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Qhov thaiv ntshav-lub hlwb thiab qhov thaiv ntshav-lub hlwb tiv thaiv cov neeg sawv cev biotherapeutic kom tsis txhob mus txog lawv lub hom phiaj hauv lub paj hlwb hauv nruab nrab, yog li ntawd cuam tshuam kev kho mob zoo ntawm cov kab mob neurological. Txhawm rau nrhiav pom cov neeg thauj neeg mob hlwb tshiab hauv vivo, peb tau qhia txog T7 phage peptide tsev qiv ntawv thiab sau cov ntshav thiab cov kua cerebrospinal (CSF) siv cov qauv loj cannulated conscious ntawm cov nas. Cov phage clones tshwj xeeb tau nplua nuj heev hauv CSF tom qab plaub qhov kev xaiv. Kev sim ntawm cov neeg sib tw peptides tau qhia ntau dua 1000-fold nplua nuj hauv CSF. Qhov bioactivity ntawm peptide-mediated xa mus rau lub hlwb tau lees paub los ntawm 40% txo qis hauv qib ntawm amyloid-β hauv cov kua cerebrospinal siv BACE1 peptide inhibitor txuas nrog cov peptide transit tshiab tau txheeb xyuas. Cov txiaj ntsig no qhia tias cov peptides txheeb xyuas los ntawm cov txheej txheem xaiv phage hauv vivo yuav yog cov tsheb muaj txiaj ntsig rau kev xa cov macromolecules mus rau lub hlwb nrog cov txiaj ntsig kho mob.
Kev tshawb fawb txog kev kho mob hauv lub paj hlwb (CNS) feem ntau tsom mus rau kev txheeb xyuas cov tshuaj thiab cov neeg sawv cev zoo tshaj plaws uas qhia txog cov khoom CNS-targeting, nrog rau kev siv zog tsawg dua los tshawb pom cov txheej txheem uas tsav kev xa tshuaj mus rau lub hlwb. Qhov no pib hloov pauv tam sim no raws li kev xa tshuaj, tshwj xeeb tshaj yog cov molecules loj, yog ib feem tseem ceeb ntawm kev tsim tshuaj neuroscience niaj hnub no. Ib puag ncig ntawm lub paj hlwb hauv nruab nrab tau tiv thaiv zoo los ntawm lub cerebrovascular barrier system, uas muaj cov ntshav-lub hlwb barrier (BBB) ​​​​thiab cov ntshav-lub hlwb barrier (BCBB)1, ua rau nws nyuaj rau xa cov tshuaj mus rau lub hlwb1,2. Nws kwv yees tias yuav luag txhua cov tshuaj molecule loj thiab ntau dua 98% ntawm cov tshuaj molecule me me raug tshem tawm ntawm lub hlwb3. Yog vim li cas nws thiaj li tseem ceeb heev los txheeb xyuas cov kab ke thauj mus los hauv lub hlwb tshiab uas muab kev xa tshuaj kho mob zoo thiab tshwj xeeb rau CNS 4,5. Txawm li cas los xij, BBB thiab BCSFB kuj tseem muaj lub sijhawm zoo rau kev xa tshuaj thaum lawv nkag mus thiab nkag mus rau txhua qhov qauv ntawm lub hlwb los ntawm nws cov vasculature dav dav. Yog li ntawd, cov kev siv zog tam sim no los siv cov txheej txheem tsis-invasive ntawm kev xa mus rau lub hlwb feem ntau yog raws li lub tshuab ntawm receptor-mediated transport (PMT) siv endogenous BBB6 receptor. Txawm hais tias muaj kev vam meej tseem ceeb tsis ntev los no siv txoj kev transferrin receptor pathway7,8, kev txhim kho ntxiv ntawm cov txheej txheem xa khoom tshiab nrog cov khoom zoo dua yog qhov yuav tsum tau ua. Txhawm rau ua tiav qhov no, peb lub hom phiaj yog txheeb xyuas cov peptides uas muaj peev xwm ua haujlwm nruab nrab ntawm CSF kev thauj mus los, vim tias lawv tuaj yeem siv los xa cov macromolecules mus rau CNS lossis qhib cov receptor tshiab. Tshwj xeeb, cov receptors thiab cov neeg thauj khoom tshwj xeeb ntawm lub cev cerebrovascular (BBB thiab BSCFB) tuaj yeem ua lub hom phiaj rau kev xa cov tshuaj biotherapeutic nquag thiab tshwj xeeb. Cerebrospinal fluid (CSF) yog cov khoom secretory ntawm choroid plexus (CS) thiab nyob rau hauv kev sib cuag ncaj qha nrog cov kua interstitial ntawm lub hlwb los ntawm subarachnoid qhov chaw thiab ventricular qhov chaw4. Tsis ntev los no nws tau pom tias subarachnoid cerebrospinal fluid diffuses ntau dhau mus rau hauv interstitium ntawm lub hlwb9. Peb vam tias yuav nkag mus rau hauv qhov chaw parenchymal siv txoj kev nkag mus rau hauv subarachnoid no lossis ncaj qha los ntawm BBB. Txhawm rau ua tiav qhov no, peb tau siv txoj kev xaiv phage hauv vivo uas muaj zog uas txheeb xyuas cov peptides uas thauj los ntawm ob txoj kev sib txawv no.
Tam sim no peb piav qhia txog kev tshuaj ntsuam xyuas phage hauv vivo nrog CSF sampling ua ke nrog kev ntsuas siab (HTS) los saib xyuas cov kev xaiv thawj zaug nrog qhov sib txawv ntawm tsev qiv ntawv siab tshaj plaws. Kev tshuaj ntsuam tau ua rau cov nas uas paub nrog lub cannula loj (CM) uas tau cog rau hauv lub cev tas mus li kom tsis txhob muaj kev kis kab mob ntshav. Qhov tseem ceeb, txoj hauv kev no xaiv ob qho tib si lub hlwb-targeting thiab peptides nrog kev thauj mus los hla lub cerebrovascular barrier. Peb siv T7 phages vim lawv qhov me me (~ 60 nm) 10 thiab qhia tias lawv tsim nyog rau kev thauj mus los ntawm cov vesicles uas tso cai rau kev hla transcellular ntawm endothelial thiab / lossis epithelial-medulla barrier. Tom qab plaub qhov kev sib tw ntawm panning, cov pej xeem phage tau raug rho tawm qhia tias muaj zog hauv vivo CSF ​​​​enrichment thiab cerebral microvessel kev koom tes. Qhov tseem ceeb, peb tau lees paub peb cov kev tshawb pom los ntawm kev qhia tias cov peptides zoo tshaj plaws thiab cov tshuaj synthesized zoo tshaj plaws muaj peev xwm thauj cov protein cargo mus rau hauv cov kua cerebrospinal. Ua ntej, cov teebmeem pharmacodynamic ntawm CNS tau tsim los ntawm kev sib txuas ua ke ntawm cov peptide transit nrog cov inhibitor ntawm BACE1 peptide. Ntxiv rau qhov ua pov thawj tias cov tswv yim tshuaj ntsuam xyuas kev ua haujlwm hauv vivo tuaj yeem txheeb xyuas cov peptides thauj lub hlwb tshiab ua cov khoom thauj protein zoo, peb xav tias cov txheej txheem xaiv ua haujlwm zoo sib xws kuj tseem ceeb hauv kev txheeb xyuas cov kev thauj lub hlwb tshiab.
Raws li cov chav tsim cov plaque (PFU), tom qab cov kauj ruam ntim phage, 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 uas yuav tsum nco ntsoov tias peb tau ua tib zoo tshuaj xyuas lub tsev qiv ntawv no ua ntej in vivo panning. PCR amplification ntawm phage tsev qiv ntawv qauv siv cov primers hloov kho tsim cov amplicons uas tau siv ncaj qha rau HTS (Supplementary Fig. 1a). Vim yog a) HTS11 sequencing yuam kev, b) cuam tshuam rau qhov zoo ntawm primers (NNK) 1-12, thiab c) qhov muaj cov tsiaj qus (wt) phage (skeleton inserts) hauv lub tsev qiv ntawv standby, cov txheej txheem lim dej tau siv los rho tawm tsuas yog cov ntaub ntawv txheeb xyuas cov txheej txheem (Supplementary Fig. 1b). Cov kauj ruam lim dej no siv rau txhua lub tsev qiv ntawv HTS sequencing. Rau lub tsev qiv ntawv txheem, tag nrho ntawm 233,868 nyeem tau txais, uas 39% dhau cov qauv lim thiab tau siv rau kev tshuaj xyuas tsev qiv ntawv thiab xaiv rau cov voj voog tom ntej (Supplementary Daim Duab 1c-e). Cov nyeem tau feem ntau yog cov lej ntawm 3 lub hauv paus khub ntev nrog lub ncov ntawm 36 nucleotides (Supplementary Daim Duab 1c), lees paub qhov tsim tsev qiv ntawv (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 ntxig, thiab yuav luag ib nrab ntawm cov kab lus (49%) muaj cov ntxig lossis rho tawm. HTS ntawm lub tsev qiv ntawv tsev qiv ntawv tau lees paub qhov muaj ntau yam peptides hauv tsev qiv ntawv: ntau dua 81% ntawm cov kab lus peptide tau pom ib zaug xwb thiab tsuas yog 1.5% tshwm sim hauv ≥4 daim ntawv theej (Supplementary Daim Duab 2a). Cov zaus ntawm cov amino acids (aa) ntawm txhua 12 qhov chaw hauv repertoire sib raug zoo nrog cov zaus xav tau rau tus lej ntawm cov codons tsim los ntawm cov NKK repertoire degenerate (Daim Duab Ntxiv 2b). Qhov zaus pom ntawm aa residues encoded los ntawm cov ntawv ntxig no sib raug zoo nrog qhov zaus xam (r = 0.893) (Daim Duab Ntxiv 2c). Kev npaj cov tsev qiv ntawv phage rau kev txhaj tshuaj suav nrog cov kauj ruam ntawm kev nthuav dav thiab kev tshem tawm ntawm endotoxin. Qhov no tau pom ua ntej kom txo tau qhov sib txawv ntawm cov tsev qiv ntawv phage12,13. Yog li ntawd, peb tau ua tiav cov tsev qiv ntawv phage uas tau raug tshem tawm endotoxin thiab piv rau nws nrog cov tsev qiv ntawv qub los kwv yees qhov zaus ntawm AA. Ib qho kev sib raug zoo (r = 0.995) tau pom ntawm lub pas dej qub thiab lub pas dej uas tau nthuav dav thiab huv (Daim Duab Ntxiv 2d), qhia tias kev sib tw ntawm cov clones uas tau nthuav dav ntawm cov phaj siv T7 phage tsis ua rau muaj kev ntxub ntxaug loj. Qhov kev sib piv no yog raws li qhov zaus ntawm tripeptide motifs hauv txhua lub tsev qiv ntawv, txij li qhov sib txawv ntawm cov tsev qiv ntawv (~ 109) tsis tuaj yeem ntes tau tag nrho txawm tias nrog HTS. Kev tshuaj xyuas zaus ntawm aa ntawm txhua qhov chaw tau qhia txog qhov kev cuam tshuam me me nyob ntawm qhov chaw hauv peb qhov chaw kawg ntawm cov repertoire nkag (Supplementary Fig. 2e). Hauv kev xaus, peb xaus lus tias qhov zoo thiab ntau haiv neeg ntawm lub tsev qiv ntawv tau txais thiab tsuas yog qhov kev hloov pauv me me hauv kev sib txawv tau pom vim yog kev nthuav dav thiab kev npaj ntawm cov tsev qiv ntawv phage ntawm ntau qhov kev xaiv.
Kev kuaj cov kua hauv lub hlwb ua ntu zus tuaj yeem ua tau los ntawm kev phais mob rau hauv CM ntawm cov nas uas paub txog kom yooj yim txheeb xyuas cov T7 phage uas tau txhaj rau hauv cov hlab ntsha (iv) ntawm BBB thiab / lossis BCSFB (Daim Duab 1a-b). Peb siv ob txhais caj npab xaiv ywj pheej (caj npab A thiab B) hauv peb qhov kev xaiv hauv vivo thawj zaug (Daim Duab 1c). Peb maj mam ua kom qhov kev xaiv nruj dua los ntawm kev txo tag nrho cov phage uas tau qhia hauv peb qhov kev xaiv thawj zaug. Rau qhov kev xaiv thib plaub ntawm kev panning, peb tau muab cov qauv los ntawm ceg A thiab B ua ke thiab ua peb qhov kev xaiv ywj pheej ntxiv. Txhawm rau kawm txog cov khoom hauv vivo ntawm T7 phage particles hauv tus qauv no, cov phage qus (PAGISRELVDKL master insert) tau txhaj rau hauv cov nas ntawm cov leeg ntawm tus Tsov tus tw. Kev rov qab tau cov phages los ntawm cov kua hauv lub hlwb thiab cov ntshav ntawm ntau lub sijhawm sib txawv qhia tau hais tias cov phages T7 icosahedral me me muaj qhov pib tshem tawm sai sai los ntawm cov ntshav (Daim Duab Ntxiv 3). Raws li cov titers uas tau muab thiab cov ntshav ntim ntawm cov nas, peb xam tias tsuas yog kwv yees li 1% wt. phage ntawm cov tshuaj uas tau muab tau pom hauv cov ntshav 10 feeb tom qab txhaj tshuaj rau hauv cov hlab ntsha. Tom qab qhov kev poob qis sai sai no, qhov kev tshem tawm thawj zaug qeeb dua tau ntsuas nrog lub sijhawm ib nrab ntawm 27.7 feeb. Qhov tseem ceeb, tsuas yog ob peb phages tau rov qab los ntawm CSF compartment, qhia txog keeb kwm yav dhau los qis rau kev tsiv teb tsaws chaw ntawm cov phage qus mus rau hauv CSF compartment (Daim Duab Ntxiv 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 uas tau txhaj thawj zaug tau pom hauv cov kua cerebrospinal thoob plaws lub sijhawm kuaj (0-250 feeb). Qhov tseem ceeb, lub sijhawm ib nrab (25.7 feeb) ntawm cov phage qus hauv cov kua cerebrospinal zoo ib yam li qhov pom hauv cov ntshav. Cov ntaub ntawv no qhia tau hais tias qhov thaiv uas cais CSF compartment ntawm cov ntshav tseem nyob ruaj khov hauv cov nas CM-cannulated, tso cai rau hauv vivo xaiv cov tsev qiv ntawv phage los txheeb xyuas cov clones uas tau yooj yim thauj los ntawm cov ntshav mus rau hauv CSF compartment.
(a) Teeb tsa ib txoj kev rau kev rov kuaj cov kua cerebrospinal (CSF) los ntawm ib lub pas dej loj. (b) Daim duab qhia qhov chaw ntawm lub paj hlwb hauv nruab nrab (CNS) thiab txoj kev xaiv siv los txheeb xyuas cov peptides uas hla cov ntshav-lub hlwb thaiv (BBB) ​​​​thiab cov ntshav-lub hlwb thaiv. (c) Hauv vivo phage zaub tshuaj ntsuam xyuas flowchart. Hauv txhua qhov kev xaiv, phages (cov cim tsiaj hauv cov xub) tau txhaj tshuaj rau hauv cov hlab ntsha. Ob ceg ywj pheej (A, B) raug khaws cia sib cais kom txog thaum qhov kev xaiv thib 4. Rau qhov kev xaiv thib 3 thiab 4, txhua lub phage clone rho tawm los ntawm CSF tau raug txheeb xyuas los ntawm tes. (d) Kinetics ntawm phage cais los ntawm cov ntshav (lub voj voog liab) thiab cov kua cerebrospinal (cov duab peb sab ntsuab) thaum lub sijhawm xaiv thawj zaug hauv ob lub nas cannulated tom qab txhaj tshuaj rau hauv cov hlab ntsha ntawm T7 peptide tsev qiv ntawv (2 x 1012 phages / tsiaj). Cov plaub fab xiav qhia txog qhov nruab nrab pib concentration ntawm phage hauv cov ntshav, xam los ntawm qhov ntau ntawm cov phage txhaj tshuaj, suav nrog tag nrho cov ntshav ntim. Cov plaub fab dub qhia txog qhov chaw sib tshuam ntawm kab y extrapolated los ntawm cov ntshav phage concentration. (e, f) Nthuav qhia qhov zaus sib piv thiab kev faib tawm ntawm txhua qhov ua tau sib tshooj tripeptide motifs pom hauv peptide. Tus lej ntawm motifs pom hauv 1000 nyeem tau pom. Tseem ceeb (p < 0.001) cov motifs nplua nuj tau cim nrog cov dots liab. (e) Correlation scatterplot piv rau qhov zaus sib piv ntawm tripeptide motif ntawm lub tsev qiv ntawv txhaj tshuaj nrog cov ntshav-tau los ntawm cov tsiaj # 1.1 thiab # 1.2. (f) Correlation scatterplot piv rau qhov zaus sib piv ntawm cov tsiaj phage tripeptide motifs # 1.1 thiab # 1.2 cais tawm hauv cov ntshav thiab cerebrospinal fluid. (g, h) Sequence ID sawv cev ntawm phage nplua nuj hauv cov ntshav (g) piv rau cov tsev qiv ntawv txhaj tshuaj thiab phage nplua nuj hauv CSF (h) piv rau cov ntshav tom qab ib puag ncig ntawm kev xaiv hauv vivo hauv ob qho tib si tsiaj. Qhov loj ntawm ib tsab ntawv code qhia tau tias cov amino acid ntawd tshwm sim ntau npaum li cas ntawm qhov chaw 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 tshuaj phage peptide tsev qiv ntawv rau hauv ob lub CM ntsuas nas (clades A thiab B) thiab cais phage los ntawm cov kua cerebrospinal thiab cov ntshav (Daim Duab 1d). Qhov pib tshem tawm sai ntawm lub tsev qiv ntawv tsis pom tseeb piv rau cov phage qus. Lub sijhawm nruab nrab ntawm lub tsev qiv ntawv txhaj tshuaj hauv ob tus tsiaj yog 24.8 feeb hauv cov ntshav, zoo ib yam li cov phage qus, thiab 38.5 feeb hauv CSF. Cov ntshav thiab cov kua cerebrospinal phage qauv los ntawm txhua tus tsiaj tau raug rau HTS thiab txhua tus peptides txheeb xyuas tau raug soj ntsuam rau qhov muaj cov qauv luv luv tripeptide. Tripeptide motifs tau xaiv vim tias lawv muab lub hauv paus tsawg kawg nkaus rau kev tsim cov qauv thiab peptide-protein sib cuam tshuam14,15. Peb pom muaj kev sib raug zoo hauv kev faib cov qauv ntawm cov tsev qiv ntawv phage txhaj tshuaj thiab cov clones rho tawm los ntawm cov ntshav ntawm ob tus tsiaj (Daim Duab 1e). Cov ntaub ntawv qhia tias cov khoom sib xyaw ntawm lub tsev qiv ntawv tsuas yog nplua nuj me ntsis hauv cov ntshav. Amino acid zaus thiab cov kab ke sib koom siab tau raug soj ntsuam ntxiv ntawm txhua qhov chaw siv kev hloov kho ntawm Weblogo16 software. Txaus siab kawg li, peb pom muaj cov glycine residues ntau ntxiv hauv cov ntshav (Daim Duab 1g). Thaum cov ntshav raug piv nrog cov clones xaiv los ntawm CSF, muaj kev xaiv zoo thiab qee qhov kev xaiv tsis zoo ntawm cov motifs tau pom (Daim Duab 1f), thiab qee cov amino acids tau muaj nyob rau ntawm qhov chaw tau teem tseg hauv 12-tus tswv cuab (Daim Duab 1h). Qhov tseem ceeb, cov tsiaj txhu sib txawv ntawm cov kua cerebrospinal, thaum cov ntshav glycine ntau ntxiv tau pom hauv ob qho tsiaj (Daim Duab Ntxiv 4a-j). Tom qab kev lim cov ntaub ntawv sib law liag hauv cov kua cerebrospinal ntawm cov tsiaj # 1.1 thiab # 1.2, tag nrho ntawm 964 thiab 420 12-mer peptides tshwj xeeb tau txais (Daim Duab Ntxiv 1d-e). Cov phage clones raug cais tawm tau raug nthuav dav thiab raug rau qhov kev xaiv thib ob hauv vivo. Cov phage uas tau rho tawm los ntawm qhov kev xaiv thib ob tau raug rau HTS hauv txhua tus tsiaj thiab txhua cov peptides uas tau txheeb xyuas tau siv ua cov tswv yim rau qhov kev pab cuam lees paub motif los tshuaj xyuas qhov tshwm sim ntawm tripeptide motifs (Daim duab 2a, b, ef). Piv rau thawj lub voj voog ntawm phage rov qab los ntawm CSF, peb tau pom kev xaiv ntxiv thiab kev tsis xaiv ntawm ntau motifs hauv CSF hauv ceg A thiab B (Daim duab 2). Ib qho algorithm txheeb xyuas network tau siv los txiav txim siab seb lawv puas sawv cev rau cov qauv sib txawv ntawm cov kab ke sib xws. Ib qho kev zoo sib xws tau pom tseeb ntawm 12-dimensional sequences rov qab los ntawm CSF hauv lwm clade A (Daim duab 2c, d) thiab clade B (Daim duab 2g, h). Kev tshuaj xyuas sib sau ua ke hauv txhua ceg tau qhia txog cov qauv xaiv sib txawv rau 12-mer peptides (Daim duab ntxiv 5c, d) thiab kev nce hauv CSF / ntshav titer piv dhau sijhawm rau cov clones sib sau ua ke tom qab qhov kev xaiv thib ob piv rau thawj qhov kev xaiv (Daim duab ntxiv 5e).
Kev nplua nuj ntawm cov motifs thiab peptides hauv cov kua dej cerebrospinal los ntawm ob qhov kev sib law liag ntawm kev xaiv cov zaub phage hauv vivo.
Txhua cov phages kua cerebrospinal uas tau rov qab los ntawm thawj qhov kev sib tw ntawm txhua tus tsiaj (tsiaj #1.1 thiab #1.2) tau muab sib sau ua ke, nthuav dav, HT-sequenced thiab rov txhaj ua ke (2 x 1010 phages/tsiaj) 2 SM cannulated nas (#1.1 → #). 2.1 thiab 2.2, 1.2 → 2.3 thiab 2.4). (a,b,e,f) Correlation scatterplots piv rau qhov zaus sib piv ntawm tripeptide motifs ntawm txhua CSF-derived phages hauv thawj thiab thib ob qhov kev xaiv. Qhov zaus sib piv thiab kev faib tawm ntawm motifs sawv cev rau txhua qhov ua tau sib tshooj tripeptides pom hauv peptides hauv ob qho kev taw qhia. Tus lej ntawm motifs pom hauv 1000 kev nyeem ntawv tau qhia. Motifs uas tau xaiv lossis tsis suav nrog (p < 0.001) hauv ib qho ntawm cov tsev qiv ntawv sib piv tau qhia nrog cov dots liab. (c, d, g, h) Cov cim qhia txog cov kab ke ntawm txhua cov kab ke ntev uas muaj CSF nplua nuj 12 amino acid raws li qhov kev xaiv thib 2 thiab thib 1 hauv vivo. Qhov loj ntawm cov lej ib tsab ntawv qhia tias cov amino acid ntawd tshwm sim ntau npaum li cas ntawm qhov chaw ntawd. Txhawm rau sawv cev rau lub logo, qhov zaus ntawm cov kab ke CSF uas tau rho tawm los ntawm cov tsiaj ib leeg ntawm ob qhov kev xaiv tau muab piv thiab cov kab ke nplua nuj hauv qhov kev xaiv thib ob tau pom: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 thiab (h) #1.2–#2.4. Cov amino acid nplua nuj tshaj plaws ntawm qhov chaw muab rau hauv (c, d) cov tsiaj tsis muaj. 2.1 thiab tsis muaj. 2.2 lossis (g, h) hauv cov tsiaj tsis muaj. 2.3 thiab tsis muaj. 2.4 tau pom hauv 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 cov kab ke peptide tshwj xeeb (# 3.1 thiab # 3.2) los ntawm 332 CSF-reconstituted phage clones cais tawm ntawm ob tus tsiaj (Supplementary Fig. 6a). Cov kab ke LGSVS (18.7%) muaj qhov sib piv siab tshaj plaws, ua raws li cov tsiaj qus-hom inserts PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), thiab SARGSWREIVSLS (2.2%). Hauv qhov kev xaiv thib plaub kawg, peb tau muab ob ceg xaiv ywj pheej los ntawm peb tus tsiaj sib cais (Daim duab 1c). Ntawm 925 cov phage clones rov qab los ntawm CSF, hauv qhov kev xaiv thib plaub peb pom 64 cov kab ke peptide tshwj xeeb (Supplementary Fig. 6b), ntawm cov uas qhov sib piv ntawm cov tsiaj qus-hom phage poob qis rau 0.8%. Cov CSF clones feem ntau nyob rau hauv qhov kev sib tw thib plaub yog LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) thiab RLSSVDSDLSGC (3, 2%). %)). Qhov ntev ntawm cov peptides xaiv yog vim muaj kev ntxig / rho tawm nucleotide lossis cov codons nres ua ntej hauv cov primers tsev qiv ntawv thaum siv cov codons degenerate rau NNK tsev qiv ntawv tsim. Cov codons nres ua ntej tsim cov peptides luv dua thiab raug xaiv vim tias lawv muaj cov motif aa zoo. Cov peptides ntev dua yuav tshwm sim los ntawm kev ntxig / rho tawm hauv cov primers ntawm cov tsev qiv ntawv tsim. Qhov no tso tus codon nres tsim sab nraum lub thav duab thiab nyeem nws kom txog thaum muaj codon nres tshiab tshwm sim hauv qab. Feem ntau, peb tau xam cov yam ntxwv nplua nuj rau tag nrho plaub qhov kev xaiv los ntawm kev sib piv cov ntaub ntawv nkag nrog cov ntaub ntawv tso zis qauv. Rau thawj qhov kev tshuaj ntsuam, peb siv cov phage titers qus ua keeb kwm yav dhau los tsis tshwj xeeb. Qhov nthuav yog, kev xaiv phage tsis zoo muaj zog heev hauv thawj CSF lub voj voog, tab sis tsis yog hauv cov ntshav (Daim duab 3a), uas tej zaum yuav yog vim muaj qhov tsis tshua muaj feem cuam tshuam ntawm feem ntau ntawm cov tswv cuab ntawm peptide tsev qiv ntawv mus rau hauv CSF compartment lossis cov phages txheeb ze feem ntau yuav khaws cia lossis tshem tawm ntawm cov ntshav ntau dua li bacteriophages. Txawm li cas los xij, hauv qhov thib ob ntawm kev panning, kev xaiv muaj zog ntawm phages hauv CSF tau pom hauv ob lub clades, qhia tias qhov kev sib tw dhau los tau nplua nuj hauv phages qhia peptides uas txhawb nqa CSF uptake (Daim duab 3a). Dua li, tsis muaj kev nplua nuj ntshav ntau. Tsis tas li ntawd hauv qhov thib peb thiab thib plaub puag ncig, cov phage clones tau nplua nuj heev hauv CSF. Piv rau qhov zaus sib piv ntawm txhua qhov sib lawv liag peptide tshwj xeeb ntawm ob qhov kev xaiv kawg, peb pom tias cov kab ke tau nplua nuj ntau dua hauv qhov kev xaiv thib plaub (Daim duab 3b). Tag nrho muaj 931 tripeptide motifs tau rho tawm los ntawm tag nrho 64 qhov sib txawv peptide siv ob qho kev taw qhia peptide. Cov motifs nplua nuj tshaj plaws hauv qhov kev sib tw thib plaub tau raug tshuaj xyuas ze dua rau lawv cov ntaub ntawv nplua nuj thoob plaws txhua qhov kev sib tw piv rau lub tsev qiv ntawv txhaj tshuaj (txiav tawm: 10% kev nplua nuj) (Daim duab ntxiv 6c). Cov qauv dav dav ntawm kev xaiv qhia tau hais tias feem ntau ntawm cov motifs kawm tau nplua nuj hauv txhua qhov kev sib tw dhau los ntawm ob ceg xaiv. Txawm li cas los xij, qee cov motifs (piv txwv li SGL, VSG, LGS GSV) feem ntau yog los ntawm lwm txoj kab A, thaum lwm tus (piv txwv li FGW, RTN, WGF, NTR) tau nplua nuj hauv lwm txoj kab B.
Kev lees paub ntawm CSF kev thauj mus los ntawm CSF-enriched phage-displayed peptides thiab biotinylated leader peptides conjugated rau streptavidin payloads.
(a) Cov piv txwv ntawm kev txhim kho tau suav rau hauv tag nrho plaub qhov kev sib tw (R1-R4) raws li kev txhaj tshuaj (tswv yim = I) phage (PFU) titers thiab txiav txim siab CSF phage titers (tso zis = O). Cov yam ntxwv txhim kho rau peb qhov kev sib tw kawg (R2-R4) tau suav los ntawm kev sib piv nrog qhov kev sib tw dhau los thiab thawj qhov kev sib tw (R1) nrog cov ntaub ntawv hnyav. Cov kab qhib yog cov kua dej cerebrospinal, cov kab ntxoov ntxoo yog plasma. (***p <0.001, raws li Tub Ntxhais Kawm t-xeem). (b) Daim ntawv teev cov phage peptides uas muaj ntau tshaj plaws, raug xaiv raws li lawv qhov sib piv rau txhua phages sau hauv CSF tom qab kev sib tw 4 ntawm kev xaiv. Rau qhov feem ntau phage clones tau hais txog xim, suav nrog thiab lawv cov yam ntxwv txhim kho ntawm kev sib tw 3 thiab 4 ntawm kev xaiv (insets). (c, d) Rau qhov feem ntau phage clones, phage khoob thiab niam txiv phage peptide tsev qiv ntawv los ntawm kev sib tw 4 tau raug tshuaj xyuas ib tus zuj zus hauv tus qauv kuaj CSF. CSF thiab cov qauv ntshav tau sau ntawm cov sijhawm qhia. (c) Cov nyiaj sib npaug ntawm 6 tus neeg sib tw phage clones (2 x 1010 phages/tsiaj), phages khoob (#1779) (2 x 1010 phages/tsiaj) thiab cov tsev qiv ntawv phage peptide hauv Tshuag (2 x 1012 phages/tsiaj) Txhaj tsawg kawg 3 CM raug muab rau tus tsiaj cannulated sib cais ntawm cov leeg nqaij tw. Cov tshuaj CSF pharmacokinetics ntawm txhua tus phage clone thiab phage peptide tsev qiv ntawv txhaj tshuaj dhau sijhawm tau qhia. (d) qhia qhov nruab nrab CSF/ntshav piv rau txhua tus phages/mL rov qab tau dhau lub sijhawm kuaj. (e) Plaub tus thawj coj peptides tsim thiab ib qho kev tswj hwm scrambled tau txuas nrog biotin rau streptavidin los ntawm lawv N-terminus (tetramer zaub) ua raws li kev txhaj tshuaj (cov leeg nqaij tw iv, 10 mg streptavidin/kg). Yam tsawg kawg peb tus nas intubated (N = 3). ). Cov qauv CSF tau sau rau ntawm cov sijhawm qhia thiab cov streptavidin concentration tau ntsuas los ntawm CSF anti-streptavidin ELISA (nd = tsis tau kuaj pom). (*p <0.05, **p <0.01, ***p <0.001, raws li kev xeem ANOVA). (f) Kev sib piv ntawm cov amino acid sequence ntawm cov phage peptide clone uas muaj ntau tshaj plaws #2002 (ntshav) nrog lwm cov phage peptide clones xaiv los ntawm qhov kev xaiv thib 4. Cov amino acid fragments zoo sib xws thiab zoo sib xws yog xim-coded.
Ntawm tag nrho cov phages nplua nuj nyob rau hauv qhov kev sib tw thib plaub (Daim duab 3b), rau tus neeg sib tw clones tau raug xaiv rau kev tshuaj xyuas tus kheej ntxiv hauv tus qauv kuaj CSF. Cov nqi sib npaug ntawm rau tus neeg sib tw phage, phage khoob (tsis muaj ntxig) thiab prophage peptide libraries tau txhaj rau hauv peb tus tsiaj CM cannulated, thiab pharmacokinetics tau txiav txim siab hauv CSF (Daim duab 3c) thiab ntshav (Supplementary Fig. 7) assays. Txhua tus phage clones uas tau sim tau tsom mus rau CSF compartment ntawm qib 10-1000 npaug siab dua li ntawm cov phage tswj khoob (# 1779). Piv txwv li, clones # 2020 thiab # 2077 muaj kwv yees li 1000 npaug siab dua CSF titers dua li phage tswj. Cov ntaub ntawv pharmacokinetic ntawm txhua tus peptide xaiv yog txawv, tab sis txhua tus ntawm lawv muaj peev xwm CSF homing siab. Peb pom tias muaj kev txo qis tas li rau cov clones #1903 thiab #2011, thaum rau cov clones #2077, #2002 thiab #2009 qhov nce ntxiv thaum 10 feeb thawj zaug yuav qhia txog kev thauj mus los tab sis yuav tsum tau txheeb xyuas. Cov Clones #2020, #2002, thiab #2077 ruaj khov ntawm cov qib siab, thaum CSF concentration ntawm clone #2009 maj mam txo qis tom qab thawj zaug nce. Tom qab ntawd peb piv rau qhov zaus sib piv ntawm txhua tus neeg sib tw CSF nrog nws cov ntshav concentration (Daim duab 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 qhia tau tias peb ntawm rau tus neeg sib tw tau nplua nuj heev hauv cov ntshav CSF. Qhov nthuav yog, clone #2077 tau qhia txog kev ruaj khov ntshav siab dua (Daim duab ntxiv 7). Txhawm rau kom paub tseeb tias cov peptides lawv tus kheej muaj peev xwm thauj cov khoom thauj tsis yog cov khoom phage mus rau hauv CSF compartment, peb tau tsim plaub tus thawj coj peptides derivatized nrog biotin ntawm N-terminus qhov twg cov peptides txuas rau cov khoom phage. Cov peptides Biotinylated (nos. 2002, 2009, 2020 thiab 2077) tau sib txuas nrog streptavidin (SA) kom tau txais cov ntawv multimeric uas zoo li phage geometry. Hom ntawv no kuj tso cai rau peb ntsuas SA kev raug mob hauv cov ntshav thiab cov kua dej cerebrospinal ua cov protein peptides thauj khoom. Qhov tseem ceeb, cov ntaub ntawv phage feem ntau tuaj yeem rov ua dua thaum cov peptides tsim tau muab tso rau hauv hom ntawv SA-conjugated no (Daim Duab 3e). Cov peptides sib xyaw muaj kev raug mob tsawg dua thiab CSF tshem tawm sai dua nrog cov qib tsis pom hauv 48 teev. Txhawm rau kom nkag siab txog txoj kev xa khoom ntawm cov peptide phage clones no mus rau hauv qhov chaw CSF, peb tau tshuaj xyuas qhov chaw ntawm cov phage peptide hits siv immunohistochemistry (IHC) los ntes cov khoom phage ncaj qha 1 teev tom qab txhaj tshuaj hauv vivo. Qhov tseem ceeb, cov clones #2002, #2077, thiab #2009 tuaj yeem pom los ntawm kev siv cov xim pleev xim rau hauv cov hlab ntsha hauv lub hlwb, thaum cov phage tswj (#1779) thiab clone #2020 tsis tau pom (Daim Duab Ntxiv 8). Qhov no qhia tau hais tias cov peptides no pab txhawb rau qhov cuam tshuam rau lub hlwb los ntawm kev hla BBB. Yuav tsum muaj kev tshuaj xyuas ntxiv kom paub meej ntxiv los sim qhov kev xav no, vim tias txoj kev BSCFB kuj tseem yuav koom nrog. Thaum piv cov amino acid sequence ntawm cov clone nplua nuj tshaj plaws (#2002) nrog lwm cov peptides xaiv, nws tau sau tseg tias qee qhov ntawm lawv muaj cov amino acid extensions zoo sib xws, uas yuav qhia txog cov txheej txheem thauj mus los zoo sib xws (Daim Duab 3f).
Vim nws cov plasma profile tshwj xeeb thiab kev nce ntxiv ntawm CSF dhau sijhawm, phage display clone #2077 tau tshawb nrhiav ntxiv rau lub sijhawm ntev dua 48 teev thiab muaj peev xwm rov ua dua qhov nce ntxiv sai ntawm CSF uas pom tias muaj feem cuam tshuam nrog cov theem SA (Daim Duab 4a). Hais txog lwm cov phage clones uas tau txheeb xyuas, #2077 tau pleev xim rau cov hlab ntsha hauv lub hlwb thiab qhia txog kev sib koom ua ke nrog capillary marker lectin thaum saib ntawm qhov kev daws teeb meem siab dua thiab tej zaum qee qhov kev pleev xim hauv qhov chaw parenchymal (Daim Duab 4b). Txhawm rau tshawb xyuas seb puas muaj peptide-mediated pharmacological effects hauv CNS, peb tau ua qhov kev sim uas cov biotinylated versions ntawm i) #2077 transit peptide thiab ii) BACE1 inhibitor peptide tau sib xyaw 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 qauv kuaj tau muab tshuaj rau hauv cov hlab ntsha thiab cov ntshav thiab cov kua qaub ncaug ntawm beta-amyloid peptide 40 (Abeta40) tau ntsuas dhau sijhawm. Abeta40 tau ntsuas hauv CSF vim nws qhia txog kev txwv BACE1 hauv lub hlwb parenchyma. Raws li xav tau, ob qho tib si complexes txo cov ntshav ntawm Abeta40 (Daim duab 4c, d). Txawm li cas los xij, tsuas yog cov qauv kuaj uas muaj cov peptide no. 2077 thiab cov inhibitor ntawm BACE1 peptide conjugated rau SA ua rau Abeta40 txo qis hauv cov kua qaub ncaug (Daim duab 4c). Cov ntaub ntawv qhia tau tias peptide no. 2077 muaj peev xwm thauj cov protein SA 60 kDa mus rau hauv CNS thiab tseem ua rau muaj cov teebmeem pharmacological nrog SA-conjugated inhibitors ntawm BACE1 peptide.
(a) Kev txhaj tshuaj clonal (2 × 10 phages/tsiaj) ntawm T7 phage qhia txog cov qauv tshuaj pharmacokinetic ntev ntawm CSF peptide #2077 (RLSSVDSDLSGC) thiab cov phage tswj tsis tau txhaj (#1779) hauv tsawg kawg peb tus nas CM-intubated. (b) Daim duab confocal microscopic ntawm cov sawv cev cortical microvessels hauv cov nas phage-injected (2 × 10 10 phages/tsiaj) qhia txog kev tiv thaiv ntawm peptide #2077 thiab cov hlab ntsha (lectin). Cov phage clones no tau muab rau 3 tus nas thiab cia kom ncig tau 1 teev ua ntej perfusion. Lub hlwb tau faib thiab pleev xim nrog polyclonal FITC-labeled antibodies tawm tsam T7 phage capsid. Kaum feeb ua ntej perfusion thiab tom qab ntawd fixation, DyLight594-labeled lectin tau muab intravenously. Cov duab fluorescent qhia txog lectin staining (liab) ntawm sab luminal ntawm microvessels thiab phages (ntsuab) hauv lumen ntawm capillaries thiab perivascular hlwb cov ntaub so ntswg. Lub scale bar sib raug rau 10 µm. (c, d) Biotinylated BACE1 inhibitory peptide ib leeg lossis ua ke nrog biotinylated transit peptide #2077 tau txuas nrog streptavidin ua raws li kev txhaj tshuaj ntawm tsawg kawg peb lub cannulated CM nas (10 mg streptavidin/kg). BACE1 peptide inhibitor-mediated kev txo qis hauv Aβ40 tau ntsuas los ntawm Aβ1-40 ELISA hauv cov ntshav (liab) thiab cerebrospinal fluid (txiv kab ntxwv) ntawm lub sijhawm qhia. Rau qhov meej dua, ib txoj kab dotted yog kos rau ntawm daim duab ntawm qhov scale ntawm 100%. (c) Feem pua ​​ntawm kev txo qis hauv Aβ40 hauv cov ntshav (cov duab peb ceg liab) thiab cov kua dej hauv lub hlwb (cov duab peb ceg txiv kab ntxwv) hauv cov nas uas kho nrog streptavidin txuas nrog transit peptide #2077 thiab BACE1 inhibitory peptide hauv qhov sib piv 3:1. (d) Feem pua ​​ntawm kev txo qis hauv cov ntshav Aβ40 (cov voj voog liab) thiab cov kua dej hauv lub hlwb (cov voj voog txiv kab ntxwv) ntawm cov nas uas kho nrog streptavidin txuas nrog BACE1 inhibitory peptide xwb. Qhov concentration ntawm Aβ hauv kev tswj hwm yog 420 pg/ml (tus qauv sib txawv = 101 pg/ml).
Phage display tau siv tau zoo hauv ntau qhov chaw ntawm kev tshawb fawb biomedical17. Txoj kev no tau siv rau kev tshawb fawb txog kev sib txawv ntawm cov hlab ntsha hauv vivo18,19 nrog rau kev tshawb fawb tsom mus rau cov hlab ntsha hauv lub hlwb20,21,22,23,24,25,26. Hauv kev tshawb fawb no, peb tau nthuav dav daim ntawv thov ntawm txoj kev xaiv no tsis yog tsuas yog rau kev txheeb xyuas ncaj qha ntawm cov peptides tsom mus rau cov hlab ntsha hauv lub hlwb, tab sis kuj rau kev tshawb pom cov neeg sib tw nrog cov khoom thauj mus los kom hla cov ntshav-lub hlwb thaiv. Tam sim no peb piav qhia txog kev txhim kho ntawm cov txheej txheem xaiv hauv vivo hauv CM intubated nas thiab qhia txog nws lub peev xwm los txheeb xyuas cov peptides nrog CSF homing khoom. Siv T7 phage uas qhia txog lub tsev qiv ntawv ntawm 12-mer random peptides, peb tau ua pov thawj tias T7 phage me txaus (kwv yees li 60 nm hauv txoj kab uas hla)10 kom hloov kho rau cov ntshav-lub hlwb thaiv, yog li ntawd ncaj qha hla cov ntshav-lub hlwb thaiv lossis choroid plexus. Peb tau pom tias kev sau CSF los ntawm cov nas CM cannulated yog ib txoj kev tshuaj ntsuam xyuas zoo hauv vivo, thiab tias cov phage uas tau rho tawm tsis yog tsuas yog khi rau cov hlab ntsha xwb tab sis kuj ua haujlwm ua tus thauj mus los hla cov ntshav-lub hlwb thaiv. Ntxiv mus, los ntawm kev sau cov ntshav thiab siv HTS rau CSF thiab cov phages uas tau los ntawm cov ntshav, peb tau lees paub tias peb qhov kev xaiv CSF tsis raug cuam tshuam los ntawm kev nplua nuj ntawm cov ntshav lossis kev ua kom nthuav dav ntawm cov kev xaiv. Txawm li cas los xij, qhov chaw ntshav yog ib feem ntawm cov txheej txheem xaiv, txij li cov phages uas muaj peev xwm ncav cuag qhov chaw CSF yuav tsum muaj sia nyob thiab ncig hauv cov ntshav ntev txaus kom nplua nuj lawv tus kheej hauv lub hlwb. Txhawm rau kom rho tawm cov ntaub ntawv sib law liag txhim khu kev qha los ntawm cov ntaub ntawv HTS raw, peb tau siv cov lim dej uas tau hloov kho rau cov yuam kev sib law liag tshwj xeeb hauv cov txheej txheem tshuaj xyuas. Los ntawm kev koom ua ke cov kev ntsuas kinetic rau hauv txoj kev tshuaj ntsuam xyuas, peb tau lees paub cov tshuaj pharmacokinetics sai ntawm cov phages T7 qus (t½ ~ 28 feeb) hauv cov ntshav24, 27, 28 thiab tseem txiav txim siab lawv lub neej ib nrab hauv cov kua cerebrospinal (t½ ~ 26 feeb) ib feeb). Txawm hais tias muaj cov qauv pharmacokinetic zoo sib xws hauv cov ntshav thiab CSF, tsuas yog 0.001% ntawm cov ntshav concentration ntawm phage tuaj yeem pom hauv CSF, qhia txog keeb kwm yav dhau los qis ntawm cov tsiaj qus T7 phage hla cov ntshav-lub hlwb thaiv. Cov haujlwm no qhia txog qhov tseem ceeb ntawm thawj qhov kev xaiv thaum siv cov tswv yim panning hauv vivo, tshwj xeeb tshaj yog rau cov kab mob phage uas tau tshem tawm sai sai ntawm kev ncig, vim tias ob peb clones tuaj yeem ncav cuag CNS compartment. Yog li, hauv thawj qhov kev hloov pauv, qhov kev txo qis hauv kev sib txawv ntawm tsev qiv ntawv yog qhov loj heev, vim tias tsuas yog muaj tsawg tus clones thaum kawg tau sau hauv tus qauv CSF nruj heev no. Qhov kev npaj panning hauv vivo no suav nrog ntau kauj ruam xaiv xws li kev sib sau ua ke hauv CSF compartment, clone ciaj sia nyob hauv cov ntshav compartment, thiab kev tshem tawm sai ntawm T7 phage clones los ntawm cov ntshav hauv 10 feeb thawj zaug (Daim duab 1d thiab Daim duab ntxiv 4M). Yog li, tom qab thawj qhov kev hloov pauv, cov phage clones sib txawv tau txheeb xyuas hauv CSF, txawm hais tias tib lub pas dej pib tau siv rau cov tsiaj ib leeg. Qhov no qhia tau hais tias ntau kauj ruam xaiv nruj rau cov tsev qiv ntawv qhov chaw uas muaj cov tswv cuab ntawm tsev qiv ntawv ntau ua rau muaj kev txo qis ntawm ntau haiv neeg. Yog li ntawd, cov xwm txheej random yuav dhau los ua ib feem tseem ceeb ntawm cov txheej txheem xaiv thawj zaug, uas cuam tshuam loj heev rau qhov tshwm sim. Nws zoo li ntau cov clones hauv tsev qiv ntawv thawj zaug muaj qhov zoo sib xws CSF enrichment propensity. Txawm li cas los xij, txawm tias nyob rau hauv tib qho kev sim, cov txiaj ntsig xaiv yuav txawv vim muaj tsawg tus ntawm txhua tus clone tshwj xeeb hauv lub pas dej thawj zaug.
Cov motifs nplua nuj hauv CSF txawv ntawm cov nyob hauv cov ntshav. Qhov nthuav yog, peb tau sau tseg thawj qhov kev hloov pauv mus rau glycine-nplua nuj peptides hauv cov ntshav ntawm cov tsiaj txhu. (Daim duab 1g, Cov duab ntxiv 4e, 4f). Phage uas muaj glycine peptides yuav ruaj khov dua thiab tsis tshua muaj feem yuav raug tshem tawm ntawm kev ncig. Txawm li cas los xij, cov glycine-nplua nuj peptides no tsis tau pom nyob rau hauv cov qauv kua cerebrospinal, qhia tias cov tsev qiv ntawv curated tau dhau los ntawm ob kauj ruam xaiv sib txawv: ib qho hauv cov ntshav thiab lwm qhov tso cai rau khaws cia hauv cov kua cerebrospinal. CSF-enriched clones los ntawm qhov kev xaiv thib plaub tau raug sim ntau heev. Yuav luag txhua tus ntawm cov clones kuaj ib tus zuj zus tau lees paub tias tau nplua nuj hauv CSF piv rau phage tswj dawb paug. Ib qho peptide ntaus (# 2077) tau kuaj xyuas ntau ntxiv. Nws qhia tau tias lub neej ib nrab ntawm cov ntshav ntev dua piv rau lwm cov hits (Daim duab 3d thiab Daim duab ntxiv 7), thiab qhov nthuav yog, cov peptide no muaj cov cysteine ​​​​residue ntawm C-terminus. Tsis ntev los no tau pom tias qhov ntxiv ntawm cysteine ​​​​rau cov peptides tuaj yeem txhim kho lawv cov khoom pharmacokinetic los ntawm kev khi rau albumin 29. Qhov no tam sim no tsis paub txog peptide # 2077 thiab xav tau kev kawm ntxiv. Qee cov peptides tau qhia txog qhov valence-dependency hauv CSF enrichment (cov ntaub ntawv tsis tau pom), uas tej zaum yuav muaj feem cuam tshuam nrog qhov geometry ntawm T7 capsid. Lub T7 system uas peb siv tau qhia 5-15 daim ntawv theej ntawm txhua peptide ib qho phage particle. IHC tau ua tiav rau cov neeg sib tw lead phage clones txhaj tshuaj rau hauv lub hlwb ntawm cov nas (Supplementary Fig. 8). Cov ntaub ntawv tau qhia tias tsawg kawg peb clones (No. 2002, No. 2009 thiab No. 2077) cuam tshuam nrog BBB. Nws tseem yuav tsum tau txiav txim siab seb qhov kev sib cuam tshuam BBB no ua rau muaj CSF lossis kev txav mus los ntawm cov clones no ncaj qha mus rau BCSFB. Qhov tseem ceeb, peb qhia tias cov peptides xaiv khaws lawv cov peev xwm thauj CSF thaum synthesized thiab khi rau cov protein cargo. Kev khi ntawm N-terminal biotinylated peptides rau SA yeej rov ua cov txiaj ntsig tau txais nrog lawv cov phage clones hauv cov ntshav thiab cov kua cerebrospinal (Daim Duab 3e). Thaum kawg, peb qhia tias cov hlau lead peptide # 2077 muaj peev xwm txhawb nqa lub hlwb ua haujlwm ntawm biotinylated peptide inhibitor ntawm BACE1 conjugated rau SA, ua rau muaj kev cuam tshuam pharmacodynamic hauv CNS los ntawm kev txo qis Abeta40 qib hauv CSF (Daim Duab 4). Peb tsis tuaj yeem txheeb xyuas cov homologues hauv cov ntaub ntawv los ntawm kev ua qhov kev tshawb nrhiav peptide sequence homology ntawm txhua qhov kev ntaus. Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias qhov loj ntawm T7 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 txhais tau tias cov peptides zoo dua tuaj yeem txheeb xyuas los ntawm kev ntsuam xyuas qhov chaw sib txuas ntawm cov kev ntaus txheeb xyuas no. Raws li kev xav, ib qho ntawm cov laj thawj uas peb tsis tau pom ib qho kev sib xws ntawm cov peptides no tej zaum yuav yog kev tsis xaiv thaum lub sijhawm hloov pauv kom tiv thaiv kev nkag mus tsis raug ntawm qee cov peptide motifs rau hauv lub hlwb.
Thaum muab ua ke, peb cov txiaj ntsig muab lub hauv paus rau kev ua haujlwm yav tom ntej los txheeb xyuas thiab piav qhia txog cov txheej txheem thauj mus los ntawm lub hlwb thaiv hauv vivo kom ntxaws ntxiv. Lub hauv paus teeb tsa ntawm txoj kev no yog raws li kev xaiv ua haujlwm uas tsis yog tsuas yog txheeb xyuas cov clones nrog cov khoom khi rau lub hlwb, tab sis kuj suav nrog ib kauj ruam tseem ceeb uas cov clones ua tiav muaj kev ua haujlwm intrinsic los hla cov teeb meem hauv lub cev hauv vivo mus rau hauv CNS compartment. yog los piav qhia txog cov txheej txheem ntawm kev thauj mus los ntawm cov peptides no thiab lawv nyiam khi rau microvasculature tshwj xeeb rau thaj tsam lub hlwb. Qhov no yuav ua rau muaj kev tshawb pom ntawm txoj hauv kev tshiab rau kev thauj mus los ntawm BBB thiab cov receptors. Peb xav tias cov peptides uas tau txheeb xyuas tuaj yeem khi ncaj qha rau cov receptors cerebrovascular lossis rau cov ligands ncig uas thauj mus los ntawm BBB lossis BCSFB. Cov peptide vectors nrog CSF kev thauj mus los 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 no 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 cov receptors lossis txoj hauv kev tshiab thiab rau kev tsim cov platforms tshiab zoo heev rau kev xa cov macromolecules, xws li biologics, mus rau lub hlwb.
Siv cov cannulate rau hauv lub cisterna loj (CM) siv kev hloov kho ntawm txoj kev piav qhia ua ntej. Cov nas Wistar uas tau siv tshuaj loog (200-350 g) tau muab tso rau ntawm lub tshuab stereotaxic thiab qhov txiav nruab nrab tau ua rau saum lub taub hau uas tau txiav thiab npaj tsis muaj kab mob kom pom lub pob txha taub hau. Muab ob lub qhov rau hauv thaj chaw ntawm lub sash sab saud thiab khi cov ntsia hlau rau hauv cov qhov. Ib lub qhov ntxiv tau muab tso rau hauv lub crest occipital sab nraud rau kev taw qhia stereotactic ntawm lub cannula stainless hlau rau hauv CM. Siv cov cement hniav ncig lub cannula thiab ruaj ntseg nrog cov ntsia hlau. Tom qab photo-curing thiab cement hardening, qhov txhab ntawm daim tawv nqaij tau kaw nrog 4/0 supramid suture. Kev tso cannula kom raug yog lees paub los ntawm kev xau ntawm cov kua cerebrospinal (CSF). Tshem tus nas ntawm lub tshuab stereotaxic, tau txais kev saib xyuas tom qab phais thiab kev tswj hwm qhov mob, thiab cia nws rov zoo li ib lub lim tiam kom txog thaum pom cov cim ntawm cov ntshav hauv cov kua cerebrospinal. Cov nas Wistar (Crl: WI / Han) tau txais los ntawm Charles River (Fabkis teb). Tag nrho cov nas tau khaws cia rau hauv cov xwm txheej tshwj xeeb uas tsis muaj kab mob. Txhua qhov kev sim tsiaj tau txais kev pom zoo los ntawm Chaw Haujlwm Saib Xyuas Tsiaj ntawm Lub Nroog Basel, Switzerland, thiab tau ua raws li Daim Ntawv Tso Cai Tsiaj No. 2474 (Kev Ntsuas Kev Thauj Mus Los Ntawm Lub Hlwb Los Ntawm Kev Ntsuas Cov Qib ntawm Cov Neeg Kho Mob hauv Cov Kua Ntshav thiab Lub Hlwb ntawm Nas).
Maj mam li ua kom tus nas tseem ceeb nrog CM cannula hauv tes. Tshem Datura tawm ntawm cannula thiab sau 10 µl ntawm cov kua cerebrospinal uas ntws los ntawm nws tus kheej. Txij li thaum qhov patency ntawm cannula thaum kawg tau raug cuam tshuam, tsuas yog cov qauv kua cerebrospinal ntshiab uas tsis muaj pov thawj ntawm kev kis kab mob ntshav lossis kev hloov pauv xim tau suav nrog hauv kev tshawb fawb no. Nyob rau tib lub sijhawm, kwv yees li 10-20 μl ntawm cov ntshav tau coj los ntawm qhov txiav me me ntawm qhov kawg ntawm tus Tsov tus tw rau hauv cov raj nrog heparin (Sigma-Aldrich). CSF thiab cov ntshav tau sau ntawm ntau lub sijhawm tom qab txhaj tshuaj T7 phage rau hauv cov hlab ntsha. Kwv yees li 5-10 μl ntawm cov kua tau pov tseg ua ntej txhua tus qauv CSF tau sau, uas sib raug rau qhov ntim tuag ntawm catheter.
Cov tsev qiv ntawv tau tsim los ntawm kev siv T7Select 10-3b vector raws li tau piav qhia hauv phau ntawv qhia T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Hauv luv luv, ib qho DNA ntxig 12-mer random tau tsim ua hom ntawv hauv qab no:
Tus NNK codon tau siv los tiv thaiv ob chav nres codons thiab amino acid overexpression hauv daim ntawv ntxig. N yog qhov sib xyaw ua ke ntawm txhua tus nucleotide, thiab K yog qhov sib xyaw ua ke ntawm adenine thiab cytosine nucleotides. Cov cheeb tsam ib txoj hlua tau hloov mus rau ob txoj hlua DNA los ntawm kev ntxiv incubation nrog dNTP (Novagen) thiab Klenow enzyme (New England Biolabs) hauv Klenow buffer (New England Biolabs) rau 3 teev ntawm 37 ° C. Tom qab qhov kev tshuaj tiv thaiv, ob txoj hlua DNA tau rov qab los ntawm EtOH precipitation. Cov DNA tau los ntawm kev zom nrog cov enzymes txwv EcoRI thiab HindIII (ob qho tib si los ntawm Roche). Daim ntawv ntxig cleaved thiab purified (QIAquick, Qiagen) (T4 ligase, New England Biolabs) ces tau ligated hauv-ncej rau hauv lub vector T7 ua ntej cleaved tom qab amino acid 348 ntawm 10B capsid gene. Ligation reactions tau incubated ntawm 16 ° C. rau 18 teev ua ntej hauv vitro ntim. Kev ntim khoom phage hauv vitro tau ua raws li cov lus qhia uas muab nrog T7Select 10-3b cloning kit (Novagen) thiab cov kua ntim tau amplified ib zaug rau lysis siv Escherichia coli (BLT5615, Novagen). Cov lysates tau centrifuged, titrated thiab khov ntawm -80 ° C. ua cov kua glycerol.
Kev nthuav dav PCR ncaj qha ntawm cov cheeb tsam hloov pauv phage uas tau nthuav dav hauv cov kua zaub lossis phaj siv cov primers fusion 454/Roche-amplicon. Lub primer fusion pem hauv ntej muaj cov kab ke uas nyob ib sab ntawm thaj chaw hloov pauv (NNK) 12 (qhia tshwj xeeb rau cov qauv), GS FLX Titanium Adapter A, thiab plaub-base library key sequence (TCAG) (Daim Duab Ntxiv 1a):
Tus reverse fusion primer kuj muaj biotin txuas rau capture beads thiab GS FLX Titanium Adapter B uas xav tau rau clonal amplification thaum lub sijhawm emulsion PCR:
Cov amplicons ces raug rau 454/Roche pyrosequencing raws li 454 GS-FLX Titanium protocol. Rau kev siv Sanger sequencing (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA tau amplified los ntawm PCR thiab sequenced nrog cov primer khub hauv qab no:
Cov ntxig los ntawm cov plaques ib leeg tau raug rau PCR amplification siv Roche Fast Start DNA Polymerase Kit (raws li cov lus qhia ntawm cov chaw tsim khoom). Ua qhov pib kub (10 feeb ntawm 95 ° C) thiab 35 lub voj voog boost (50 s ntawm 95 ° C, 1 feeb ntawm 50 ° C, thiab 1 feeb ntawm 72 ° C).
Phage los ntawm cov tsev qiv ntawv, phage qus, phage cawm los ntawm CSF thiab ntshav, lossis cov clones ib leeg tau nthuav dav hauv Escherichia coli BL5615 hauv TB kua zaub (Sigma Aldrich) lossis hauv 500 cm2 tais diav (Thermo Scientific) rau 4 teev ntawm 37 ° C. Phage tau rho tawm ntawm cov phaj los ntawm kev yaug cov phaj nrog Tris-EDTA buffer (Fluka Analytical) lossis los ntawm kev sau cov phaj nrog cov pipette sterile. Phage tau rho tawm ntawm cov kab lis kev cai supernatant lossis extraction buffer nrog ib puag ncig ntawm polyethylene glycol (PEG 8000) precipitation (Promega) thiab resuspended hauv Tris-EDTA buffer.
Cov phage uas tau nthuav dav tau raug rau 2-3 qhov kev tshem tawm endotoxin siv cov hlaws tshem tawm endotoxin (Miltenyi Biotec) ua ntej txhaj tshuaj rau hauv cov hlab ntsha (IV) (500 μl/tsiaj). Hauv thawj qhov kev txhaj tshuaj, 2 × 1012 phages tau muab tso rau hauv; hauv qhov thib ob, 2 × 1010 phages; hauv qhov kev xaiv thib peb thiab thib plaub, 2 × 109 phages ib tus tsiaj. Cov ntsiab lus ntawm Phage hauv CSF thiab cov qauv ntshav uas tau sau rau ntawm lub sijhawm tau txiav txim siab los ntawm kev suav cov plaque raws li cov lus qhia ntawm cov chaw tsim khoom (T7Select system manual). Kev xaiv Phage tau ua los ntawm kev txhaj tshuaj rau hauv cov hlab ntsha ntawm cov tsev qiv ntawv huv si rau hauv cov leeg ntawm tus Tsov tus tw lossis los ntawm kev txhaj tshuaj dua ntawm phage uas tau rho tawm los ntawm CSF los ntawm qhov kev xaiv dhau los, thiab kev sau qoob loo tom qab tau ua tiav ntawm 10 feeb, 30 feeb, 60 feeb, 90 feeb, 120 feeb, 180 feeb, thiab 240 feeb raws li CSF thiab cov qauv ntshav. Tag nrho plaub qhov kev sib tw ntawm kev sib tw hauv vivo tau ua tiav uas ob ceg xaiv tau khaws cia thiab tshuaj xyuas sib cais thaum lub sijhawm peb qhov kev xaiv thawj zaug. Txhua daim phage inserts rho tawm los ntawm CSF los ntawm ob qhov kev xaiv thawj zaug tau raug rau 454/Roche pyrosequencing, thaum txhua daim clones rho tawm los ntawm CSF los ntawm ob qhov kev xaiv kawg tau raug sequenced manually. Txhua daim ntshav phages los ntawm thawj qhov kev xaiv kuj tau raug rau 454/Roche pyrosequencing. Rau kev txhaj tshuaj ntawm phage clones, cov phages xaiv tau raug nthuav dav hauv E. coli (BL5615) ntawm 500 cm2 phaj ntawm 37 ° C rau 4 teev. Cov clones xaiv ib tus zuj zus thiab manually sequenced tau propagated hauv TB medium. Tom qab phage extraction, purification thiab tshem tawm endotoxin (raws li tau piav qhia saum toj no), 2 × 1010 phages / tsiaj hauv 300 μl tau txhaj tshuaj rau hauv ib txoj hlab ntsha tail.
Kev ua ua ntej thiab kev lim cov ntaub ntawv raws li qhov zoo. Cov ntaub ntawv raw 454/Roche tau hloov los ntawm hom ntawv qhia dej binary (sff) mus rau hom ntawv Pearson uas nyeem tau tib neeg (fasta) siv cov software muag khoom. Kev ua ntxiv ntawm cov kab ke nucleotide tau ua tiav siv cov kev pab cuam C thiab cov ntawv sau tshwj xeeb (cov software tsis tau tso tawm) raws li tau piav qhia hauv qab no. Kev tshuaj xyuas cov ntaub ntawv tseem ceeb suav nrog cov txheej txheem lim dej ntau theem. Txhawm rau lim cov ntawv nyeem uas tsis muaj cov kab ke DNA 12mer uas siv tau, cov ntawv nyeem tau sib dhos ua ke rau daim ntawv pib (GTGATGTCGGGGATCCGAATTCT), daim ntawv nres (TAAGCTTGCGGCCGCACTCGAGTA) thiab daim ntawv tom qab (CCCTGCAGGGATATCCCGGGAGCTCGGAC) siv qhov kev xeem thoob ntiaj teb Needleman-Wunsch. kev sib dhos tso cai rau txog 2 qhov tsis sib xws ib qho kev sib dhos31. Yog li ntawd, kev nyeem tsis muaj cov cim pib thiab nres thiab kev nyeem uas muaj cov ntawv ntxig tom qab, piv txwv li, kev sib dhos uas tshaj qhov tus lej tso cai ntawm cov mismatches, tau raug tshem tawm ntawm lub tsev qiv ntawv. Raws li qhov nyeem seem, cov kab lus N-mer DNA txuas ntxiv los ntawm qhov pib thiab xaus ua ntej qhov nres tau raug tshem tawm ntawm qhov nyeem thawj kab lus thiab ua tiav ntxiv (tom qab no hu ua "ntxig"). Tom qab txhais cov ntawv ntxig, qhov seem tom qab thawj qhov codon nres ntawm 5′ kawg ntawm tus primer raug tshem tawm ntawm cov ntawv ntxig. Tsis tas li ntawd, cov nucleotides ua rau cov codon tsis tiav ntawm 3′ kawg ntawm tus primer kuj raug tshem tawm. Txhawm rau tshem tawm cov ntawv ntxig uas muaj cov kab lus keeb kwm yav dhau los, cov ntawv ntxig txhais uas pib nrog cov qauv amino acid "PAG" kuj raug tshem tawm. Peptides nrog qhov ntev tom qab txhais lus tsawg dua 3 amino acids raug tshem tawm ntawm lub tsev qiv ntawv. Thaum kawg, tshem tawm cov redundancy hauv lub pas dej ntxig thiab txiav txim siab qhov zaus ntawm txhua qhov ntxig tshwj xeeb. Cov txiaj ntsig ntawm qhov kev tshuaj xyuas no suav nrog cov npe ntawm cov kab lus nucleotide (ntxig) thiab lawv cov zaus (nyeem) (Cov Duab Ntxiv 1c thiab 2).
Cov N-mer DNA pawg ntxig los ntawm kev sib xws: Txhawm rau tshem tawm cov yuam kev sequencing 454/Roche-specific (xws li teeb meem nrog kev txuas ntxiv homopolymer sequencing) thiab tshem tawm cov redundancies tsis tseem ceeb, cov N-mer DNA sequence inserts (insers) uas tau lim ua ntej raug txheeb los ntawm kev sib xws. insertions (txog li 2 lub hauv paus tsis sib xws tso cai) siv cov algorithm iterative txhais raws li hauv qab no: insertions raug txheeb ua ntej los ntawm lawv qhov zaus (siab tshaj plaws mus rau qis tshaj plaws), thiab yog tias lawv zoo ib yam, los ntawm lawv qhov kev txheeb thib ob los ntawm qhov ntev (ntev tshaj plaws mus rau luv tshaj plaws)). Yog li, cov insertions ntau zaus thiab ntev tshaj plaws txhais thawj "pawg". Lub zaus ntawm pawg tau teeb tsa rau lub zaus tseem ceeb. Tom qab ntawd, txhua qhov insertions uas tseem nyob hauv daim ntawv teev npe tau sim ntxiv rau pawg los ntawm kev sib phim Needleman-Wunsch. Yog tias tus lej ntawm cov mismatches, insertions, lossis deletions hauv kev sib phim tsis tshaj qhov txwv ntawm 2, ib qho insertions raug ntxiv rau pawg, thiab tag nrho cov zaus ntawm pawg tau nce los ntawm ntau npaum li cas insertions tau ntxiv. Inserts ntxiv rau ib pawg tau cim tias siv thiab tshem tawm ntawm kev ua ntxiv. Yog tias cov kab lus ntxig tsis tuaj yeem ntxiv rau hauv ib pawg uas twb muaj lawm, cov kab lus ntxig yuav siv los tsim ib pawg tshiab nrog qhov zaus ntxig tsim nyog thiab cim tias siv lawm. Qhov rov ua dua xaus thaum txhua qhov kab lus ntxig tau siv los tsim ib pawg tshiab lossis tuaj yeem suav nrog hauv ib pawg uas twb muaj lawm. Tom qab tag nrho, cov kab lus ntxig uas muaj cov nucleotides thaum kawg raug txhais ua cov kab lus peptide (peptide libraries). Qhov tshwm sim ntawm qhov kev tshuaj xyuas no yog ib pawg ntawm cov kab lus ntxig thiab lawv cov zaus sib xws uas ua rau muaj pes tsawg tus nyeem sib law liag (Daim Duab Ntxiv 2).
Tsim Motif: Raws li daim ntawv teev cov peptides tshwj xeeb, ib lub tsev qiv ntawv tau tsim uas muaj txhua yam amino acid qauv (aa) raws li qhia hauv qab no. Txhua qhov qauv uas muaj qhov ntev 3 tau rho tawm ntawm peptide thiab nws cov qauv rov qab tau ntxiv nrog rau lub tsev qiv ntawv motif uas muaj txhua yam qauv (tripeptides). Cov tsev qiv ntawv ntawm cov motifs rov ua dua tau raug 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, zaus ntawm peptide uas muaj cov motif tripeptide pom tau ntxiv thiab muab rau motif hauv lub tsev qiv ntawv motif ("tus lej ntawm motifs"). Qhov tshwm sim ntawm kev tsim motif yog ib qho array ob-seem uas muaj txhua qhov tshwm sim ntawm tripeptides (motifs) thiab lawv cov nqi, uas yog tus lej ntawm kev nyeem sequencing uas ua rau motif sib xws thaum cov nyeem tau lim, pawg, thiab txhais. Cov ntsuas raws li tau piav qhia ntxaws saum toj no.
Kev ua kom zoo ntawm tus lej ntawm cov motifs thiab cov scatterplots sib xws: Tus lej ntawm cov motifs rau txhua tus qauv tau ua kom zoo siv
qhov twg ni yog tus lej ntawm cov nyeem uas muaj lub ntsiab lus i. Yog li, vi sawv cev rau feem pua ​​​​​​ntawm cov nyeem (lossis peptides) uas muaj motif i hauv cov qauv. P-tus nqi ​​​​rau tus lej tsis-normalized ntawm motifs tau suav siv Fisher qhov kev xeem tseeb. Hais txog correlograms ntawm tus lej ntawm motives, Spearman's correlations tau suav siv tus lej normalized ntawm motives nrog R.
Txhawm rau pom cov ntsiab lus ntawm cov amino acids ntawm txhua qhov chaw hauv lub tsev qiv ntawv peptide, web logograms 32, 33 (http://weblogo.threeplusone.com) tau tsim. Ua ntej, cov ntsiab lus ntawm cov amino acids ntawm txhua qhov chaw ntawm 12-mer peptide yog khaws cia rau hauv 20 × 12 matrix. Tom qab ntawd, ib pawg ntawm 1000 peptides uas muaj cov ntsiab lus amino acid sib xws ntawm txhua qhov chaw yog tsim los ntawm hom ntawv fasta-sequence thiab muab ua cov tswv yim rau web-logo 3, uas tsim cov duab sawv cev ntawm cov ntsiab lus amino acid sib xws ntawm txhua qhov chaw. rau ib lub tsev qiv ntawv peptide muab. Txhawm rau pom cov ntaub ntawv ntau qhov ntev, cov ntawv qhia cua sov tau tsim los ntawm kev siv cov cuab yeej tsim sab hauv hauv R (biosHeatmap, ib pob R uas tseem tsis tau tso tawm). Cov dendrograms uas tau nthuav tawm hauv cov ntawv qhia cua sov tau suav siv Ward txoj kev hierarchical clustering nrog Euclidean qhov deb metric. Rau kev tshuaj xyuas cov ntaub ntawv motif scoring, P tus nqi rau kev ntsuas tsis zoo tau suav siv Fisher qhov kev xeem tseeb. Cov nqi P rau lwm cov ntaub ntawv tau suav hauv R siv Student's t-test lossis ANOVA.
Cov phage clones thiab phages uas tsis muaj cov ntxig tau txhaj rau hauv cov hlab ntsha ntawm cov leeg ntawm tus tw (2 × 1010 phages/tsiaj hauv 300 μl PBS). Kaum feeb ua ntej perfusion thiab tom qab ntawd fixation, cov tsiaj tib yam tau txhaj rau hauv cov hlab ntsha nrog 100 μl ntawm DyLight594-labeled lectin (Vector Laboratories Inc., DL-1177). 60 feeb tom qab phage txhaj, cov nas tau perfused los ntawm lub plawv nrog 50 ml PBS ua raws li 50 ml 4% PFA/PBS. Cov qauv hlwb kuj tau kho dua ib hmos hauv 4% PFA/PBS thiab tsau rau hauv 30% sucrose ib hmos ntawm 4°C. Cov qauv tau khov flash hauv OCT sib tov. Kev tshuaj ntsuam xyuas immunohistochemical ntawm cov qauv khov tau ua tiav ntawm chav tsev kub ntawm 30 µm cryosections thaiv nrog 1% BSA thiab incubated nrog polyclonal FITC-labeled antibodies tawm tsam T7 phage (Novus NB 600-376A) ntawm 4 ° C. Incubated ib hmos. Thaum kawg, cov seem tau ntxuav 3 zaug nrog PBS thiab kuaj xyuas nrog lub tshuab confocal laser microscope (Leica TCS SP5).
Tag nrho cov peptides uas muaj qhov huv tsawg kawg nkaus ntawm 98% tau tsim los ntawm GenScript USA, biotinylated thiab lyophilized. Biotin raug khi los ntawm ib qho triple glycine spacer ntxiv ntawm N-terminus. Tshawb xyuas tag nrho cov peptides siv mass spectrometry.
Streptavidin (Sigma S0677) tau sib xyaw nrog 5- npaug equimolar ntau dhau ntawm biotinylated peptide, biotinylated BACE1 inhibitory peptide, lossis kev sib xyaw ua ke (3:1 piv) ntawm biotinylated BACE1 inhibitory peptide thiab BACE1 inhibitory peptide hauv 5-10% DMSO/incubated hauv PBS. 1 teev ntawm chav sov ua ntej txhaj tshuaj. Streptavidin-conjugated peptides tau txhaj tshuaj rau hauv cov hlab ntsha ntawm qhov koob tshuaj ntawm 10 mg / kg rau hauv ib qho ntawm cov leeg ntawm cov nas uas muaj lub hlwb.
Qhov kev ntsuas ntawm cov tshuaj streptavidin-peptide tau raug soj ntsuam los ntawm ELISA. Cov phaj Nunc Maxisorp microtiter (Sigma) tau coated hmo ntuj ntawm 4 ° C nrog 1.5 μg / ml nas anti-streptavidin antibody (Thermo, MA1-20011). Tom qab thaiv (blocking buffer: 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 buffer) rau 3 Thib Ob, CSF thiab plasma qauv tau ntxiv rau cov qhov dej diluted nrog blocking buffer (plasma 1: 10,000, CSF 1: 115). Lub phaj ces tau incubated hmo ntuj ntawm 4 ° C nrog kev kuaj pom antibody (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239). Tom qab ntxuav peb kauj ruam, streptavidin raug kuaj pom los ntawm kev incubation hauv TMB substrate solution (Roche) txog li 20 feeb. Tom qab nres kev tsim xim nrog 1M H2SO4, ntsuas qhov absorbance ntawm 450 nm.
Lub luag haujlwm ntawm cov tshuaj streptavidin-peptide-BACE1 inhibitor complex tau soj ntsuam los ntawm Aβ(1-40) ELISA raws li cov txheej txheem ntawm cov chaw tsim khoom (Wako, 294-64701). Hauv luv luv, cov qauv CSF tau diluted hauv tus qauv diluent (1:23) thiab incubated hmo ntuj ntawm 4°C hauv 96-qhov phaj coated nrog BNT77 capture antibody. Tom qab tsib kauj ruam ntxuav, HRP-conjugated BA27 antibody tau ntxiv thiab incubated rau 2 teev ntawm 4° C., ua raws li tsib kauj ruam ntxuav. Aβ(1–40) tau kuaj pom los ntawm kev incubation hauv TMB kua rau 30 feeb ntawm chav tsev kub. Tom qab kev tsim xim tau raug nres nrog cov kua nres, ntsuas qhov absorbance ntawm 450 nm. Cov qauv plasma tau raug rau cov khoom khov kho extraction ua ntej Aβ(1–40) ELISA. Plasma tau ntxiv rau 0.2% DEA (Sigma) hauv 96-qhov phaj thiab incubated ntawm chav tsev kub rau 30 feeb. Tom qab ntxuav cov phaj SPE (Oasis, 186000679) nrog dej thiab 100% methanol, cov qauv plasma tau ntxiv rau cov phaj SPE thiab tshem tawm tag nrho cov kua. Cov qauv tau ntxuav (ua ntej nrog 5% methanol ces 30% methanol) thiab eluted nrog 2% NH4OH/90% methanol. Tom qab ziab cov eluate ntawm 55°C rau 99 feeb ntawm N2 tam sim no, cov qauv tau txo qis hauv cov tshuaj diluents txheem thiab Aβ (1–40) tau ntsuas raws li tau piav qhia saum toj no.
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