tshwm sim ntawm motif tiam yog ob-dimensional array uas muaj tag nrho cov tshwm sim ntawm tripeptides (motifs) thiab lawv cov txiaj ntsig, uas yog tus naj npawb ntawm cov ntawv nyeem uas ua rau cov motif sib xws thaum nyeem tau lim, pab pawg, thiab txhais lus.Metrics raws li tau piav qhia hauv qab no.
Normalization ntawm tus naj npawb ntawm motifs thiab sib thooj scatterplots: Tus naj npawb ntawm motifs rau txhua tus qauv yog normalized siv
qhov twg ni yog tus naj npawb ntawm cov nyeem uas muaj cov ncauj lus kuv.Yog li, vi sawv cev rau feem pua ​​​​ntau zaus ntawm kev nyeem (lossis peptides) uas muaj motif kuv hauv cov qauv.P-tus nqi rau cov tsis muaj tus lej ntawm cov motifs tau suav nrog Fisher qhov kev xeem.Hais txog correlograms ntawm tus naj npawb ntawm motives, Spearman qhov kev sib raug zoo tau suav nrog siv tus naj npawb ntawm cov kev xav nrog R.
Txhawm rau pom cov ntsiab lus ntawm cov amino acids ntawm txhua txoj haujlwm hauv lub tsev qiv ntawv peptide, lub vev xaib logograms 32, 33 (http://weblogo.threeplusone.com) tau tsim.Ua ntej, cov ntsiab lus ntawm cov amino acids ntawm txhua txoj haujlwm ntawm 12-mer peptide yog khaws cia hauv 20 × 12 matrix.Tom qab ntawd, ib txheej ntawm 1000 peptides uas muaj tib cov ntsiab lus ntawm cov amino acid cov ntsiab lus ntawm txhua txoj hauj lwm yog tsim nyob rau hauv fasta-sequence hom thiab muab raws li cov tswv yim rau web-logo 3, uas generates ib tug graphical sawv cev ntawm cov txheeb ze amino acid cov ntsiab lus ntawm txhua txoj hauj lwm.rau lub tsev qiv ntawv peptide muab.Txhawm rau pom cov ntaub ntawv sib txawv, cov ntawv qhia tshav kub tau tsim los siv cov cuab yeej tsim kho sab hauv hauv R (biosHeatmap, tseem-rau-tso-tso R pob).Cov dendrograms nthuav tawm hauv daim ntawv qhia tshav kub tau suav nrog Ward's hierarchical clustering method with the Euclidean distance metric.Rau kev txheeb xyuas cov ntaub ntawv motif scoring, P qhov tseem ceeb rau qhov tsis zoo ntawm cov qhab nia raug suav nrog siv Fisher qhov kev ntsuas tseeb.P-tus nqi rau lwm cov ntaub ntawv tau suav nrog hauv R siv Student's t-test los yog ANOVA.
Xaiv phage clones thiab phages yam tsis muaj qhov ntxig tau txhaj tshuaj intravenously ntawm tus Tsov tus tw leeg (2 × 1010 phages / tsiaj hauv 300 μl PBS).Kaum feeb ua ntej perfusion thiab tom qab fixation, tib cov tsiaj tau txhaj tshuaj intravenously nrog 100 μl ntawm DyLight594-labeled lectin (Vector Laboratories Inc., DL-1177).60 feeb tom qab phage txhaj, nas tau perfused hauv plawv nrog 50 ml PBS tom qab 50 ml 4% PFA / PBS.Kev kuaj mob hlwb tau ntxiv rau ib hmos hauv 4% PFA / PBS thiab soaked hauv 30% sucrose thaum hmo ntuj ntawm 4 ° C.Cov qauv yog flash khov hauv OCT sib tov.Immunohistochemical tsom xam ntawm cov qauv khov tau ua nyob rau hauv chav tsev kub ntawm 30 µm cryosections thaiv nrog 1% BSA thiab incubated nrog polyclonal FITC-labeled antibodies tiv thaiv T7 phage (Novus NB 600-376A) ntawm 4 ° C.Incubate hmo ntuj.Thaum kawg, cov seem tau ntxuav 3 zaug nrog PBS thiab tshuaj xyuas nrog lub tshuab ntsuas hluav taws xob confocal (Leica TCS SP5).
Tag nrho cov peptides nrog qhov tsawg kawg nkaus purity ntawm 98% tau tsim los ntawm GenScript USA, biotinylated thiab lyophilized.Biotin yog khi ntawm ib qho ntxiv triple glycine spacer ntawm N-terminus.Txheeb xyuas tag nrho cov peptides siv cov spectrometry loj.
Streptavidin (Sigma S0677) tau sib xyaw nrog 5-fold equimolar tshaj ntawm biotinylated peptide, biotinylated BACE1 inhibitory peptide, los yog kev sib xyaw ua ke (3: 1 piv) ntawm biotinylated BACE1 inhibitory peptide thiab BACE1 inhibitory peptide hauv 5-10% DMSO / .BS1 teev ntawm chav tsev kub ua ntej txhaj tshuaj.Streptavidin-conjugated peptides tau txhaj tshuaj intravenously ntawm ib koob ntawm 10 mg / kg rau hauv ib qho ntawm cov leeg leeg ntawm nas nrog lub paj hlwb.
Qhov concentration ntawm streptavidin-peptide complexes tau soj ntsuam los ntawm ELISA.Nunc Maxisorp microtiter daim hlau (Sigma) tau coated thaum hmo ntuj ntawm 4 ° C nrog 1.5 μg / ml nas anti-streptavidin antibody (Thermo, MA1-20011).Tom qab thaiv kev thaiv (kev thaiv tsis pub muaj: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ntawm chav tsev kub rau 2 teev, ntxuav lub phaj nrog 0.05% Tween-20 / PBS (ntxuav tsis huv) rau 3 Thib ob plalute blocks (Csfb) ntxiv rau qhov dej zoo sma 1:10,000, CSF 1:115).Tom qab ntawd lub phaj tau incubated thaum hmo ntuj ntawm 4 ° C nrog kuaj pom cov tshuaj tiv thaiv (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239).Tom qab peb kauj ruam ntxuav, streptavidin tau kuaj pom los ntawm incubation hauv TMB substrate tov (Roche) txog li 20 min.Tom qab nres xim kev loj hlob nrog 1M H2SO4, ntsuas qhov nqus ntawm 450 nm.
Kev ua haujlwm ntawm streptavidin-peptide-BACE1 inhibitor complex tau soj ntsuam los ntawm Aβ(1-40) ELISA raws li cov chaw tsim khoom raws tu qauv (Wako, 294-64701).Luv luv, CSF cov qauv tau diluted nyob rau hauv tus qauv diluent (1:23) thiab incubated hmo ntuj ntawm 4 ° C nyob rau hauv 96-zoo daim hlau coated nrog BNT77 capture antibody.Tom qab tsib kauj ruam ntxuav, HRP-conjugated BA27 antibody ntxiv thiab incubated rau 2 teev ntawm 4 ° C., ua raws li tsib kauj ruam ntxuav.Aβ(1–40) tau kuaj pom los ntawm incubation hauv TMB tov rau 30 feeb ntawm chav tsev kub.Tom qab kev txhim kho xim tau raug tso tseg nrog kev daws teeb meem, ntsuas qhov nqus ntawm 450 nm.Cov qauv ntshav plasma tau raug rau cov txheej txheem rho tawm ua ntej Aβ(1-40) ELISA.Plasma tau ntxiv rau 0.2% DEA (Sigma) hauv 96-zoo daim hlau thiab incubated ntawm chav tsev kub rau 30 feeb.Tom qab ua tiav kev ntxuav SPE daim hlau (Oasis, 186000679) nrog dej thiab 100% methanol, cov plasma kuaj tau ntxiv rau SPE daim hlau thiab tag nrho cov kua raug tshem tawm.Cov qauv raug ntxuav (thawj nrog 5% methanol ces 30% methanol) thiab eluted nrog 2% NH4OH / 90% methanol.Tom qab ziab cov eluate ntawm 55 ° C rau 99 min ntawm qhov N2 tam sim no, cov qauv raug txo qis hauv cov qauv diluents thiab Aβ(1-40) tau ntsuas raws li tau piav qhia saum toj no.
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