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Tšitiso ea mali-boko le mokoallo oa mali-boko li thibela li-biotherapeutic agents ho finyella lipakane tsa tsona tsamaisong ea methapo e bohareng, kahoo li sitisa phekolo e sebetsang ea mafu a methapo.Ho sibolla lipalangoang tse ncha tsa boko ka vivo, re hlahisitse laeborari ea T7 phage peptide mme ra bokella mali le cerebrospinal fluid (CSF) re sebelisa mofuta o moholo oa letamo oa likhoto.Li-clone tse khethehileng tsa phage li ruisitsoe haholo ho CSF ​​ka mor'a likhetho tse 'nè tsa khetho.Teko ea li-peptide tsa mokhethoa ka bomong e senotse ntlafatso e fetang 1000 ho CSF.The bioactivity ea peptide-mediated pelehi bokong e netefalitsoe ke phokotso ea 40% ea boemo ba amyloid-β ho cerebrospinal fluid ho sebelisa BACE1 peptide inhibitor e hokahaneng le peptide e tsejoang ea novel transit.Liphetho tsena li fana ka maikutlo a hore li-peptide tse khethiloeng ka mekhoa ea khetho ea in vivo phage e ka ba likoloi tse sebetsang bakeng sa ho tsamaisa li-macromolecules bokong ka phello ea kalafo.
Patlisiso e lebisitsoeng ea tsamaiso ea methapo e bohareng (CNS) e tsepamisitse maikutlo haholo ho khethoeng litlhare tse ntlafalitsoeng le liakhente tse bonts'ang thepa e lebisitsoeng ho CNS, ka boiteko bo fokolang ba ho fumana mekhoa e tsamaisang phepelo e sebetsang ea lithethefatsi bokong.Sena se qala ho fetoha hona joale kaha phano ea lithethefatsi, haholo-holo limolek'hule tse kholo, e le karolo ea bohlokoa ntlafatsong ea sejoale-joale ea mahlale a methapo ea kutlo.Tikoloho ea tsamaiso ea methapo e bohareng e sirelelitsoe hantle ke tsamaiso ea cerebrovascular barriers, e nang le mokoallo oa mali-boko (BBB) ​​le mokoallo oa mali-boko (BCBB) 1, e leng se etsang hore ho be thata ho isa lithethefatsi bokong1,2.Ho hakanngoa hore hoo e batlang e le lithethefatsi tsohle tse kholo tsa molek'hule le ho feta 98% ea lithethefatsi tse nyenyane tsa molek'hule li tlosoa bokong3.Ke ka lebaka leo ho leng bohlokoa haholo ho khetholla mekhoa e mecha ea lipalangoang tsa boko tse fanang ka phepelo e nepahetseng le e tobileng ea meriana ea phekolo ho CNS 4,5.Leha ho le joalo, BBB le BCSFB le tsona li fana ka monyetla o babatsehang oa ho fana ka lithethefatsi ha li ntse li phunyeletsa le ho kena likarolong tsohle tsa boko ka methapo ea eona e pharaletseng.Ka hona, boiteko ba hona joale ba ho sebelisa mekhoa e sa tsitsang ea ho pepa bokong bo itšetlehile haholo ka mochine oa lipalangoang tsa receptor-mediated (PMT) ho sebelisa BBB6 receptor e sa feleng.Ho sa tsotellehe tsoelo-pele ea morao-rao ea bohlokoa ka ho sebelisa mokhoa oa ho amohela li-transferrin7,8, nts'etsopele e eketsehileng ea mekhoa e mecha ea ho fana ka thepa e nang le thepa e ntlafetseng ea hlokahala.Ho fihlela sena, sepheo sa rona e ne e le ho tsebahatsa li-peptide tse khonang ho hokahanya lipalangoang tsa CSF, kaha ha e le hantle li ka sebelisoa ho isa li-macromolecules ho CNS kapa ho bula litsela tse ncha tsa ho amohela.Ka ho khetheha, li-receptors tse itseng le lipalangoang tsa tsamaiso ea cerebrovascular (BBB le BSCFB) li ka sebetsa e le lipakane tse ka bang teng bakeng sa phano e sebetsang le e tobileng ea lithethefatsi tsa biotherapeutic.Cerebrospinal fluid (CSF) ke sehlahisoa sa sephiri sa plexus ea choroid (CS) 'me e amana ka ho toba le mokelikeli o kenang bokong ka sebaka sa subbarachnoid le ventricular space4.Haufinyane tjena ho bonts'itsoe hore subarachnoid cerebrospinal fluid e hasana ka mokhoa o feteletseng ho interstitium ea boko9.Re tšepa ho fihlella sebaka sa parenchymal ho sebelisa pampitšana ena ea phallo ea subbarachnoid kapa ka ho toba ka BBB.Ho fihlela sena, re kentse ts'ebetsong leano le matla la khetho ea vivo phage le khethollang li-peptide tse tsamaisoang ke e 'ngoe ea litsela tsena tse peli tse fapaneng.
Hona joale re hlalosa mokhoa oa ho hlahloba li-vivo phage o nang le sampuli ea CSF e kopantsoeng le tatellano e phahameng ea throughput (HTS) ho lekola likhetho tsa pele tse nang le mefuta e mengata e phahameng ka ho fetisisa ea laeborari.Tlhahlobo e ile ea etsoa ho likhoto tse lemohileng tse nang le cannula e kholo e kentsoeng ka ho sa feleng (CM) ho qoba tšoaetso ea mali.Habohlokoa, mokhoa ona o khetha tse lebisang bokong le li-peptide tse nang le ts'ebetso ea lipalangoang ho pholletsa le mokoallo oa cerebrovascular.Re sebelisitse li-phages tsa T7 ka lebaka la boholo ba tsona bo nyane (~ 60 nm) 10 mme re fana ka tlhahiso ea hore li loketse ho tsamaisa li-vesicles tse lumellang transcellular crossing ea endothelial le / kapa epithelial-medulla barrier.Kamora mekhahlelo e mene ea ho ts'oara, palo ea batho ba phage e ile ea qheleloa ka thoko e bonts'a matla ho kholiso ea CSF ea vivo le cerebral microvessel association.Habohlokoa, re khonne ho netefatsa seo re se fumaneng ka ho bontša hore li-peptide tse khethiloeng ka ho fetisisa le tse entsoeng ka lik'hemik'hale li khona ho tsamaisa thepa ea protheine ka har'a cerebrospinal fluid.Taba ea pele, litlamorao tsa pharmacodynamic tsa CNS li thehiloe ka ho kopanya peptide e etellang pele ea lipalangoang le inhibitor ea BACE1 peptide.Ntle le ho bonts'a hore maqheka a ts'ebetso ea ts'ebetso ea vivo a ka tsebahatsa li-peptide tse ncha tsa lipalangoang tsa boko e le bajari ba liprotheine tse sebetsang hantle, re lebelletse hore mekhoa e ts'oanang ea khetho le eona e tla ba ea bohlokoa ho tsebahatseng litsela tse ncha tsa lipalangoang tsa boko.
E ipapisitse le li-plaque-forming units (PFU), kamora mohato oa ho paka phage, laebrari ea li-peptide tsa phage tsa 12-mer linear T7 tse nang le mefuta e fapaneng ea hoo e ka bang 109 e entsoe le ho bōptjoa (sheba Lisebelisoa le Mekhoa).Ho bohlokoa ho hlokomela hore re ile ra hlahlobisisa laeborari ena ka hloko pele ho panning ea vivo.PCR amplification ea lisampole laebraring ea phage ho sebelisa li-primers tse fetotsoeng tse hlahisitsoeng li-amplicon tse neng li sebetsa ka ho toba ho HTS (Supplementary Fig. 1a).Ka lebaka la a) HTS11 liphoso tsa tatellano, b) tšusumetso ho boleng ba li-primers (NNK) 1-12, le c) ho ba teng ha mofuta o hlaha (wt) phage (li-skeleton inserts) laebraring ea standby, mokhoa oa ho sefa ka tatellano o ile oa kenngoa ts'ebetsong ho ntša boitsebiso bo netefalitsoeng ba tatellano (Supplementary Fig. 1b).Mehato ena ea ho sefa e sebetsa ho lilaebrari tsohle tsa tatellano ea HTS.Bakeng sa laeborari e tloaelehileng, ho ile ha fumanoa palo e baloang e 233,868, eo 39% ea eona e ileng ea feta mokhoa oa ho sefa 'me ea sebelisoa bakeng sa tlhahlobo ea laeborari le khetho bakeng sa lipotoloho tse latelang (Setšoantšo sa Tlatsetso 1c–e).Ho bala ho ne ho e-na le li-multiples tsa li-pair tsa motheo tsa 3 ka bolelele le tlhōrō ea li-nucleotides tsa 36 (Supplementary Fig. 1c), e tiisang moralo oa laebrari (NNK) 1-12.Haholo-holo, hoo e ka bang 11% ea litho tsa laeborari li ne li e-na le mokokotlo oa 12-dimensional wild-type (wt) PAGISRELVDKL, 'me hoo e ka bang halofo ea tatellano (49%) e ne e na le tse kentsoeng kapa tse hlakotsoeng.HTS ea laebrari e netefalitse phapang e phahameng ea li-peptide ka laebraring: ho feta 81% ea tatellano ea peptide e fumanoe hanngoe feela 'me ke 1.5% feela e etsahetseng likopi tsa ≥4 (Supplementary Fig. 2a).Maqhubu a li-amino acid (aa) libakeng tsohle tsa 12 ka har'a repertoire a amana hantle le maqhubu a lebeletsoeng bakeng sa palo ea li-codon tse hlahisoang ke ho senyeha ha NKK repertoire (Supplementary Fig. 2b).Maqhubu a hlokometsoeng a masala a aa a kentsoeng ke lisebelisoa tsena a kopantsoe hantle le maqhubu a baloang (r = 0.893) (Supplementary Fig. 2c).Tokiso ea lilaebrari tsa phage bakeng sa ente e kenyelletsa mehato ea ho holisa le ho tlosa endotoxin.Sena se bonts'itsoe pele hore se ka fokotsa mefuta-futa ea lilaebrari tsa phage12,13.Ka hona, re ile ra thathamisa laebrari ea phage e entsoeng ka poleiti e neng e tlositsoe endotoxin mme ra e bapisa le laeborari ea pele ho hakanya makhetlo a AA.Khokahano e matla (r = 0.995) e ile ea hlokomeloa pakeng tsa letamo la pele le letamo le matlafalitsoeng le le hloekisitsoeng (Supplementary Fig. 2d), e bontšang hore tlhōlisano pakeng tsa li-clones tse matlafalitsoeng lipoleiti tse sebelisang T7 phage ha lia ka tsa baka khethollo e kholo.Papiso ena e ipapisitse le makhetlo a li-tripeptide motifs laeboraring ka 'ngoe, kaha mefuta-futa ea lilaebrari (~109) e ke ke ea nkuoa ka botlalo leha e le ka HTS.Tlhahlobo ea khafetsa ea aa sebakeng se seng le se seng e senotse leeme le lenyenyane le itšetlehileng ka boemo libakeng tse tharo tsa ho qetela tsa repertoire e kentsoeng (Supplementary Fig. 2e).Qetellong, re ile ra etsa qeto ea hore boleng le mefuta-futa ea laebrari li ne li amoheleha 'me ho ile ha hlokomeloa liphetoho tse nyenyane feela tsa mefuta-futa ka lebaka la ho holisa le ho lokisetsa lilaebrari tsa phage pakeng tsa likhetho tse' maloa.
Seli cerebrospinal fluid sampling e ka etsoa ka ho buoa ka ho kenya cannula ho CM ea likhoto tse tsebang ho thusa ho tsebahatsa T7 phage e kenngoa ka methapo (iv) ka BBB le / kapa BCSFB (Fig. 1a-b).Re sebelisitse matsoho a mabeli a ikemetseng a khetho (matsoho A le B) likhethong tse tharo tsa pele tsa khetho ea in vivo (setšoantšo sa 1c).Re ile ra eketsa butle-butle matla a khetho ka ho fokotsa palo eohle ea phage e hlahisitsoeng likhethong tse tharo tsa pele tsa khetho.Bakeng sa potoloho ea bone ea panning, re ile ra kopanya lisampole tse tsoang makaleng A le B mme ra etsa likhetho tse ling tse tharo tse ikemetseng.Ho ithuta ka in vivo thepa ea likaroloana tsa T7 phage mofuteng ona, phage ea mofuta o hlaha (PAGISRELVDKL master insert) e ne e kenngoa ka likhoto ka mothapo oa mohatla.Ho tsosolosoa ha phages ho tloha cerebrospinal fluid le mali ka nako e fapaneng ho bontšitse hore karolo e batlang e le nyenyane ea T7 icosahedral phages e ne e e-na le mohato o potlakileng oa ho tlosoa ho tloha karolong ea mali (Supplementary Fig. 3).Ho latela li-titer tse tsamaisoang le bongata ba mali a likhoto, re ile ra bala hore ke hoo e ka bang 1% wt.phage e tsoang ho lethal dose e fanoeng e ile ea fumanoa maling metsotso e 10 ka mor'a ente ea methapo.Ka mor'a ho theoha hona ha pele ka potlako, tumello ea mantlha e fokolang e ile ea lekanyetsoa ka halofo ea bophelo ea metsotso ea 27.7.Habohlokoa, ke li-phages tse fokolang feela tse ileng tsa fumanoa ka kamoreng ea CSF, e bontšang semelo se tlaase bakeng sa ho falla ha phage ea mefuta e hlaha ka kamoreng ea CSF (Supplementary Fig. 3).Ka karolelano, ke hoo e ka bang 1 x 10-3% li-titers tsa T7 phage maling le 4 x 10-8% ea phages e kentsoeng qalong e ile ea fumanoa ka cerebrospinal fluid nakong eohle ea sampole (0-250 min).Haholo-holo, halofo ea bophelo (25.7 min) ea mofuta o hlaha oa phage ka cerebrospinal fluid e ne e tšoana le e bonoang maling.Lintlha tsena li bonts'a hore mokoallo o arolang karolo ea CSF ho tsoa maling o ntse o le teng ka har'a likhoto tsa CM-cannulated, e leng se lumellang ka vivo khetho ea lilaebrari tsa phage ho khetholla li-clone tse tsamaisoang habonolo ho tloha maling ho ea kamoreng ea CSF.
(a) Ho theha mokhoa oa ho etsa sampole bocha cerebrospinal fluid (CSF) ho tsoa letamong le leholo.(b) Setšoantšo se bonts'ang sebaka sa cellular se thibelang tsamaiso ea methapo e bohareng (CNS) le leano la khetho le sebelisetsoang ho khetholla li-peptide tse tšelang mokoallo oa mali-bokong (BBB) ​​le mokoallo oa boko ba mali.(c) In vivo phage bontša flowchart screening.Karolong e 'ngoe le e' ngoe ea khetho, phages (li-identifiers tsa liphoofolo ka har'a metsu) li ne li kenngoa ka methapo.Makala a mabeli a ikemetseng a fapaneng (A, B) a bolokiloe a arohane ho fihlela potoloho ea 4 ea khetho.Bakeng sa likhetho tsa 3 le 4, clone e 'ngoe le e' ngoe ea phage e ntšitsoeng ho CSF ​​e ne e hlophisitsoe ka letsoho.(d) Kinetics ea phage e arohaneng le mali (li-circles tse khubelu) le cerebrospinal fluid (li-triangles tse tala) nakong ea lekhetlo la pele la khetho ka likhoto tse peli tse nang le cannulated ka mor'a ho kenngoa ka methapo ea laebrari ea T7 peptide (2 x 1012 phages / phoofolo).Li-square tse putsoa li bonts'a kakaretso ea pele ea phage maling, e baloang ho tloha ho palo ea phage e kentsoeng, ho nahanoa ka kakaretso ea mali.Mabala a matšo a bonts'a ntlha ea mateano ea mola oa y o fetisitsoeng ho tloha ho phage ea mali.(e,f) Hlahisa makhetlo a lekanyelitsoeng le kabo ea li-tripeptide tse ka bang teng tse fumanehang ho peptide.Palo ea li-motifs tse fumanoang lipapisong tse 1000 li bontšoa.Habohlokoa (p <0.001) li-motif tse ntlafalitsoeng li tšoailoe ka matheba a mafubelu.(e) Correlation scatterplot bapisa maqhubu a amanang le tripeptide motif ea laebrari e entetsoeng le phage e nkiloeng ka mali ho tsoa liphoofolong #1.1 le #1.2.(f) Correlation scatterplot ho bapisa maqhubu a amanang le liphoofolo tsa phage tripeptide motifs #1.1 le #1.2 tse arohaneng maling le cerebrospinal fluid.(g, h) Boemeli ba ID ea tatellano ea phage e ruisitsoeng ka mali (g) khahlanong le lilaebrari tse entetsoeng le phage e matlafalitsoeng ho CSF ​​(h) khahlanong le mali ka mor'a hore ho be le khetho ea in vivo liphoofolong ka bobeli.Boholo ba khoutu ea tlhaku e le 'ngoe bo bontša hore na amino acid e etsahala hangata hakae sebakeng seo.Botala = polar, pherese = neutral, blue = motheo, khubelu = acidic le e ntšo = hydrophobic amino acid.Setšoantšo sa 1a, b se entsoe le ho hlahisoa ke Eduard Urich.
Re kentse laeborari ea phage peptide ho likhoto tse peli tsa CM (clades A le B) le phage e ka thoko ho tsoa ho cerebrospinal fluid le mali (Setšoantšo sa 1d).Tlhokomelo ea pele e potlakileng ea laebrari e ne e fokola haholo ha e bapisoa le phage ea mofuta o hlaha.Kakaretso ea halofo ea bophelo ea laebrari e kentsoeng liphoofolong ka bobeli e ne e le metsotso ea 24.8 maling, e tšoanang le phage ea mofuta o hlaha, le metsotso ea 38.5 ho CSF.Mehlala ea mali le cerebrospinal fluid phage ho tsoa ho phoofolo e 'ngoe le e' ngoe e ne e le tlas'a HTS 'me li-peptide tsohle tse khethiloeng li ile tsa hlahlojoa bakeng sa boteng ba motif e khutšoanyane ea tripeptide.Li-tripeptide motifs li ile tsa khethoa hobane li fana ka motheo o fokolang oa sebopeho sa sebopeho le litšebelisano tsa protheine ea peptide14,15.Re fumane kamano e ntle ka ho ajoa ha motifs pakeng tsa laebrari ea phage e entsoeng ka ente le li-clone tse nkiloeng maling a liphoofolo tse peli (setšoantšo sa 1e).Lintlha li bontša hore sebopeho sa laebrari se ruisitsoe ka mokhoa o fokolang feela karolong ea mali.Li-frequency tsa amino acid le tatelano ea tumellano li ile tsa hlahlobjoa hape sebakeng se seng le se seng ho sebelisoa mokhoa oa ho ikamahanya le software ea Weblogo16.Hoa thahasellisa hore ebe re ile ra fumana matlafatso e matla maling a masala a glycine (setšoantšo sa 1g).Ha mali a bapisoa le li-clone tse khethiloeng ho tloha CSF, khetho e matla le ho tlosoa ho itseng ha li-motifs ho ile ha hlokomeloa (setšoantšo sa 1f), 'me li-amino acid tse itseng li ne li le teng ka ho khetheha libakeng tse reriloeng esale pele ho setho sa 12 (setšoantšo sa 1h).Ka ho hlakileng, liphoofolo ka bomong li ne li fapane haholo ka cerebrospinal fluid, athe mali a glycine e ruisang a ile a bonoa liphoofolong ka bobeli (Supplementary Fig. 4a-j).Ka mor'a ho sefa ka matla ha data ea tatellano ka cerebrospinal fluid ea liphoofolo #1.1 le #1.2, kakaretso ea 964 le 420 e ikhethang ea 12-mer peptide e ile ea fumanoa (Supplementary Fig. 1d-e).Li-clone tsa phage tse arohaneng li ile tsa holisoa 'me tsa kenngoa mokhahlelong oa bobeli oa khetho ea in vivo.Phage e ntšitsoeng mokhahlelong oa bobeli oa khetho e ne e le tlas'a HTS ho phoofolo e 'ngoe le e' ngoe 'me li-peptide tsohle tse khethiloeng li ne li sebelisoa e le ho kenya letsoho lenaneong la ho lemoha motif ho hlahloba ketsahalo ea li-tripeptide motifs (Fig. 2a, b, ef).Ha ho bapisoa le potoloho ea pele ea phage e fumanoeng ho tloha CSF, re bone khetho e eketsehileng le ho tlosoa ha mekhoa e mengata ho CSF ​​makaleng A le B (Setšoantšo sa 2).Ho ile ha sebelisoa algorithm ea ho tsebahatsa marang-rang ho fumana hore na li emela mekhoa e fapaneng ea tatellano e tsitsitseng.Ho tšoana ho hlakileng ho ile ha hlokomeloa pakeng tsa tatellano ea 12-dimensional e fumanoeng ke CSF ka mokhoa o mong oa clade A (Fig. 2c, d) le clade B (Fig. 2g, h).Tlhahlobo e kopantsoeng lekaleng le leng le le leng e ile ea senola litlaleho tse fapaneng tsa khetho bakeng sa li-peptide tsa 12-mer (Supplementary Fig. 5c, d) le keketseho ea tekanyo ea CSF / mali ea titer ka nako bakeng sa li-clones tse kopantsoeng ka mor'a khetho ea bobeli ho bapisoa le lekhetlo la pele la khetho (Supplementary Fig. 5e).).
Ntlafatso ea li-motifs le li-peptide ka har'a cerebrospinal fluid ka mekhahlelo e 'meli e latellanang ea khetho ea pontšo ea phage e sebetsang ka har'a vivo.
Li-phages tsohle tsa cerebrospinal fluid li ile tsa hlaphoheloa ho tloha ka lekhetlo la pele la phoofolo e 'ngoe le e' ngoe (liphoofolo #1.1 le #1.2) li ne li kopantsoe, li holisa, tsa tatellano ea HT le ho kenngoa hape hammoho (2 x 1010 phages / phoofolo) 2 SM litoeba tsa cannulated (#1.1 → #).2.1 le 2.2, 1.2 → 2.3 le 2.4).(a,b,e,f) Correlation scatterplots bapisa maqhubu a lekanyelitsoeng a tripeptide motifs tsa phages tsohle tse nkiloeng ho CSF ​​likhethong tsa pele le tsa bobeli tsa khetho.Maqhubu a amanang le ho ajoa ha li-motifs tse emelang li-tripeptide tsohle tse ka bang teng tse fumanehang ho li-peptide ka mahlakoreng ka bobeli.Palo ea li-motifs tse fumanoang lipapisong tse 1000 li bontšoa.Maikutlo a neng a khethiloe haholo (p <0.001) a khethiloe kapa a sa kenyelletsoa ho e 'ngoe ea lilaebrari tse bapisitsoeng a totobatsoa ka matheba a mafubelu.(c, d, g, h) Letšoao la tatellano la tatellano ea tatellano ea tatellano e telele ea li-amino acid tse 12 tsa CSF tse thehiloeng ho li-round 2 le 1 tsa khetho ea in vivo.Boholo ba khoutu ea tlhaku e le 'ngoe bo bontša hore na amino acid e etsahala hangata hakae sebakeng seo.Ho emela letšoao, makhetlo a mangata a CSF a ntšitsoeng ho liphoofolo ka bomong pakeng tsa mekhahlelo e 'meli ea khetho e bapisoa le tatellano e ntlafalitsoeng ea potoloho ea bobeli e bontšoa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 le (h) #1.2–#2.4.Li-amino acid tse matlafalitsoeng ka ho fetisisa boemong bo fanoeng ho (c, d) liphoofolo no.2.1 le che.2.2 kapa (g, h) liphoofolong no.2.3 le che.2.4 li bonts'oa ka 'mala.Botala = polar, pherese = neutral, blue = motheo, khubelu = acidic le e ntšo = hydrophobic amino acid.
Ka mor'a khetho ea boraro ea khetho, re ile ra khetholla li-peptide tse ikhethang tsa 124 (#3.1 le #3.2) ho tloha ho 332 CSF-reconstituted phage clones e arohaneng le liphoofolo tse peli (Supplementary Fig. 6a).Tatelano ya LGSVS (18.7%) e ne e na le karolo e phahameng ka ho fetisisa e lekanyeditsweng, e latelwang ke mefuta e hlaha PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), le SARGSWREIVSLS (2.2%).Karolong ea bone ea ho qetela, re ile ra kopanya makala a mabeli a khethiloeng ka boikemelo ho tsoa liphoofolong tse tharo tse arohaneng (Setšoantšo sa 1c).Har'a li-clone tsa 925 tse hlophisitsoeng tsa phage tse fumanoeng ho tloha CSF, karolong ea bone re ile ra fumana li-peptide tse ikhethang tsa 64 (Supplementary Fig. 6b), eo ho eona karolo e lekanyelitsoeng ea mofuta o hlaha oa phage e theohetse ho 0.8%.Li-clone tsa CSF tse atileng haholo molemong oa bone e ne e le LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) le RLSSVDSDLSGC (3, 2%).%)).Bolelele ba li-peptide tse khethiloeng bo bakoa ke ho kenngoa / ho tlosoa ha li-nucleotide kapa li-codon tsa ho emisa pele ho nako libukeng tsa laebrari ha u sebelisa li-codon tse senyehileng bakeng sa moralo oa laeborari ea NNK.Li-codons tsa ho emisa pele ho nako li hlahisa li-peptide tse khuts'oane 'me lia khethoa hobane li na le aa motif e ntle.Li-peptide tse telele li ka bakoa ke ho kenya / ho hlakoloa ho li-primer tsa lilaebrari tsa maiketsetso.Sena se beha "stop codon" e hlophisitsoeng ka ntle ho foreimi 'me e e bale ho fihlela setopo se secha se hlaha tlase.Ka kakaretso, re balile lintlha tsa ntlafatso bakeng sa mekhahlelo eohle e mene ea khetho ka ho bapisa lintlha tse kentsoeng le sampole e hlahisoang.Bakeng sa potoloho ea pele ea tlhahlobo, re sebelisitse li-titer tsa mofuta o hlaha e le sesupo sa morao-rao se sa tobang.Hoa thahasellisa hore khetho e mpe ea phage e ne e le matla haholo potolohong ea pele ea CSF, empa eseng maling (setšoantšo sa 3a), e ka 'nang ea e-ba ka lebaka la monyetla o fokolang oa ho hasana ha litho tse ngata tsa laebrari ea peptide ka kamoreng ea CSF kapa phages e amanang le eona e atisa ho bolokoa ka mokhoa o atlehileng kapa o tlosoa maling ho feta bacteriophages.Leha ho le joalo, karolong ea bobeli ea panning, khetho e matla ea phages ho CSF ​​e ile ea hlokomeloa ka lihlopha tse peli, ho fana ka maikutlo a hore potoloho e fetileng e ne e ruisitsoe ka phages e bontšang li-peptide tse khothalletsang ho kenngoa ha CSF (Fig. 3a).Hape, ntle le matlafatso ea bohlokoa ea mali.Hape karolong ea boraro le ea bone, li-clone tsa phage li ile tsa ntlafatsoa haholo ho CSF.Ha re bapisa lebelo le lekanyelitsoeng la tatellano e 'ngoe le e' ngoe e ikhethang ea peptide pakeng tsa likhetho tse peli tsa ho qetela tsa khetho, re fumane hore tatellano e ne e ruisitsoe le ho feta karolong ea bone ea khetho (setšoantšo sa 3b).Kakaretso ea li-tripeptide motifs tse 931 li ntšitsoe ho tatellano eohle e ikhethang ea li-peptide tse 64 ho sebelisoa lipeptide tse peli.Li-motif tse ntlafalitsoeng ka ho fetesisa papaling ea bone li ile tsa hlahlobjoa ka hloko bakeng sa lintlha tsa bona tsa ho ithuisa ho pota-pota ka hohle ha li bapisoa le laebrari e kentsoeng (e khaotsoeng: 10% e ruisang) (Fig. 6c).Mekhoa e akaretsang ea khetho e bontšitse hore boholo ba merero e ithutoang e ruisitsoe mekhahlelong eohle e fetileng ea makala a mabeli a khetho.Leha ho le joalo, lihloliloeng tse ling (mohlala, SGL, VSG, LGS GSV) li ne li tsoa ho sehlopha se seng sa A, athe tse ling (mohlala, FGW, RTN, WGF, NTR) li ne li ruisitsoe ho sehlopha se seng sa B.
Ho netefatsoa ha lipalangoang tsa CSF tsa CSF-enriched phage-displayed peptide le biotinylated leader peptide conjugated to streptavidin payloads.
(a) Likarolo tsa ntlafatso tse baloang ka mekhahlelo eohle e mene (R1-R4) ho ipapisitsoe le li-titers tse kentsoeng (tse kenang = I) phage (PFU) le li-phage titers tsa CSF (sephetho = O).Lintlha tsa ntlafatso bakeng sa mekhahlelo e meraro ea ho qetela (R2-R4) li ne li baloa ka ho bapisoa le potoloho e fetileng le ea pele (R1) e nang le boitsebiso ba boima.Libaka tse bulehileng ke cerebrospinal fluid, li-bar tse nang le moriti ke plasma.(***p<0.001, e ipapisitse le tlhahlobo ea t ea Seithuti).(b) Lethathamo la li-peptide tse ngata ka ho fetisisa tsa phage, tse behiloeng ho ea ka karolo ea tsona e lekanyelitsoeng ho lihlopha tsohle tse bokelitsoeng ho CSF ​​ka mor'a khetho ea 4.Li-clone tse tšeletseng tse tloaelehileng haholo tsa phage li totobalitsoe ka 'mala, li baloa le lintlha tsa tsona tsa ho ntlafatsa lipakeng tsa 3 le 4 ea khetho (inset).(c, d) Li-clone tse tšeletseng tse ntlafalitsoeng ka ho fetesisa, lilaebrari tse se nang letho le lilaebrari tsa phage peptide ho tloha ho 4 li ile tsa hlahlobjoa ka bonngoe ho latela mohlala oa mohlala oa CSF.CSF le lisampole tsa mali li ile tsa bokelloa ka nako e bontšitsoeng.(c) Likarolo tse lekanang tsa li-clone tsa mokhethoa oa 6 (2 x 1010 phages / liphoofolo), phages e se nang letho (#1779) (2 x 1010 phages / liphoofolo) le lilaebrari tsa li-stock phage peptide (2 x 1012 phages / liphoofolo) Enta bonyane 3 CM e tsamaisoa ka mohatla ka har'a sekoti.Pharmacokinetics ea CSF ea ente e 'ngoe le e' ngoe ea phage clone le laeborari ea phage peptide ha nako e ntse e ea e bonts'oa.(d) e bonts'a karo-karolelano ea karo-karolelano ea CSF / mali bakeng sa phages/mL tsohle tse hlaphohetsoeng nakong ea lisampole.(e) Li-peptide tse 'nè tsa moeta-pele oa maiketsetso le taolo e le' ngoe ea scrambled li ne li amahanngoa le biotin ho streptavidin ka N-terminus ea bona (pontšo ea tetramer) e lateloa ke ente (mohatla oa mohatla iv, 10 mg streptavidin / kg).Bonyane likhoto tse tharo tse intubated (N = 3).).Li-sampuli tsa CSF li ile tsa bokelloa ka linako tse bontšitsoeng 'me likhahla tsa streptavidin li lekantsoe ke CSF anti-streptavidin ELISA (nd = ha e fumanoe).(*p<0.05, **p<0.01, ***p<0.001, e ipapisitse le teko ea ANOVA).(f) Papiso ea tatellano ea amino acid ea phage peptide clone #2002 (pherese) e nang le li-clone tse ling tse khethiloeng tsa phage peptide ho tloha mokhahlelong oa 4 oa khetho.Likhechana tse tšoanang le tse tšoanang tsa amino acid li na le mebala.
Har'a likarolo tsohle tse ntlafalitsoeng molemong oa bone (setšoantšo sa 3b), li-clone tse tšeletseng tsa bonkgetheng li ile tsa khethoa bakeng sa tlhahlobo e eketsehileng ea motho ka mong mofuteng oa mohlala oa CSF.Likarolo tse lekanang tsa phage ea mokhethoa e tšeletseng, phage e se nang letho (ha e na ho kenya) le lilaebrari tsa peptide ea prophage li ile tsa kenngoa liphoofolong tse tharo tsa CM tse nang le cannulated, 'me li-pharmacokinetics li ile tsa khethoa ho CSF ​​(Fig. 3c) le litlhahlobo tsa mali (Supplementary Fig. 7).Li-clone tsohle tsa phage tse lekiloeng li ne li lebisitse kamoreng ea CSF maemong a 10-1000 ho feta ea phage e se nang letho (#1779).Mohlala, li-clones #2020 le #2077 li ne li e-na le li-titer tsa CSF tse phahameng ka makhetlo a 1000 ho feta phage ea taolo.Boemo ba pharmacokinetic ba peptide e 'ngoe le e' ngoe e khethiloeng e fapane, empa kaofela ha bona ba na le bokhoni bo phahameng ba ho hohela ba CSF.Re bone phokotseho e sa khaotseng ha nako ea li-clones tsa #1903 le #2011, ha li-clone tsa #2077, #2002 le #2009 keketseho nakong ea metsotso e 10 ea pele e ka bonts'a lipalangoang tse sebetsang empa li hloka ho netefatsoa.Li-Clones #2020, #2002, le #2077 li tsitsitse maemong a holimo, athe CSF ea clone #2009 e ile ea fokotseha butle kamora keketseho ea pele.Ka mor'a moo re ile ra bapisa lebelo le lekanyelitsoeng la mokhethoa e mong le e mong oa CSF le khatello ea mali ea hae (setšoantšo sa 3d).Khokahano ea moelelo oa mokhethoa e mong le e mong oa CSF le karolo ea eona ea mali ka linako tsohle tsa lisampole e bonts'itse hore ba bararo ho ba tšeletseng ba ne ba ruisitsoe haholo maling a CSF.Hoa thahasellisa hore clone #2077 e bontšitse botsitso bo phahameng ba mali (Setšoantšo sa Tlatsetso 7).Ho netefatsa hore li-peptide ka botsona li khona ho tsamaisa thepa ntle le likaroloana tsa phage ka phaposing ea CSF, re ile ra kopanya li-peptide tse 'ne tse nkiloeng ka biotin ho N-terminus moo li-peptide li hokelang karoloana ea phage.Li-peptide tsa biotinylated (nos. 2002, 2009, 2020 le 2077) li ne li kopantsoe le streptavidin (SA) ho fumana mefuta e mengata ea mefuta e batlang e etsisa phage geometry.Sebopeho sena se boetse se re lumelletse ho lekanya ho pepeseha ha SA maling le mokelikeli oa cerebrospinal e le li-peptide tsa protheine tse tsamaisang thepa.Habohlokoa, data ea phage e ne e ka atisa ho hlahisoa ha li-peptide tsa maiketsetso li ne li tsamaisoa ka mokhoa ona oa SA-conjugated (Fig. 3e).Li-peptide tse senyehileng li ne li e-na le ponahalo e fokolang ea pele le tumello e potlakileng ea CSF ka maemo a sa bonahaleng nakong ea lihora tse 48.Ho fumana temohisiso mabapi le litsela tsa ho fana ka li-clone tsena tsa peptide phage sebakeng sa CSF, re ile ra hlahloba sebaka sa sebaka sa motho ka mong sa phage peptide hits ho sebelisa immunohistochemistry (IHC) ho lemoha ka ho toba likaroloana tsa phage 1 hora ka mor'a ho kenngoa ka methapo ka har'a vivo.Haholo-holo, li-clone #2002, #2077, le #2009 li ne li ka bonoa ka ho silafatsa ka matla ho li-capillaries tsa boko, ha phage ea taolo (#1779) le clone #2020 e sa fumanwe (Setšoantšo sa Tlatsetso 8).Sena se fana ka maikutlo a hore li-peptide tsena li kenya letsoho ho phello bokong hantle ka ho tšela BBB.Ho hlokahala tlhahlobo e felletseng ho leka khopolo-taba ena, kaha tsela ea BSCFB e kanna ea ameha.Ha ho bapisoa tatellano ea amino acid ea clone e ntlafalitsoeng ka ho fetisisa (#2002) le li-peptide tse ling tse khethiloeng, ho ile ha hlokomeloa hore tse ling tsa tsona li na le li-extensions tse tšoanang tsa amino acid, tse ka bontšang mokhoa o tšoanang oa lipalangoang (setšoantšo sa 3f).
Ka lebaka la boemo ba eona bo ikhethang ba plasma le keketseho e kholo ea CSF ha nako e ntse e feta, phage display clone #2077 e ile ea hlahlojoa hape ka nako e telele ea lihora tsa 48 'me ea khona ho hlahisa keketseho e potlakileng ea CSF e hlokometsoeng ka ho kopana le maemo a tsitsitseng a SA (Fig. 4a).Mabapi le li-clone tse ling tse khethiloeng tsa phage, #2077 e ne e silafetse ka matla bakeng sa li-capillaries tsa boko 'me e bontšitse colocalization e kholo ka lectin ea capillary ha e shejoa ka qeto e phahameng' me mohlomong le litšila sebakeng sa parenchymal (Setšoantšo sa 4b).Ho etsa lipatlisiso tsa hore na litlamorao tsa pharmacological tsa peptide li ka fumaneha ho CNS, re entse liteko moo mefuta ea biotinylated ea i) #2077 transit peptide le ii) BACE1 inhibitor peptide e kopantsoe le SA ka likarohano tse peli tse fapaneng.Bakeng sa motsoako o le mong re sebelisitse BACE1 peptide inhibitor feela 'me ho e' ngoe re sebelisitse tekanyo ea 1:3 ea BACE1 peptide inhibitor ho #2077 peptide.Mehlala ka bobeli e ile ea tsamaisoa ka methapo 'me maemo a mali le cerebrospinal fluid a beta-amyloid peptide 40 (Abeta40) a lekantsoe ka nako.Abeta40 e lekantsoe ho CSF ​​kaha e bonts'a inhibition ea BACE1 bokong ba parenchyma.Joalokaha ho ne ho lebelletsoe, li-complexes ka bobeli li fokolitse haholo maemo a mali a Abeta40 (setšoantšo sa 4c, d).Leha ho le joalo, ke mehlala feela e nang le motsoako oa peptide no.2077 le inhibitor ea BACE1 peptide conjugated to SA e bakile ho fokotseha ho hoholo ho Abeta40 ho cerebrospinal fluid (Fig. 4c).Lintlha li bontša hore peptide no.2077 e khona ho tsamaisa protheine ea 60 kDa SA ho CNS hape e hlahisa litlamorao tsa meriana ka SA-conjugated inhibitors ea BACE1 peptide.
(a) Clonal injection (2 × 10 phages / phoofolo) ea T7 phage e bontšang nako e telele ea pharmacokinetic profiles ea CSF peptide #2077 (RLSSVDSDLSGC) le phage ea ho laola e sa etsoang (#1779) bonyane likhoto tse tharo tse nang le CM-intubated.(b) Setšoantšo sa microscopic sa confocal sa li-microvessels tsa cortical tse nang le litoeba tse entsoeng ka phage (2 × 10 10 phages / phoofolo) e bontšang counterstaining ea peptide #2077 le likepe (lectin).Li-clone tsena tsa phage li ne li tsamaisoa ho likhoto tsa 3 'me tsa lumelloa ho potoloha hora ea 1 pele ho perfusion.Boko bo ile ba aroloa le ho silafatsoa ka li-antibodies tse ngotsoeng ke polyclonal FITC khahlanong le T7 phage capsid.Metsotso e leshome pele ho mokelikeli le ho lokisa ka mor'a moo, DyLight594-lectin e ngotsoeng e ne e kenngoa ka methapo.Litšoantšo tsa fluorescent tse bontšang lectin staining (e khubelu) ea lehlakore le khanyang la li-microvessels le phages (e tala) ka har'a lumen ea li-capillaries le lisele tsa boko ba perivascular.Sekala sa sekhahla se lumellana le 10 µm.(c, d) Biotinylated BACE1 inhibitory peptide e le 'ngoe kapa e kopantsoe le peptide ea transit ea biotinylated #2077 e ne e kopantsoe le streptavidin e lateloa ke ente e kenang ka methapo ea bonyane likhoto tse tharo tsa cannulated tsa CM (10 mg streptavidin/kg).Phokotso ea BACE1 peptide inhibitor-mediated ho Aβ40 e lekantsoe ke Aβ1-40 ELISA maling (e khubelu) le cerebrospinal fluid (orange) ka nako e bontšitsoeng.Bakeng sa ho hlakisa hantle, mola o nang le matheba o toroa kerafong ka tekanyo ea 100%.(c) Phokotso ea liperesente tsa Aβ40 maling (likhutlotharo tse khubelu) le cerebrospinal fluid (likhutlotharo tsa lamunu) likhotong tse tšoaroang ka streptavidin conjugated to transit peptide #2077 le BACE1 inhibitory peptide ka 3: 1 ratio.(d) Phokotso ea liperesente tsa mali Aβ40 (li-circles tse khubelu) le cerebrospinal fluid (li-orange circles) tsa likhoto tse tšoaroang ka streptavidin hammoho le peptide e thibelang BACE1 feela.Motsoako oa Aβ taolong e ne e le 420 pg / ml (ho kheloha ho tloaelehileng = 101 pg / ml).
Pontšo ea Phage e sebelisitsoe ka katleho libakeng tse 'maloa tsa lipatlisiso tsa biomedical17.Mokhoa ona o 'nile oa sebelisoa bakeng sa lithuto tsa mefuta-futa ea li-vascular18,19 hammoho le lithuto tse lebisang likepeng tsa boko20,21,22,23,24,25,26.Phuputsong ena, re atolositse ts'ebeliso ea mokhoa ona oa khetho eseng feela ho tsebahatsa ka ho toba li-peptide tse lebisang likepeng tsa boko, empa hape le ho sibolloa ha batho ba nang le mekhoa e sebetsang ea lipalangoang ho tšela mokoallo oa mali-boko.Hona joale re hlalosa nts'etsopele ea ts'ebetso ea khetho ea in vivo ho likhoto tse intubated tsa CM mme re bonts'a bokhoni ba eona ba ho khetholla li-peptide tse nang le thepa ea homing ea CSF.Re sebelisa phage ea T7 e bontšang laebrari ea li-peptide tse sa tloaelehang tsa 12-mer, re ile ra khona ho bontša hore phage ea T7 e nyenyane ka ho lekaneng (hoo e ka bang 60 nm ka bophara) 10 hore e tloaelehe ho thibela mali-boko, kahoo e tšela ka ho toba mokoallo oa mali-boko kapa plexus ea choroid.Re hlokometse hore kotulo ea CSF ho tsoa ho likhoto tsa CM tse nang le cannulated e ne e le mokhoa o laoloang hantle ka mokhoa oa ho hlahloba ts'ebetso ea vivo, le hore phage e ntšitsoeng e ne e sa tlameletsoe feela ho vasculature empa hape e ne e sebetsa e le sepalangoang ho pholletsa le mokoallo oa mali-boko.Ho feta moo, ka ho bokella mali ka nako e le 'ngoe le ho sebelisa HTS ho CSF ​​le phages e nkiloeng maling, re tiisitse hore khetho ea rona ea CSF ha ea ka ea susumetsoa ke ho matlafatsa mali kapa ho lekana bakeng sa katoloso pakeng tsa likhetho.Leha ho le joalo, phaposi ea mali ke karolo ea mokhoa oa ho khetha, kaha phages e khonang ho fihla phaposing ea CSF e tlameha ho phela le ho potoloha maling nako e telele e lekaneng ho ithuisa bokong.Bakeng sa ho ntša tlhahisoleseling e tšepahalang ea tatellano ho data e tala ea HTS, re sebelisitse li-filters tse ikamahanyang le liphoso tsa tatellano e khethehileng ea sethala tlhahlobong ea mosebetsi.Ka ho kenyelletsa li-parameter tsa kinetic mokhoeng oa ho hlahloba, re netefalitse pharmacokinetics e potlakileng ea mofuta oa hlaha oa T7 phages (t½ ~ 28 min) maling24, 27, 28 mme hape re entse qeto ea halofo ea bophelo ba bona ho cerebrospinal fluid (t½ ~ 26 min) ka motsotso).Leha ho na le litlaleho tse tšoanang tsa pharmacokinetic maling le CSF, ke 0.001% feela ea khatello ea mali ea phage e ka fumanoang ho CSF, e bontšang ho tsamaea ha mokokotlo oa mofuta oa T7 phage ho pholletsa le mokoallo oa mali-boko.Mosebetsi ona o totobatsa bohlokoa ba potoloho ea pele ea khetho ha ho sebelisoa maqheka a panning a vivo, haholo-holo bakeng sa mekhoa ea phage e tlosoa ka potlako ho tloha ho potoloha, kaha li-clone tse seng kae li khona ho fihla kamoreng ea CNS.Kahoo, karolong ea pele, phokotso ea mefuta-futa ea libuka e ne e le khōlō haholo, kaha ke palo e fokolang feela ea li-clone tse ileng tsa qetella li bokelloa ka mokhoa ona o thata haholo oa CSF.Leano lena la panning ea vivo le ne le kenyelletsa mehato e mengata ea khetho e kang ho bokella ka mafolofolo ka kamoreng ea CSF, ho pholoha ha clone karolong ea mali, le ho tlosoa ka potlako ha li-clone tsa T7 phage maling nakong ea metsotso ea pele ea 10 (Fig. 1d le Supplementary Fig. 4M).).Kahoo, ka mor'a potoloho ea pele, li-clone tse fapaneng tsa phage li ile tsa khetholloa ho CSF, le hoja letamo le tšoanang la pele le ne le sebelisetsoa liphoofolo ka bomong.Sena se fana ka maikutlo a hore mehato e mengata e thata ea khetho bakeng sa lilaebrari tsa mehloli e nang le palo e kholo ea litho tsa laeborari e fella ka phokotso e kholo ea mefuta-futa.Ka hona, liketsahalo tse sa reroang li tla fetoha karolo ea bohlokoa ea ts'ebetso ea pele ea khetho, e tla ama sephetho haholo.Ho ka etsahala hore ebe li-clone tse ngata ka laeboraring ea mantlha li ne li na le tšekamelo e tšoanang ea ntlafatso ea CSF.Leha ho le joalo, esita le tlas'a maemo a tšoanang a liteko, liphetho tsa khetho li ka 'na tsa fapana ka lebaka la palo e nyenyane ea clone e' ngoe le e 'ngoe ka letamo la pele.
Melemo e matlafalitsoeng ho CSF ​​e fapane le e maling.Ho khahlisang, re hlokometse phetoho ea pele ho li-peptide tse nang le glycine maling a phoofolo ka 'ngoe.(Setšoantšo sa 1g, Figs tse tlatselletsang. 4e, 4f).Phage e nang le li-peptide tsa glycine e kanna ea tsitsa, 'me ha se na monyetla oa hore e tlosoe ho potoloha.Leha ho le joalo, li-peptide tsena tse ruileng tsa glycine ha lia ka tsa fumanoa lisampoleng tsa mokelikeli oa cerebrospinal, e leng se bontšang hore lilaebrari tse koaletsoeng li ile tsa feta mehatong e 'meli e fapaneng ea khetho: e 'ngoe maling' me e 'ngoe e lumelloa ho bokella mokelikeli oa cerebrospinal.Li-clones tse matlafalitsoeng ke CSF tse bakoang ke potoloho ea bone ea khetho li lekoa haholo.Hoo e ka bang li-clone tsohle tse lekiloeng ka bomong li netefalitsoe hore li ruisitsoe ho CSF ​​ha li bapisoa le phage e se nang letho ea taolo.Peptide e le 'ngoe e otla (#2077) e ile ea hlahlojoa ka botlalo.E ne e bonts'a halofo ea bophelo bo bolelele ba plasma ha e bapisoa le tse ling tse otlang (Setšoantšo sa 3d le Setšoantšo sa Tlatsetso 7), mme ho khahlisang, peptide ena e ne e na le masala a cysteine ​​​​ho C-terminus.Ho sa tsoa bontšoa hore tlatsetso ea cysteine ​​​​ho li-peptide e ka ntlafatsa thepa ea bona ea pharmacokinetic ka ho tlama albumin 29.Sena ha se tsejoe bakeng sa peptide #2077 mme se hloka boithuto bo bong.Li-peptide tse ling li bonts'itse ho its'etleha ho valence ho ntlafatso ea CSF (data e sa bonts'itsoeng), e kanna ea amana le geometry e bonts'itsoeng ea T7 capsid.Sistimi ea T7 eo re e sebelisitseng e bonts'itse likopi tse 5-15 tsa peptide ka 'ngoe ka phage.IHC e ne e etsoa ka li-clone tsa phage tsa mokhethoa tse kenngoeng ka methapo ka har'a cerebral cortex ea likhoto (Supplementary Fig. 8).Lintlha li bontšitse hore bonyane li-clone tse tharo (No. 2002, No. 2009 le No. 2077) li sebelisana le BBB.Ho sa ntse ho tla etsoa qeto ea hore na tšebelisano ena ea BBB e fella ka ho bokellana ha CSF kapa motsamao oa li-clone tsena ka kotloloho ho BCSFB.Habohlokoa, re bonts'a hore li-peptide tse khethiloeng li boloka matla a tsona a lipalangoang tsa CSF ha li kopantsoe 'me li tlameletsoe ho thepa ea protheine.Ho tlama ha N-terminal biotinylated peptides ho SA ha e le hantle ho pheta liphetho tse fumanoeng ka li-clone tsa bona tsa phage maling le cerebrospinal fluid (Setšoantšo sa 3e).Qetellong, re bonts'a hore lead peptide #2077 e khona ho khothaletsa ts'ebetso ea boko ba biotinylated peptide inhibitor ea BACE1 conjugated to SA, e bakang litlamorao tsa pharmacodynamic ho CNS ka ho fokotsa haholo maemo a Abeta40 ho CSF ​​(Fig. 4).Ha rea ​​khona ho tsebahatsa li-homologues sebakeng sa polokelo ea litaba ka ho etsa lipatlisiso tsa tatellano ea li-peptide tsa li-hits tsohle.Ho bohlokoa ho hlokomela hore boholo ba laeborari ea T7 e batla e le 109, athe boholo ba laeborari ea theory bakeng sa 12-mers ke 4 x 1015. Ka hona, re khethile karoloana e nyane feela ea sebaka sa mefuta-futa ea laebrari ea 12-mer peptide, e ka bolelang hore li-peptide tse ntlafalitsoeng haholoanyane li ka khetholloa ka ho lekola sebaka se haufi sa libaka tsena tse khahlano.Ka boikaketsi, le leng la mabaka a entseng hore re se fumane li-homologue tsa tlhaho tsa li-peptide tsena e kanna ea se khethoe nakong ea ho iphetola ha lintho ho thibela ho kena ho sa laoleheng ha li-peptide motifs tse itseng bokong.
Ha re kopane, liphetho tsa rona li fana ka motheo oa mosebetsi oa nako e tlang ho tsebahatsa le ho khetholla litsamaiso tsa lipalangoang tsa mokoallo oa cerebrovascular in vivo ka botlalo.Setupo sa mantlha sa mokhoa ona se ipapisitse le leano la khetho le sebetsang le sa khetholleng feela li-clone tse nang le thepa e tlamang methapo ea methapo ea boko, empa hape e kenyelletsa mohato o mahlonoko oo li-clone tse atlehileng li nang le ts'ebetso ea mantlha ea ho tšela mekoallo ea bioloji ho kena ka phaposing ea CNS.ke ho hlakisa mokhoa oa ho tsamaisa li-peptide tsena le khetho ea tsona ea ho tlama ho microvasculature e khethehileng sebakeng sa boko.Sena se ka lebisa ho sibolloe litsela tse ncha tsa ho tsamaisa BBB le li-receptors.Re lebeletse hore li-peptide tse khethiloeng li ka tlama ka ho toba ho li-cerebrovascular receptors kapa li-ligands tse potolohang tse tsamaisoang ka BBB kapa BCSFB.Li-vector tsa peptide tse nang le ts'ebetso ea lipalangoang tsa CSF tse fumanoeng mosebetsing ona li tla tsoela pele ho etsoa lipatlisiso.Hajoale re ntse re batlisisa ka botlalo ba boko ba li-peptide tsena bakeng sa bokhoni ba tsona ba ho tšela BBB le/kapa BCSFB.Li-peptide tsena tse ncha e tla ba lisebelisoa tsa bohlokoa haholo bakeng sa ho sibolloa ha li-receptor tse ncha kapa litsela le bakeng sa nts'etsopele ea liforomo tse ncha tse sebetsang hantle haholo bakeng sa phano ea li-macromolecules, joalo ka biologics, bokong.
Cannulate cisterna e kholo (CM) ho sebelisa phetoho ea mokhoa o hlalositsoeng pele.Litoeba tsa Wistar tse hlabositsoeng (200-350 g) li ne li kenngoa holim'a sesebelisoa sa stereotaxic 'me ho ile ha etsoa sekhahla se bohareng holim'a letlalo la hlooho le betliloeng le ho lokisoa ka mokhoa o hlakileng ho pepesa lehata.Hlakola masoba a mabeli sebakeng sa lesela le ka holimo 'me u kenye li-screws tsa ho lokisa mekoting.Ho ile ha phunngoa lesoba le leng ka har'a lateral occipital crest bakeng sa tataiso ea stereotactic ea cannula ea tšepe e sa hloekang ho kena CM.Tšela samente ea meno ho potoloha cannula 'me u sireletse ka li-screw.Ka mor'a ho phekola foto le ho thatafala ha samente, leqeba la letlalo le ne le koetsoe ka 4/0 supramid suture.Ho beoa ka nepo ha cannula ho netefatsoa ke ho lutla ho sa tloaelehang ha cerebrospinal fluid (CSF).Tlosa rat ho lisebelisoa tsa stereotaxic, fumana tlhokomelo e nepahetseng ka mor'a ts'ebetso le taolo ea bohloko, 'me u e lumelle hore e fole bonyane beke e le' ngoe ho fihlela matšoao a mali a bonoa ka cerebrospinal fluid.Litoeba tsa Wistar (Crl: WI/Han) li ile tsa fumanoa Charles River (Fora).Likhoto tsohle li ne li bolokoa tlas'a maemo a itseng a se nang likokoana-hloko.Liteko tsohle tsa liphoofolo li ile tsa amoheloa ke Ofisi ea Liphoofolo tsa Motse oa Basel, Switzerland, 'me li entsoe ho latela License ea Liphoofolo No.
Ka bonolo hlokomela rat ka CM cannula letsohong.Tlosa Datura ho cannula 'me u bokelle 10 µl ea mokelikeli oa cerebrospinal o phallang.Kaha patency ea cannula e ile ea qetella e senyehile, ke feela lisampole tse hlakileng tsa cerebrospinal fluid tse se nang bopaki ba tšilafalo ea mali kapa ho fetoha ha 'mala li kenyelelitsoe thutong ena.Ka mokhoa o ts'oanang, hoo e ka bang 10-20 μl ea mali e nkiloe ho tloha ho sehoe se senyenyane ntlheng ea mohatla ka har'a li-tubes tse nang le heparin (Sigma-Aldrich).CSF le mali li ile tsa bokelloa ka linako tse fapaneng ka mor'a ente ea methapo ea T7 phage.Hoo e ka bang 5-10 μl ea mokelikeli o ile oa lahloa pele sampuli e 'ngoe le e' ngoe ea CSF e bokelloa, e lumellanang le molumo o shoeleng oa catheter.
Lilaebrari li ile tsa hlahisoa ho sebelisoa vector ea T7Select 10-3b joalokaha ho hlalositsoe bukeng ea tsamaiso ea T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Ka bokhuts'oane, 12-mer DNA e kentsoeng e entsoe ka mokhoa o latelang:
NNK codon e ne e sebelisetsoa ho qoba li-codon tse peli tsa ho emisa le ho feteletsa ha amino acid ho kenya letsoho.N ke karo-karolelano ea equimolar e kopantsoeng ka letsoho ea nucleotide e 'ngoe le e' ngoe, 'me K ke tekanyo e kopantsoeng ea equimolar ea adenine le cytosine nucleotide.Libaka tse le 'ngoe tse qakaneng li ile tsa fetoleloa ho DNA e mahlakore a mabeli ka ho kenngoa hape ka dNTP (Novagen) le enzyme ea Klenow (New England Biolabs) sebakeng sa Klenow buffer (New England Biolabs) ka lihora tse 3 ka 37°C.Kamora karabelo, DNA e nang le likhoele tse peli e ile ea fumanoa ke pula ea EtOH.DNA e hlahisitsoeng e ile ea chekoa ka li-enzyme tse thibelang EcoRI le HindIII (ka bobeli ho tsoa Roche).The petsoha le ho hloekisoa (QIAquick, Qiagen) kenya (T4 ligase, New England Biolabs) ka nako eo ligated in-frame ho pre-cleaved T7 vector ka mor'a amino acid 348 ea 10B capsid gene.Li-Ligation reactions li ile tsa kenngoa ka 16 ° C. bakeng sa lihora tse 18 pele ho pakete ea in vitro.Phage packaging in vitro e entsoe ho latela litaelo tse fanoeng le T7Select 10-3b cloning kit (Novagen) mme tharollo ea ho paka e ile ea phahamisoa hang ho lysis ho sebelisa Escherichia coli (BLT5615, Novagen).Li-lysates li ne li le bohareng, li titrated 'me li leqhoa ho -80 ° C. e le tharollo ea stock ea glycerol.
Kholiso e tobileng ea PCR ea libaka tse fapaneng tsa phage tse matlafalitsoeng ka moro kapa poleiti ho sebelisa li-primers tsa 454 / Roche-amplicon fusion.Sebapali sa pele sa fusion se na le tatellano e pota-potileng sebaka se feto-fetohang (NNK) 12 (template-specific), GS FLX Titanium Adapter A, le tatellano ea linotlolo tsa laeborari tse nne (TCAG) (Setšoantšo sa Tlatsetso 1a):
Reverse fusion primer e boetse e na le biotin e khomaretsoeng ho hapa lifaha le GS FLX Titanium Adapter B e hlokahalang bakeng sa amplification ea clonal nakong ea emulsion PCR:
Joale li-amplicon li ile tsa behoa tlas'a 454 / Roche pyrosequencing ho ea ka 454 GS-FLX Titanium protocol.Bakeng sa tatellano ea litaelo tsa Sanger (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA e ile ea holisoa ke PCR 'me ea hlophisoa ka lipara tse latelang:
Ho kenya li-plaque ka bomong ho ile ha etsoa ka matla a PCR ho sebelisoa Roche Fast Start DNA Polymerase Kit (ho ea ka litaelo tsa moetsi).Etsa qalo e chesang (metsotso e 10 ho 95 °C) le lipotoloho tse matlafatsang tse 35 (50 s ho 95 °C, 1 min ho 50 °C, le 1 min ho 72 °C).
Phage e tsoang lilaebraring, phage ea mofuta o hlaha, phage e pholositsoeng ho tloha CSF le mali, kapa li-clone ka bomong li ile tsa phahamisoa ho Escherichia coli BL5615 ka moro oa lefuba (Sigma Aldrich) kapa lijana tsa 500 cm2 (Thermo Scientific) bakeng sa 4 h ho 37 ° C.Phage e ile ea ntšoa lipoleiting ka ho hlatsoa lipoleiti ka Tris-EDTA buffer (Fluka Analytical) kapa ka ho bokella li-plaque tse nang le malebela a nyopa a pipette.Phage e ne e arohane le setso sa supernatant kapa extraction buffer ka polyethylene glycol (PEG 8000) precipitation (PEG 8000) precipitation (Promega) 'me ea khutlisetsoa ho Tris-EDTA buffer.
Phage e matlafalitsoeng e ne e le tlas'a mekhahlelo ea 2-3 ea ho tlosoa ha endotoxin ho sebelisoa lifaha tsa ho tlosa endotoxin (Miltenyi Biotec) pele ho ente ea intravenous (IV) (500 μl / phoofolo).Karolong ea pele, 2 × 1012 phages e ile ea hlahisoa;ea bobeli, 2×1010 phages;karolong ea boraro le ea bone ea khetho, 2×109 phages phoofolo ka 'ngoe.Likahare tsa phage ho CSF ​​le lisampole tsa mali tse bokelletsoeng ka nako e bontšitsoeng li ile tsa khethoa ka ho bala plaque ho latela litaelo tsa moetsi (T7Select system manual).Khetho ea phage e ne e etsoa ka ente ea methapo ea lilaebrari tse hloekisitsoeng ka har'a mothapo oa mohatla kapa ka ho kenngoa hape ha phage e nkiloeng ho CSF ​​ho tloha nakong e fetileng ea khetho, 'me kotulo e latelang e ile ea etsoa ka 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, le 240 mins le sampuli ea mali ea CSF ka ho latellana.Kakaretso ea mekhahlelo e mene ea in vivo panning e ile ea etsoa moo makala a mabeli a khethiloeng a ileng a bolokoa ka thoko le ho hlahlojoa nakong ea mekhahlelo e meraro ea pele ea khetho.Lisebelisoa tsohle tsa phage tse ntšitsoeng ho CSF ​​ho tloha likhethong tse peli tsa pele tsa khetho li ne li le tlas'a 454 / Roche pyrosequencing, ha li-clone tsohle tse nkiloeng ho CSF ​​ho tloha likhethong tse peli tsa ho qetela tsa khetho li ne li hlophisitsoe ka letsoho.Liphage tsohle tsa mali ho tloha mokhahlelong oa pele oa khetho le tsona li ne li le tlas'a 454 / Roche pyrosequencing.Bakeng sa ente ea li-clone tsa phage, phages e khethiloeng e ile ea matlafatsoa ho E. coli (BL5615) ka lipoleiti tsa 500 cm2 ho 37 ° C bakeng sa lihora tse 4.Li-clone tse khethiloeng ka bomong le tse latellanang ka letsoho li ile tsa phatlalatsoa ka mokhoa oa lefuba.Ka mor'a ho ntšoa ha phage, ho hloekisoa le ho tlosoa ha endotoxin (joalokaha ho hlalositsoe ka holimo), 2 × 1010 phages / phoofolo ka 300 μl e ne e kenngoa ka methapo ka har'a mothapo o le mong oa mohatla.
Ho hlophisoa esale pele le ho sefa ha boleng ba data ea tatellano.Lintlha tsa Raw 454/Roche li ile tsa fetoloa ho tloha ho binary standard stream map format (sff) ho ea ho Pearson human human format (fasta) ho sebelisoa software ea barekisi.Ts'ebetso e tsoelang pele ea tatellano ea nucleotide e ile ea etsoa ho sebelisoa mananeo a C a nang le thepa le mangolo (sephutheloana sa software se sa lokolloang) joalokaha ho hlalositsoe ka tlase.Tlhahlobo ea lintlha tsa mantlha e kenyelletsa mekhoa e tiileng ea ho sefa ka mekhahlelo e mengata.Ho sefa litaba tse neng li se na tatellano e nepahetseng ea 12mer kenya DNA, lipalo li ile tsa hlophisoa ka tatellano ho qala label (GTGATGTCGGGGATCCGAATTCT), stop label (TAAGCTTGCGGCCGCACTCGAGTA) le background insert (CCCTGCAGGGATATCCCGGAGCTCTCGTCGAC) ho sebelisoa globaled Need.tekatekano e dumellang ho se tsamaellane ho tse 2 ka tolamiso31.Ka hona, ho bala ntle le ho qala le ho emisa li-tag le ho bala tse nang le lintlha tse ka morao, ke hore, li-alignments tse fetang palo e lumelletsoeng ea ho se lumellane, li ile tsa tlosoa laebraring.Ha e le lipalo tse setseng, tatellano ea N-mer DNA e tlohang ho letšoao la ho qala le ho fela pele letšoao la ho emisa le ntšoa ho tloha tatelanong ea pele ea ho bala le ho tsoela pele ho sebetsoa (eo hamorao e tla bitsoa "insert").Ka mor'a ho fetolela ho kenngoa, karolo ka mor'a hore codon ea pele ea ho emisa qetellong ea 5 'ea primer e tlosoe ho kenya.Ho phaella moo, li-nucleotide tse lebisang ho li-codon tse sa fellang qetellong ea 3 'ea primer le tsona li ile tsa tlosoa.Ho qhelela ka thoko tse kentsoeng tse nang le tatellano ea bokamorao feela, tse fetoletsoeng tse qalang ka paterone ea amino acid "PAG" le tsona li ile tsa tlosoa.Li-peptide tse nang le bolelele ba morao-rao ba li-amino acid tse ka tlase ho 3 li ile tsa ntšoa laebraring.Qetellong, tlosa redundancy ka letamo la ho kenya 'me u tsebe hore na ho na le makhetlo a mangata hakae a kenyelletsoeng e' ngoe le e 'ngoe e ikhethang.Liphello tsa tlhahlobo ena li kenyelelitse lethathamo la li-nucleotide tse latellanang (tse kenyang) le maqhubu a tsona (bala) (Lipalo tsa Tlatsetso 1c le 2).
Sehlopha sa N-mer DNA se kenya ka tatellano ea tatellano: Ho felisa liphoso tsa tatellano ea 454 / Roche-tse khethehileng (joalo ka mathata a ho latellana ha li-homopolymer extensions) le ho tlosa li- redundancies tse seng bohlokoa haholo, tse kileng tsa hlophisoa N-mer DNA tatellano ea ho kenya (kenya) e hlophisoa ka ho tšoana.ho kenya (ho fihlela ho 2 metheo e sa lumellaneng e lumelletsoeng) ho sebelisa algorithm e pheta-phetoang e hlalosoang ka tsela e latelang: ho kenngoa ho hlophisoa pele ka makhetlo a bona (ho tloha holimo ho ea tlaase), 'me haeba ho tšoana, ka mefuta ea bona ea bobeli ka bolelele (e telele ho ea ho e khutšoanyane) ).Ka hona, ho kenyelletsa khafetsa le nako e telele ka ho fetisisa ho hlalosa "sehlopha" sa pele.Maqhubu a sehlopha a behiloe ho maqhubu a senotlolo.Joale, kenyelletso e 'ngoe le e' ngoe e setseng lethathamong le hlophisitsoeng e ile ea lekoa ho kenyelletsoa sehlopheng ka ho tsamaisana le Needleman-Wunsch ka bobeli.Haeba palo ea lintho tse sa lumellaneng, tse kentsoeng, kapa tse hlakotsoeng ka ho latellana ha li fete moeli oa 2, ho kenyelletsoa ho kenyelletsoa sehlopheng, 'me palo ea kakaretso ea sehlopha e eketsoa ke hore na ho kenyelletsoa hangata hakae.Maikutlo a kenyellelitsoeng sehlopheng a tšoauoa e le a sebelisitsoeng 'me ha a kenyelletsoe tšebetsong e tsoelang pele.Haeba tatellano ea ho kenya e ke ke ea eketsoa sehlopheng se seng se ntse se le teng, tatellano ea ho kenya e sebelisoa ho theha sehlopha se secha se nang le maqhubu a loketseng a ho kenya le ho tšoauoa joalo ka ha se sebelisitsoe.Poeletso e fela ha tatelano e nngwe le e nngwe ya ho kenya e se e sebediswa ho theha sehlopha se setjha kapa e ka kenyeletswa sehlopheng se seng se ntse se le teng.Ntle le moo, tse kentsoeng ka lihlopha tse nang le li-nucleotide qetellong li fetoleloa ka tatellano ea peptide (lilaebrari tsa peptide).Sephetho sa tlhahlobo ena ke sete sa ho kenya le maqhubu a bona a lumellanang a etsang palo ea lipalo tse latellanang (Supplementary Fig. 2).
Moloko oa Motif: Ho ipapisitsoe le lethathamo la li-peptide tse ikhethang, ho entsoe laeborari e nang le mekhoa eohle e ka bang teng ea amino acid (aa) joalo ka ha ho bonts'itsoe ka tlase.Mokhoa o mong le o mong o ka khonehang oa bolelele ba 3 o ile oa ntšoa ho peptide 'me mokhoa oa eona o fapaneng o ile oa kenyelletsoa hammoho le laebrari e tloaelehileng ea motif e nang le mekhoa eohle (tripeptides).Lilaebrari tse nang le litšupiso tse pheta-phetoang haholo li ile tsa hlophisoa ka tatellano le ho tlosoa ho sa hlokahaleng.Joale, bakeng sa tripeptide e 'ngoe le e 'ngoe e laebraring ea motif, re ne re hlahloba boteng ba eona laebraring re sebelisa lisebelisoa tsa computational.Tabeng ena, makhetlo a mangata a peptide e nang le "motif tripeptide" e fumanoeng e eketsoa 'me e behiloe ho motif laebraring ea motif ("palo ea motifs").Sephetho sa tlhahiso ea motif ke lethathamo la mahlakore a mabeli le nang le liketsahalo tsohle tsa tripeptides (motifs) le boleng ba tsona, e leng palo ea ho bala ka tatellano e fellang ka motif e tsamaellanang ha ho baloa, ho hlophisoa, le ho fetoleloa.Metrics joalo ka ha e hlalositsoe ka botlalo ka holimo.
Ho tloaeleha ha palo ea motifs le li-scatterplots tse tsamaellanang: Palo ea motifs bakeng sa sampole ka 'ngoe e ne e tloaelehile ho sebelisoa.
moo e leng palo ea tse baloang tse nang le sehlooho i.Kahoo, vi e emela palo ea liperesente tse baloang (kapa li-peptide) tse nang le motif i sampole.Litekanyetso tsa P bakeng sa palo e sa tloaelehang ea motifs e baloa ho sebelisoa tlhahlobo e nepahetseng ea Fisher.Mabapi le li-correlograms tsa palo ea sepheo, likhokahano tsa Spearman li ile tsa baloa ho sebelisoa palo e tloaelehileng ea sepheo le R.
Ho bona ka mahlo a kelello litaba tsa li-amino acid sebakeng se seng le se seng sa laebrari ea peptide, li-logograms tsa websaete 32, 33 (http://weblogo.threeplusone.com) li ile tsa bōptjoa.Taba ea pele, litaba tsa li-amino acid sebakeng se seng le se seng sa 12-mer peptide li bolokiloe ka har'a matrix a 20 × 12.Ebe, sehlopha sa li-peptide tse 1000 tse nang le li-amino acid tse lekanang sebakeng se seng le se seng li hlahisoa ka mokhoa oa tatellano ea fasta 'me li fanoa e le kenyelletso ho logo ea marang-rang ea 3, e hlahisang setšoantšo sa setšoantšo sa litaba tsa amino acid sebakeng ka seng.bakeng sa laeborari ea peptide e fanoeng.Ho bona ka mahlo a kelello li-dataset tsa mefuta-futa, limmapa tsa mocheso li entsoe ka sesebelisoa se entsoeng ka hare ho R (biosHeatmap, sephutheloana sa R ​​se seng se tla lokolloa).Li-dendrogram tse hlahisitsoeng 'mapeng oa mocheso li ile tsa baloa ho sebelisoa mokhoa oa Ward oa ho bokellana ka maemo a fapaneng ka metric ea sebaka sa Euclidean.Bakeng sa tlhahlobo ea lipalo-palo ea lintlha tsa lintlha tsa sepheo, litekanyetso tsa P bakeng sa lintlha tse sa tloaelehang li ne li baloa ho sebelisoa tlhahlobo e nepahetseng ea Fisher.Litekanyetso tsa P bakeng sa li-dataset tse ling li baloa ka R ho sebelisoa tlhahlobo ea t ea Seithuti kapa ANOVA.
Li-clone tsa phage tse khethiloeng le phages ntle le ho kenngoa li ne li kenngoa ka methapo ka mothapo oa mohatla (2 × 1010 phages / phoofolo ka 300 μl PBS).Metsotso e leshome pele ho mokelikeli le ho lokisa ka mor'a moo, liphoofolo tse tšoanang li ne li kenngoa ka methapo ea 100 μl ea DyLight594 e ngotsoeng lectin (Vector Laboratories Inc., DL-1177).Metsotso ea 60 ka mor'a ente ea phage, likhoto li ne li tšeloa ka pelo ka 50 ml PBS e lateloa ke 50 ml 4% PFA / PBS.Mehlala ea boko e ile ea boela ea ts'oaroa bosiu bo le bong ho 4% PFA/PBS mme ea kolobisoa ka 30% sucrose bosiu bo le bong ho 4 ° C.Mehlala e hatselitsoe ka har'a motsoako oa OCT.Tlhahlobo ea Immunohistochemical ea lisampole tse hatselitsoeng e entsoe ka mocheso oa kamore ho 30 µm cryosections e koetsoeng ka 1% BSA le ho kenngoa ka li-antibodies tse ngotsoeng ke polyclonal FITC khahlanong le T7 phage (Novus NB 600-376A) ho 4 ° C.E tšele ka bosiu bo le bong.Qetellong, likarolo li ile tsa hlatsuoa ka makhetlo a 3 ka PBS 'me tsa hlahlojoa ka microscope ea laser confocal (Leica TCS SP5).
Li-peptide tsohle tse nang le bonyane bohloeki ba 98% li entsoe ke GenScript USA, biotinylated le lyophilized.Biotin e tlamisoa ka sebaka se eketsehileng sa triple glycine ho N-terminus.Sheba li-peptide tsohle u sebelisa mass spectrometry.
Streptavidin (Sigma S0677) e ne e kopantsoe le ho feta ha 5-fold equimolar ea peptide ea biotinylated, biotinylated BACE1 inhibitory peptide, kapa motsoako (3: 1 ratio) ea biotinylated BACE1 inhibitory peptide le BACE1 inhibitory peptide ho 5-10% ea PBS e nang le PSO.Hora e 1 mocheso oa kamore pele ho ente.Li-peptide tsa Streptavidin-conjugated li ne li kenngoa ka methapo ka tekanyo ea 10 mg / kg ho e 'ngoe ea methapo ea mohatla oa likhoto tse nang le mokokotlo oa boko.
Khatiso ea streptavidin-peptide complexes e ile ea hlahlojoa ke ELISA.Lipoleiti tsa Nunc Maxisorp microtiter (Sigma) li ne li koahetsoe ka bosiu bo le bong ho 4 ° C le 1.5 μg / ml mouse anti-streptavidin antibody (Thermo, MA1-20011).Ka mor'a ho thibela (thibela buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) mocheso oa kamore bakeng sa lihora tsa 2, hlatsoa poleiti ka 0.05% Tween-20 / PBS (hlatsoa buffer) bakeng sa 3 Second, CluSF e kenyelitsoe 0 plasma e fapaneng le plasma1 plasma: 0, CSF 1:115).Joale poleiti e ne e kenngoa ka bosiu bo le bong ho 4 ° C ka ho lemoha antibody (1 μg / ml, anti-streptavidin-HRP, Novus NB120-7239).Ka mor'a mehato e meraro ea ho hlatsoa, ​​​​streptavidin e ile ea fumanoa ka ho kenngoa ka TMB substrate solution (Roche) ho fihlela ho 20 min.Kamora ho emisa nts'etsopele ea mebala ka 1M H2SO4, lekanya ho monya ho 450 nm.
Mosebetsi oa streptavidin-peptide-BACE1 inhibitor complex o ile oa hlahlojoa ke Aβ (1-40) ELISA ho latela protocol ea moetsi (Wako, 294-64701).Ka bokhutšoane, lisampole tsa CSF li ile tsa hlapolloa ka hlapollo e tloaelehileng (1:23) 'me tsa kenngoa bosiu bo le bong ho 4°C ka har'a lipoleiti tsa liliba tse 96 tse tlositsoeng ka antibody ea BNT77.Ka mor'a mehato e mehlano ea ho hlatsoa, ​​​​lesole la 'mele la HRP-conjugated BA27 le ile la kenngoa 'me la kenngoa lihora tse 2 ka 4° C., le lateloa ke mehato e mehlano ea ho hlatsoa.Aβ(1–40) e ile ea fumanoa ka ho kenngoa ka har'a tharollo ea TMB metsotso e 30 mocheso oa kamore.Kamora hore nts'etsopele ea mebala e emisoe ka tharollo ea ho emisa, lekanya monyetla ho 450 nm.Mehlala ea plasma e ne e le tlas'a ts'ebetso e tiileng ea karolo pele ho Aβ (1-40) ELISA.Plasma e ile ea eketsoa ho 0.2% DEA (Sigma) ka lipoleiti tsa liliba tse 96 'me e kenngoa mocheso oa kamore ka metsotso e 30.Ka mor'a ho hlatsoa lipoleiti tsa SPE ka ho latellana (Oasis, 186000679) ka metsi le 100% methanol, lisampole tsa plasma li ile tsa kenngoa lipoleiting tsa SPE 'me metsi 'ohle a tlosoa.Mehlala e ile ea hlatsuoa (pele ka 5% methanol joale 30% methanol) 'me ea hlatsuoa ka 2% NH4OH/90% methanol.Ka mor'a ho omisa li-eluate ho 55 ° C bakeng sa metsotso ea 99 ka nako e sa khaotseng ea N2 hona joale, lisampole li ile tsa fokotsoa ka li-diluents tse tloaelehileng 'me Aβ (1-40) e lekantsoe joalokaha ho hlalositsoe ka holimo.
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