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Umgoqo wegazi nobuchopho kanye nesithiyo segazi-nobuchopho kuvimbela ama-biotherapeutic agents ukuthi afinyelele imigomo yawo ohlelweni lwezinzwa oluphakathi, ngaleyo ndlela avimbele ukwelashwa okuphumelelayo kwezifo zemizwa.Ukuthola izithuthi zobuchopho ezinoveli ku-vivo, sethule umtapo wezincwadi we-T7 phage peptide futhi saqoqa igazi kanye noketshezi lwe-cerebrospinal (CSF) sisebenzisa imodeli yechibi elikhulu elaziwayo lamagundane.Ama-clones athile e-phage acetshiswe kakhulu ku-CSF ngemuva kwemijikelezo emine yokukhetha.Ukuhlolwa kwama-peptide omuntu ngamunye kwembula ukunothisa okuphindwe kayi-1000 ku-CSF.I-bioactivity yokulethwa kwe-peptide-mediated ebuchosheni yaqinisekiswa ngokuncipha okungama-40% kwezinga le-amyloid-β kuketshezi lwe-cerebrospinal kusetshenziswa i-BACE1 peptide inhibitor exhunywe ku-peptide yokuthutha yenoveli ehlonziwe.Le miphumela iphakamisa ukuthi ama-peptide ahlonzwe ngezindlela zokukhetha i-vivo phage angase abe izimoto eziwusizo zokulethwa okuhlelekile kwama-macromolecules ebuchosheni anomphumela wokwelapha.
Ucwaningo lokwelapha oluqondiswe kusistimu yezinzwa emaphakathi (CNS) lugxile kakhulu ekuhlonzeni izidakamizwa ezithuthukisiwe nama-ejenti abonisa izakhiwo eziqondiswe ku-CNS, ngomzamo omncane wokuthola izindlela ezishayela ukulethwa kwezidakamizwa okusebenzayo ebuchosheni.Lokhu sekuqala ukushintsha manje njengoba ukulethwa kwezidakamizwa, ikakhulukazi ama-molecule amakhulu, kuyingxenye ebalulekile yokuthuthukiswa kwezidakamizwa ze-neuroscience.Imvelo yesimiso sezinzwa esimaphakathi ivikelwe kahle uhlelo lwe-cerebrovascular barrier, oluhlanganisa umgoqo wegazi nobuchopho (BBB) ​​kanye nesithiyo sobuchopho begazi (BCBB)1, okwenza kube inselele ukuletha izidakamizwa ebuchosheni1,2.Kulinganiselwa ukuthi cishe zonke izidakamizwa ezinkulu ze-molecule kanye nezidakamizwa ezincane ezingaphezu kwama-98% zikhishwa ebuchosheni3.Yingakho kubaluleke kakhulu ukukhomba izinhlelo ezintsha zokuthutha ubuchopho ezinikeza ukulethwa okuphumelelayo nokuqondile kwemithi yokwelapha ku-CNS 4,5.Kodwa-ke, i-BBB ne-BCSFB nazo ziveza ithuba elihle kakhulu lokulethwa kwezidakamizwa njengoba zingena futhi zingena kuzo zonke izakhiwo zobuchopho ngokusebenzisa imithambo yayo ebanzi.Ngakho-ke, imizamo yamanje yokusebenzisa izindlela ezingezona ezihlaselayo zokulethwa ebuchosheni ngokuyinhloko isekelwe endleleni yokuthutha i-receptor-mediated (PMT) isebenzisa i-BBB6 receptor engapheli.Naphezu kwentuthuko yakamuva ebalulekile kusetshenziswa i-transferrin receptor pathway7,8, ukuthuthukiswa okuqhubekayo kwezinhlelo ezintsha zokulethwa ezinezakhiwo ezithuthukisiwe kuyadingeka.Kuze kube manje, umgomo wethu kwakuwukubona ama-peptide akwazi ukulamula ezokuthutha ze-CSF, njengoba empeleni ayengase asetshenziswe ukuletha ama-macromolecules ku-CNS noma ukuvula izindlela ezintsha zokwamukela.Ikakhulukazi, ama-receptors athile kanye nabathuthi bohlelo lwe-cerebrovascular (BBB kanye ne-BSCFB) bangasebenza njengezinhloso ezingaba khona zokulethwa okusebenzayo nokuqondile kwezidakamizwa ze-biotherapeutic.I-Cerebrospinal fluid (CSF) iwumkhiqizo oyimfihlo we-choroid plexus (CS) futhi ithintana ngqo noketshezi oluphakathi kobuchopho ngesikhala esingaphansi kanye nesikhala se-ventricular4.Muva nje kuboniswe ukuthi i-subarachnoid cerebrospinal fluid ihlakazeka ngokweqile ku-interstitium yobuchopho9.Sithemba ukuthi sizofinyelela isikhala se-parenchymal sisebenzisa leli pheshana lokungena kwe-subarachnoid noma ngokuqondile nge-BBB.Ukufeza lokhu, sisebenzise isu eliqinile lokukhetha i-vivo phage elihlonza kahle ama-peptide ahanjiswa ngenye yalezi zindlela ezimbili ezihlukene.
Manje sesichaza indlela yokuhlola elandelanayo ye-vivo phage enesampula ye-CSF ehambisana nokulandelana kwe-throughput (HTS) ukuqapha imizuliswano yokukhetha yokuqala enokwehluka okuphezulu kakhulu komtapo wolwazi.Ukuhlolwa kwenziwa kumagundane aqaphelayo nge-cannula enkulu ye-cisterna (CM) efakwe unomphela ukuze kugwenywe ukungcoliswa kwegazi.Okubalulekile, le ndlela ikhetha kokubili okuqondiswe ebuchosheni nama-peptide ngomsebenzi wokuthutha kuwo wonke umgoqo wobuchopho.Sisebenzise ama-T7 phages ngenxa yobukhulu bawo obuncane (~60 nm) 10 futhi saphakamisa ukuthi afanele ukuthuthwa kwama-vesicles avumela ukuwela kwe-transcellular kwe-endothelial kanye/noma i-epithelial-medulla barrier.Ngemuva kwemijikelezo emine ye-panning, i-phage populations yahlukaniswa ikhombisa ukuqina kwe-vivo CSF ​​​​enothisa kanye ne-cerebral microvessel association.Okubalulekile, sikwazile ukuqinisekisa lokho esikutholile ngokubonisa ukuthi ama-peptide ekhandidethi akhethwayo futhi ahlanganiswe ngamakhemikhali ayakwazi ukuthutha imithwalo yamaprotheni ku-cerebrospinal fluid.Okokuqala, imiphumela ye-pharmacodynamic ye-CNS yasungulwa ngokuhlanganisa i-peptide yokuhamba ehamba phambili ne-inhibitor ye-BACE1 peptide.Ngokungeziwe ekuboniseni ukuthi amasu okuhlola ukusebenza kwe-vivo angakwazi ukuhlonza ama-peptide amasha okuthutha ubuchopho njengabathwali bempahla yamaprotheni abasebenzayo, silindele izindlela ezifanayo zokukhetha ezisebenzayo ukuthi nazo zibaluleke ekuhlonzeni izindlela ezintsha zokuthutha ubuchopho.
Ngokusekelwe kumayunithi okwenza uqwembe (i-PFU), ngemva kwesinyathelo sokupakisha i-phage, umtapo wezincwadi wama-peptides we-T7 phage angahleliwe we-12-mer enokwehlukahlukana okucishe kube yi-109 waklanywa futhi wadalwa (bheka Izinto Zokusebenza kanye Nezindlela).Kubalulekile ukuqaphela ukuthi sihlaziye ngokucophelela le labhulali ngaphambi kwe-vivo panning.I-PCR yokukhulisa amasampula welabhulali ye-phage kusetshenziswa ama-primer akhiqizwe ama-amplicon ayesebenza ngokuqondile ku-HTS (I-Supplementary Fig. 1a).Ngenxa a) amaphutha okulandelana kwe-HTS11, b) umthelela kukhwalithi yeziqalo (NNK)1-12, kanye c) nokuba khona kohlobo lwasendle (wt) i-phage (ukufakwa kwamathambo) kulabhulali yokulinda, inqubo yokuhlunga ukulandelana yasetshenziswa ukuze kukhishwe ulwazi lokulandelana oluqinisekisiwe kuphela (I-Fig. 1b eyengeziwe).Lezi zinyathelo zokuhlunga zisebenza kuwo wonke amalabhulali alandelanayo e-HTS.Emtapweni wolwazi ojwayelekile, kutholwe isamba esingu-233,868 esifundiwe, lapho u-39% uphumelele umbandela wokuhlunga futhi wasetshenziselwa ukuhlaziya umtapo wolwazi nokukhetha imizuliswano eyalandela (Umfanekiso Owengeziwe 1c–e).Ukufundwa bekuyiziphindaphinda ngokuyinhloko zamapheya esisekelo angu-3 ubude nenani eliphakeme kuma-nucleotides angu-36 (I-Supplementary Fig. 1c), eqinisekisa umklamo welabhulali (NNK) 1-12.Ngokuphawulekayo, cishe u-11% wamalungu omtapo wolwazi aqukethe umgogodla we-PAGISRELVDKL ongumgogodla we-12-dimensional (wt), futhi cishe uhhafu wokulandelana kwawo (49%) wawuqukethe okufakiwe noma okususiwe.I-HTS yomtapo wolwazi iqinisekise ukuhlukahluka okuphezulu kwama-peptide kulabhulali: ngaphezu kuka-81% wokulandelana kwe-peptide kutholwe kanye kuphela futhi u-1.5% kuphela owenzekile kumakhophi angu-≥4 (I-Supplementary Fig. 2a).Amafrikhwensi ama-amino acid (aa) kuzo zonke izikhundla ze-12 ku-repertoire ahlotshaniswa kahle namafrikhwensi alindeleke enanini lamakhodoni akhiqizwe i-repertoire ye-NKK ewohlokayo (I-Supplementary Fig. 2b).Imvamisa ephawuliwe yezinsalela ze-aa ezifakwe yilokhu okufakiwe kuhlotshaniswa kahle nemvamisa ebaliwe (r = 0.893) (I-Supplementary Fig. 2c).Ukulungiswa kwemitapo ye-phage yomjovo kufaka phakathi izinyathelo zokukhulisa nokususwa kwe-endotoxin.Lokhu ngaphambilini kuboniswe ukuthi kungase kunciphise ukuhlukahluka kwemitapo yolwazi yamaphage12,13.Ngakho-ke, silandele umtapo wezincwadi we-plate-amplified phage osususwe i-endotoxin futhi sawuqhathanisa nomtapo wolwazi wangempela ukuze silinganisele imvamisa ye-AA.Ukuxhumana okuqinile (r = 0.995) kubonwe phakathi kwechibi langempela kanye nedamu elikhulisiwe nelihlanjululwe (I-Supplementary Fig. 2d), okubonisa ukuthi ukuncintisana phakathi kwama-clones akhuliswe kumapuleti asebenzisa i-T7 phage akuzange kubangele ukuchema okukhulu.Lokhu kuqhathanisa kusekelwe ezikhathini eziningi ze-tripeptide motifs kulabhulali ngayinye, njengoba ukuhlukahluka kwemitapo yolwazi (~109) akukwazi ukuthwebula ngokugcwele ngisho nange-HTS.Ukuhlaziywa kwemvamisa ye-aa endaweni ngayinye kwembule ukuchema okuncane okuncike endaweni ezindaweni ezintathu zokugcina ze-repertoire efakiwe (I-Supplementary Fig. 2e).Sengiphetha, siphethe ngokuthi ikhwalithi nokwehlukahlukana komtapo wolwazi kwamukelekile futhi izinguquko ezincane kuphela ezinhlobonhlobo eziye zabonwa ngenxa yokukhulisa nokulungiswa kwemitapo yolwazi yefeji phakathi kwemizuliswano embalwa yokukhetha.
Isampula ye-serial cerebrospinal fluid ingenziwa ngokuhlinza i-cannula ku-CM yamagundane aqaphile ukuze kube lula ukuhlonzwa kwe-T7 phage ejovwe nge-intravenously (iv) nge-BBB kanye/noma i-BCSFB (Fig. 1a-b).Sisebenzise izingalo ezimbili zokukhetha ezizimele (izingalo A no-B) emizuliswaneni emithathu yokuqala yokukhetha i-vivo (Fig. 1c).Sikhulise kancane kancane ukuqina kokukhetha ngokunciphisa inani eliphelele le-phage elethulwe emizuliswaneni emithathu yokuqala yokukhetha.Emzuliswaneni wesine wokuphena, sihlanganise amasampula avela egatsheni A no-B futhi senza ukukhetha okuzimele okuthathu okwengeziwe.Ukutadisha izakhiwo ze-vivo zezinhlayiya ze-T7 phage kulo modeli, i-wild-type phage (i-PAGISRELVDKL master insert) ijovwe kumagundane ngomthambo womsila.Ukubuyiselwa kwe-phages kusuka ku-cerebrospinal fluid kanye negazi ngezikhathi ezahlukene kubonise ukuthi i-T7 icosahedral phages encane yayinesigaba sokuqala sokugunyazwa esisheshayo esivela engxenyeni yegazi (I-Supplementary Fig. 3).Ngokusekelwe kuma-titers alawulwayo kanye nevolumu yegazi lamagundane, sibale ukuthi cishe i-1% wt kuphela.I-phage kusuka kumthamo olawulwayo itholwe egazini imizuzu eyi-10 ngemuva kokujova ngomjovo.Ngemva kwalokhu kuncipha okusheshayo kokuqala, imvume eyisisekelo ehamba kancane ikalwa ngesigamu sempilo yemizuzu engama-27.7.Okubalulekile, ama-phage ambalwa kakhulu kuphela abuyiselwe endaweni ye-CSF, ebonisa isizinda esiphansi sokufuduka kwe-phage yohlobo lwasendle endaweni ye-CSF (I-Supplementary Fig. 3).Ngokwesilinganiso, kuphela i-1 x 10-3% titer ye-T7 phage egazini kanye ne-4 x 10-8% yama-phages afakwe ekuqaleni atholwe ku-cerebrospinal fluid phakathi nayo yonke inkathi yesampula (0-250 min).Ngokuphawulekayo, uhhafu wempilo (imizuzu engu-25.7) ye-phage yohlobo lwasendle ku-cerebrospinal fluid yayifana naleyo ebonwa egazini.Le datha ibonisa ukuthi umgoqo ohlukanisa indawo ye-CSF egazini uphelele kumagundane e-CM-cannulated, okuvumela ukukhethwa kwe-vivo kwemitapo yolwazi ye-phage ukukhomba ama-clones ahanjiswa kalula esuka egazini aye egumbini le-CSF.
(a) Ukusetha indlela yokuphinda kusampulalwe uketshezi lwe-cerebrospinal (CSF) kusuka echibini elikhulu.(a)(c) Ishadi lokugeleza lokuhlola le-vivo phage.Emzuliswaneni ngamunye wokukhetha, ama-phage (izihlonzi zezilwane ngaphakathi kwemicibisholo) ajovwa ngomthambo.Amagatsha amabili ahlukile azimele (A, B) agcinwa ngokuhlukana kuze kube umzuliswano wesi-4 wokukhetha.Ngomjikelezo wokukhetha wesi-3 nowesi-4, i-clone yephage ngayinye ekhishwe ku-CSF yahlelwa ngesandla.(d) I-Kinetics ye-phage ehlukanisiwe negazi (imibuthano ebomvu) kanye noketshezi lwe-cerebrospinal (onxantathu abaluhlaza) phakathi nomzuliswano wokuqala wokukhetha kumagundane amabili afakwe amathini ngemva kokujova ngomjovo womtapo wezincwadi we-T7 peptide (2 x 1012 phages/isilwane).Izikwele eziluhlaza okwesibhakabhaka zibonisa isilinganiso sokuqala sokuhlushwa kwe-phage egazini, kubalwa inani le-phage elijovwe, kucatshangelwa inani legazi eliphelele.Izikwele ezimnyama zibonisa iphuzu lokuphambana komugqa ongu-y okhishwe ekugxilweni kwephage yegazi.(e,f) Yethula imvamisa ehlobene nokusabalalisa kwawo wonke ama-tripeptide motifs okungenzeka agqagqene atholakala ku-peptide.Inani le-motifs elitholakala ekufundweni kwe-1000 liyaboniswa.Ngokuphawulekayo (p <0.001) ama-motif anothisiwe amakwe ngamachashazi abomvu.(e) I-Correlation scatterplot eqhathanisa imvamisa ehlobene ye-tripeptide motif yomtapo wolwazi ojovwe nephage ethathwe egazini ezilwaneni #1.1 kanye ne-#1.2.(f) I-Correlation scatterplot eqhathanisa amafrikhwensi ahlobene e-animal phage tripeptide motifs #1.1 kanye ne-#1.2 ehlukene egazini kanye noketshezi lwe-cerebrospinal.(g, h) Ukumelwa kwe-ID yokulandelana kwephage elinothiswe egazini (g) uma kuqhathaniswa nemitapo yolwazi ejovwe kanye nephage elinothiswe ku-CSF (h) uma kuqhathaniswa negazi ngemva komjikelezo wokukhethwa kwe-vivo kuzo zombili izilwane.Usayizi wekhodi yohlamvu olulodwa ubonisa ukuthi leyo amino acid yenzeka kaningi kangakanani kuleso simo.Okuluhlaza = i-polar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acids.Umfanekiso 1a, b waklanywa futhi wakhiqizwa ngu-Eduard Urich.
Sijove umtapo wezincwadi we-phage peptide kumagundane ensimbi amabili e-CM (i-clades A no-B) kanye ne-phage ehlukanisiwe ku-cerebrospinal fluid kanye negazi (Umfanekiso 1d).Ukususwa ngokushesha komtapo wolwazi kwakungacaci kangako uma kuqhathaniswa ne-fage yohlobo lwasendle.Isilinganiso sempilo yesigamu selabhulali ejovwe kuzo zombili izilwane kwakuyimizuzu engu-24.8 egazini, efana ne-wild-type phage, kanye nemizuzu engu-38.5 ku-CSF.Amasampula egazi kanye ne-cerebrospinal fluid phage esilwaneni ngasinye afakwa ngaphansi kwe-HTS futhi wonke ama-peptide ahlonziwe ahlaziywa ukuba khona kwe-tripeptide motif emfushane.I-Tripeptide motifs yakhethwa ngoba ihlinzeka ngesisekelo esincane sokwakheka kwesakhiwo kanye nokusebenzisana kwe-peptide-protein14,15.Sithole ukuhlobana okuhle ekusabalaliseni ama-motifs phakathi komtapo wezincwadi we-phage ojojowe kanye nama-clones akhishwe egazini lezilwane zombili (Fig. 1e).Imininingwane ikhombisa ukuthi ukwakheka komtapo wezincwadi kucetshiswe kancane kuphela endaweni yegazi.Amafrikhwensi e-amino acid nokulandelana kokuvumelana kwabuye kwahlaziywa endaweni ngayinye kusetshenziswa ukuguqulwa kwesoftware yeWeblogo16.Ngokuthakazelisayo, sithole ukucebisa okunamandla ezinsalela zegazi ze-glycine (Fig. 1g).Lapho igazi liqhathaniswa nama-clones akhethwe ku-CSF, ukukhethwa okuqinile kanye nokungakhethi okuthile kwe-motifs kwabonwa (Umfanekiso we-1f), futhi ama-amino acid athile ayekhona ngokukhethayo ezikhundleni ezinqunywe kusengaphambili ku-12-ilungu (Fig. 1h).Ngokuphawulekayo, izilwane ngazinye zihluke kakhulu ku-cerebrospinal fluid, kanti ukucebisa kwe-glycine yegazi kwabonwa kuzo zombili izilwane (I-Supplementary Fig. 4a-j).Ngemva kokuhlunga okuqinile kwedatha yokulandelana ku-cerebrospinal fluid yezilwane #1.1 kanye ne-#1.2, isamba esingu-964 kanye ne-420 eyingqayizivele ye-12-mer peptide yatholwa (I-Supplementary Fig. 1d-e).Ama-clone e-phage ahlukanisiwe akhulisiwe futhi angaphansi komzuliswano wesibili wokukhethwa kwe-vivo.I-Phage ekhishwe emzuliswaneni wesibili wokukhetha yayingaphansi kwe-HTS esilwaneni ngasinye futhi wonke ama-peptide ahlonziwe asetshenziswa njengokufakwa kuhlelo lokuqashelwa kwe-motif ukuze kuhlaziywe ukuvela kwe-tripeptide motifs (Fig. 2a, b, ef).Uma kuqhathaniswa nomjikelezo wokuqala we-phage etholwe ku-CSF, sabona ukukhethwa okuqhubekayo nokungakhethwa kwama-motifs amaningi ku-CSF emagatsheni A no-B (Fig. 2).Kusetshenziswe i-algorithm yokuhlonza inethiwekhi ukuze kunqunywe ukuthi imele amaphethini ahlukene okulandelana okungaguquki.Ukufana okucacile kwabonwa phakathi kokulandelana kwe-12-dimensional okutholwe yi-CSF kwenye i-clade A (Fig. 2c, d) kanye ne-clade B (Fig. 2g, h).Ukuhlaziywa okuhlanganisiwe egatsheni ngalinye kwembule amaphrofayili ahlukene okukhetha ama-peptide angu-12-mer (I-Supplementary Fig. 5c,d) kanye nokwenyuka kwesilinganiso se-CSF/blood titer ratio ngokuhamba kwesikhathi kuma-clones ahlanganisiwe ngemva komjikelezo wesibili wokukhetha uma kuqhathaniswa nomzuliswano wokuqala wokukhetha (I-Supplementary Fig. 5e).).
Ukucebisa ama-motifs nama-peptides ku-cerebrospinal fluid ngemijikelezo emibili elandelanayo yokukhetha isibonisi sephage esisebenzayo se-vivo.
Wonke ama-cerebrospinal fluid phage alulame emzuliswaneni wokuqala wesilwane ngasinye (izilwane #1.1 kanye ne-#1.2) zahlanganiswa, zakhuliswa, zahlelwa nge-HT futhi zaphinde zafakwa ndawonye (2 x 1010 phages/isilwane) amagundane angama-2 SM (#1.1 → #).2.1 kanye no-2.2, 1.2 → 2.3 kanye no-2.4).(a,b,e,f) I-Correlation scatterplots eqhathanisa imvamisa ehlobene ye-tripeptide motifs yawo wonke ama-phage asuselwa ku-CSF emzuliswaneni wokuqala nowesibili wokukhetha.Imvamisa ehlobene nokusatshalaliswa kwama-motifs amele wonke ama-tripeptide agqagqene atholakala kuma-peptide kuzo zombili izindlela.Inani le-motifs elitholakala ekufundweni kwe-1000 liyaboniswa.Ama-Motif aye akhethwa kakhulu (p <0.001) noma angafakwanga kwenye yelabhulali eqhathanisiwe agqanyiswe ngamachashazi abomvu.(c, d, g, h) Hlela ukumelwa kwelogo yakho konke ukulandelana okude kwe-amino acid eyi-CSF ecebile engu-12 ngokusekelwe kumjikelezo 2 no-1 wokukhethwa kwe-vivo.Usayizi wekhodi yohlamvu olulodwa ubonisa ukuthi leyo amino acid yenzeka kaningi kangakanani kuleso simo.Ukumela ilogo, imvamisa yokulandelana kwe-CSF ekhishwe esilwaneni ngasinye phakathi kwemizuliswano emibili yokukhetha iyaqhathaniswa futhi ukulandelana okunothisiwe emzuliswaneni wesibili kuyaboniswa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 kanye (h) #1.2–#2.4.Ama-amino acid acetshiswe kakhulu endaweni enikeziwe ku-(c, d) izilwane no.2.1 kanye no.2.2 noma (g, h) ezilwaneni no.2.3 futhi no.2.4 kukhonjiswa ngombala.Okuluhlaza = i-polar, purple = neutral, blue = basic, red = acidic and black = hydrophobic amino acids.
Ngemuva komzuliswano wesithathu wokukhetha, sihlonze ukulandelana kwe-peptide eyingqayizivele ye-124 (#3.1 kanye ne-#3.2) kusuka kuma-clone we-phage akhiwe kabusha we-332 CSF ahlukaniswe nezilwane ezimbili (I-Supplementary Fig. 6a).Ukulandelana kwe-LGSVS (18.7%) kube nengxenye ephezulu kakhulu ehlobene, elandelwa ukufakwa kohlobo lwasendle PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), kanye ne-SARGSWREIVSLS ( 2.2%).Emzuliswaneni wesine wokugcina, sihlanganise amagatsha amabili akhethwe ngokuzimela ezilwaneni ezintathu ezihlukene (Fig. 1c).Kuma-clones we-phage alandelanayo we-925 atholwe ku-CSF, emzuliswaneni wesine sathola ukulandelana kwe-peptide eyingqayizivele ye-64 (I-Supplementary Fig. 6b), phakathi kwayo ingxenye ehlobene ye-phage yohlobo lwasendle yehla ku-0.8%.Ama-clone e-CSF ajwayeleke kakhulu emzuliswaneni wesine kube yi-LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) kanye ne-RLSSVDSDLSGC (3, 2%).%)).Ubude bebanga lama-peptide akhethiwe bungenxa yokufakwa/ukususwa kwe-nucleotide noma amakhodoni okumisa ngaphambi kwesikhathi kuziqalisi zomtapo wolwazi lapho kusetshenziswa amakhodoni awohlokayo ekwakhiweni komtapo wolwazi we-NNK.Amakhodoni okumisa ngaphambi kwesikhathi akhiqiza ama-peptide amafushane futhi akhethwa ngoba aqukethe i-aa motif evumayo.Ama-peptide amade angase abe umphumela wokufakwa/ukususwa kuzingqalasizinda zamalabhulali okwenziwa.Lokhu kubeka ikhodon yokumisa edizayinelwe ngaphandle kohlaka futhi iyifunda kuze kube yilapho kuvela ikhodon entsha yesitobhi phansi komfula.Ngokuvamile, sibale izici zokucebisa zayo yonke imizuliswano emine yokukhetha ngokuqhathanisa idatha yokufaka nedatha yesampula yokuphumayo.Emzuliswaneni wokuqala wokuhlola, sisebenzise iziqu ze-phage zohlobo lwasendle njengereferensi yangemuva engaqondile.Kuyathakazelisa ukuthi ukukhethwa kwe-phage okungalungile kwakunamandla kakhulu kumjikelezo wokuqala we-CSF, kodwa hhayi egazini (Fig. 3a), okungenzeka kube ngenxa yamathuba aphansi okusabalalisa okungahambi kahle kwamalungu amaningi omtapo we-peptide engxenyeni ye-CSF noma ama-phages ahlobene avame ukugcinwa ngokuphumelelayo noma asuswe egazini kune-bacteriophages.Kodwa-ke, emzuliswaneni wesibili we-panning, ukukhethwa okunamandla kwama-phages ku-CSF kwabonwa kuzo zombili izigaba, okuphakamisa ukuthi umjikelezo wangaphambili wawucetshiswe kuma-phages abonisa ama-peptide akhuthaza ukutholwa kwe-CSF (Fig. 3a).Futhi, ngaphandle kokucebisa igazi okubalulekile.Futhi emzuliswaneni wesithathu nowesine, ama-clone e-phage acetshiswa kakhulu ku-CSF.Uma siqhathanisa imvamisa ehlobene yokulandelana kwe-peptide ngayinye eyingqayizivele phakathi kwemizuliswano emibili yokugcina yokukhetha, sithole ukuthi ukulandelana kwacetshiswa nakakhulu emzuliswaneni wesine wokukhetha (Fig. 3b).Ingqikithi yama-tripeptide motifs angu-931 yakhishwa kuwo wonke ama-peptide ayingqayizivele alandelanayo angu-64 kusetshenziswa kokubili ukuqondiswa kwe-peptide.Ama-motif anothiswe kakhulu emzuliswaneni wesine ahlolisiswa kabanzi mayelana namaphrofayili azo okunothisa kuyo yonke imizuliswano uma kuqhathaniswa nomtapo wolwazi ojovwe (okunqunyiwe: ukunothisa okungu-10%) (I-Supplementary Fig. 6c).Amaphethini ajwayelekile okukhethwa abonise ukuthi iningi lezisusa ezihlolisisiwe zacetshiswa kuyo yonke imizuliswano edlule yawo womabili amagatsha okukhetha.Nokho, ezinye izihlokwana (isb. i-SGL, i-VSG, i-LGS GSV) zaziphuma kakhulu ku-clade A, kuyilapho ezinye (isb. i-FGW, i-RTN, i-WGF, i-NTR) yanothiswa kwenye indawo engu-B.
Ukuqinisekiswa kokuthuthwa kwe-CSF kwe-CSF-enothiswe i-phage-displayed peptide kanye nama-peptide omholi we-biotinylated ahlanganiswe nemithwalo yokukhokha ye-streptavidin.
(a) Izilinganiso zokunothisa ezibalwe kuyo yonke imizuliswano emine (R1-R4) ngokusekelwe kumathitha ajovwe (okufakwayo = I) kwephage (PFU) kanye neziqu zephage ze-CSF (okukhiphayo = O).Izici zokucebisa emizuliswaneni emithathu yokugcina (R2-R4) zibalwe ngokuqhathanisa nomzuliswano odlule kanye nomzuliswano wokuqala (R1) nedatha yesisindo.Imigoqo evulekile iwuketshezi lwe-cerebrospinal, imigoqo enomthunzi i-plasma.(***p<0.001, ngokusekelwe kusivivinyo sika-t soMfundi).(b) Uhlu lwama-phage peptide oluningi kakhulu, olubalwa ngokulingana kwawo wonke ama-phage aqoqwe ku-CSF ngemva komjikelezo wesi-4 wokukhethwa.Ama-clone e-phage ayisithupha avame ukugqanyiswa ngombala, anezinombolo kanye nezici zawo zokucebisa phakathi komzuliswano wesi-3 nowesi-4 wokukhetha (izithonjana).(c,d) Ama-clone e-phage acetshiswe kakhulu, ama-phage angenalutho kanye nemitapo yolwazi ye-phage peptide evela kumjikelezo wesi-4 ahlaziywa ngawodwana kumodeli yesampula ye-CSF.I-CSF kanye namasampula egazi aqoqwe ngesikhathi esibonisiwe.(c) Inani elilinganayo lamaphage amaphage angu-6 (2 x 1010 amaphage/izilwane), amaphage angenalutho (#1779) (2 x 1010 amaphage/izilwane) kanye nemitapo yolwazi ye-stock phage peptide (2 x 1012 phage/izilwane) Jova okungenani u-3 CM ujova isilwane ngokuhlukene ngekani.I-pharmacokinetics ye-CSF ye-phage clone ngayinye ejovwe kanye nomtapo wezincwadi we-phage peptide ngokuhamba kwesikhathi ikhonjisiwe.(d) ikhombisa isilinganiso se-CSF/isilinganiso segazi sawo wonke ama-phages/mL atholiwe ngesikhathi sokusampula.(e) Ama-peptide aphambili okwenziwa amane kanye nokulawula okukodwa okuklwetshiwe kwaxhunyaniswa ne-biotin ku-streptavidin ngokusebenzisa i-N-terminus (isibonisi se-tetramer) elandelwa umjovo (i-tail vein iv, 10 mg streptavidin/kg).Okungenani amagundane amathathu afakwe ngaphakathi (N = 3).).Amasampula e-CSF aqoqwe kumaphoyinti esikhathi abonisiwe futhi ukugxila kwe-streptavidin kukalwa yi-CSF anti-streptavidin ELISA (nd = ayitholakali).(*p<0.05, **p<0.01, ***p<0.001, ngokusekelwe ekuhlolweni kwe-ANOVA).(f) Ukuqhathaniswa kokulandelana kwe-amino acid ye-phage peptide clone #2002 (onsomi) namanye ama-clone akhethiwe we-phage peptide kusukela kumzuliswano wesi-4 wokukhetha.Izingcezwana ze-amino acid ezifanayo nezifanayo zinekhodi yombala.
Kuwo wonke ama-phage athuthukisiwe emzuliswaneni wesine (Fig. 3b), ama-clones ayisithupha akhethiwe ukuze kuqhutshekwe nokuhlaziywa komuntu ngamunye kumodeli yesampula ye-CSF.Amanani alinganayo e-phage yamakhandidethi ayisithupha, i-phage engenalutho (akukho ukufaka) kanye nemitapo yolwazi ye-prophage peptide yafakwa ezilwaneni ezintathu ze-CM ezinama-cannulated, futhi i-pharmacokinetics yanqunywa ku-CSF (Fig. 3c) kanye negazi (I-Supplementary Fig. 7) ukuhlolwa.Wonke ama-clone e-phage ahlolwe aqondise indawo ye-CSF ezingeni eliphindwe izikhathi ezingu-10-1000 kunalelo lephage elingenalutho lokulawula (#1779).Isibonelo, ama-clones #2020 kanye ne-#2077 aneziqu ze-CSF eziphindwe izikhathi eziyi-1000 kunephage yokulawula.Iphrofayili ye-pharmacokinetic ye-peptide ngayinye ekhethiwe ihlukile, kodwa wonke anekhono eliphezulu le-CSF homing.Sibone ukwehla okuqhubekayo ngokuhamba kwesikhathi kwama-clones #1903 kanye no-#2011, kuyilapho kuma-clones angu-#2077, #2002 kanye no-#2009 ukwenyuka phakathi nemizuzu eyi-10 yokuqala kungase kubonise ukuthutha okusebenzayo kodwa kudinga ukuqinisekiswa.Ama-Clones #2020, #2002, kanye ne-#2077 azinza emazingeni aphezulu, kuyilapho ukugxila kwe-CSF ye-clone #2009 yehla kancane ngemva kokunyuka kokuqala.Sabe sesiqhathanisa imvamisa ehlobene yekhandidethi ngayinye ye-CSF ne-concentration yayo yegazi (Fig. 3d).Ukuhlotshaniswa kwenani eliphakathi kwekhandidethi ngalinye le-CSF negama legazi ngazo zonke izikhathi zesampula kubonise ukuthi abathathu kwabayisithupha abakhethiwe bacetshiswe kakhulu egazini le-CSF.Kuyathakazelisa ukuthi i-clone #2077 ibonise ukuzinza okuphezulu kwegazi (Umfanekiso Ongeziwe 7).Ukuqinisekisa ukuthi ama-peptide ngokwawo ayakwazi ukuthutha impahla ngaphandle kwezinhlayiya zephage endaweni ye-CSF, sihlanganise ama-peptide abaholi amane aphuma ku-biotin ku-N-terminus lapho ama-peptide anamathela ezinhlayiyeni zephage.Ama-peptides e-Biotinylated (nos. 2002, 2009, 2020 and 2077) ahlanganiswa ne-streptavidin (SA) ukuze athole amafomu e-multimeric alingisa i-phage geometry.Le fomethi iphinde yasivumela ukuthi silinganise ukuchayeka kwe-SA egazini kanye noketshezi lwe-cerebrospinal njengama-peptide amaprotheni athwala impahla.Okubalulekile, idatha ye-phage yayingase iphinde ikhiqizwe lapho ama-peptide okwenziwa asetshenziswa kule fomethi ye-SA-conjugated (Fig. 3e).Ama-peptide ahlahliwe abe nokuchayeka okuncane kokuqala kanye nokuvunyelwa kwe-CSF ngokushesha ngamazinga angabonakali phakathi namahora angama-48.Ukuze sithole ukuqonda emigwaqweni yokulethwa kwalawa ma-clones e-peptide phage esikhaleni se-CSF, sihlaziye ukutholakala kwe-phage peptide hits kusetshenziswa i-immunohistochemistry (IHC) ukuthola ngokuqondile izinhlayiya ze-phage ihora le-1 ngemuva komjovo we-intravenous in vivo.Ngokuphawulekayo, ama-clones #2002, #2077, kanye ne-#2009 angatholwa ngokufaka amabala aqinile kuma-capillaries obuchopho, kuyilapho i-phage yokulawula (#1779) kanye ne-clone #2020 ayizange ibonwe (Umfanekiso Owengeziwe 8).Lokhu kusikisela ukuthi lawa ma-peptide aneqhaza emthelela ebuchosheni ngokuqondile ngokuwela i-BBB.Ukuhlaziywa okunemininingwane eyengeziwe kuyadingeka ukuhlola le nkolelo-mbono, njengoba umzila we-BSCFB ungase uhileleke.Lapho kuqhathaniswa ukulandelana kwe-amino acid ye-clone ecetshiswe kakhulu (#2002) namanye ama-peptide akhethiwe, kwaphawulwa ukuthi amanye awo anezandiso ze-amino acid ezifanayo, ezingase zibonise indlela yokuthutha efanayo (Fig. 3f).
Ngenxa yephrofayili yayo eyingqayizivele ye-plasma kanye nokwanda okuphawulekayo kwe-CSF ngokuhamba kwesikhathi, i-clone yokubonisa i-phage #2077 yabuye yahlolisiswa esikhathini eside samahora angu-48 futhi yakwazi ukukhiqiza kabusha ukwanda okusheshayo kwe-CSF ebonwe ngokubambisana namazinga e-SA aqhubekayo (Fig. 4a).Ngokuphathelene namanye ama-clone e-phage ahlonziwe, i-#2077 yangcoliswa kakhulu ama-capillaries obuchopho futhi yabonisa ukuhlangana okubalulekile kwe-capillary marker lectin lapho ibhekwa ngokulungiswa okuphezulu futhi ngokunokwenzeka namabala athile endaweni ye-parenchymal (Umfanekiso 4b).Ukuphenya ukuthi ingabe imiphumela yemithi ye-peptide-mediated ingatholakala ku-CNS, senze ukuhlola lapho izinguqulo ze-biotinylated ze-i) i-peptide yokuthutha engu-#2077 kanye nee) i-BACE1 inhibitor peptide zixutshwe ne-SA ngezilinganiso ezimbili ezihlukene.Enhlanganisela eyodwa sasebenzisa kuphela i-BACE1 peptide inhibitor kanti kwenye sasebenzisa isilinganiso esingu-1:3 se-BACE1 peptide inhibitor kuya ku-#2077 peptide.Womabili amasampuli alawulwa ngomthambo wegazi futhi amazinga egazi ne-cerebrospinal fluid we-beta-amyloid peptide 40 (Abeta40) akalwa ngokuhamba kwesikhathi.I-Abeta40 ikalwe ku-CSF njengoba ikhombisa ukuvimbela kwe-BACE1 ku-parenchyma yobuchopho.Njengoba kulindelekile, zombili izinkimbinkimbi zanciphisa kakhulu amazinga egazi e-Abeta40 (Fig. 4c, d).Kodwa-ke, amasampula kuphela aqukethe ingxube ye-peptide no.2077 kanye ne-inhibitor ye-BACE1 peptide conjugated to SA kubangele ukwehla okukhulu ku-Abeta40 ku-cerebrospinal fluid (Fig. 4c).Idatha ibonisa ukuthi i-peptide no.I-2077 iyakwazi ukuthutha iphrotheni engu-60 kDa SA iyise ku-CNS futhi iphinde idale imiphumela yezemithi ngama-SA-conjugated inhibitors e-BACE1 peptide.
(a) Umjovo we-Clonal (2 × 10 phage/isilwane) we-T7 phage obonisa amaphrofayili e-pharmacokinetic wesikhathi eside we-CSF peptide #2077 (RLSSVDSDLSGC) kanye nephage yokulawula engajovwe (#1779) okungenani amagundane amathathu e-CM-intubated.(b) Isithombe se-Confocal microscopic se-cortical microvessels emele amagundane ajovwe nge-phage (2 × 10 10 phages/isilwane) esibonisa ukuphikiswa kwe-peptide #2077 kanye nemikhumbi (lectin).Lawa ma-clones we-phage anikezwe amagundane angu-3 futhi avunyelwe ukujikeleza ihora le-1 ngaphambi kokugcotshwa.Ubuchopho bahlukaniswa futhi bangcoliswa amasosha omzimba abhalwe nge-polyclonal FITC ngokumelene ne-T7 phage capsid.Imizuzu eyishumi ngaphambi kokugcotshwa kanye nokulungiswa okulandelayo, i-lectin ebhalwe i-DyLight594 yayifakwa ngomthambo.Izithombe ezikhanyayo ezibonisa i-lectin staining (ebomvu) yohlangothi olukhanyayo lwama-microvessels nama-phages (oluhlaza) kulume lwama-capillaries kanye nezicubu zobuchopho ze-perivascular.Ibha yesikali ihambisana no-10 µm.(c, d) I-Biotinylated BACE1 i-peptide yokuvimbela inhibitory iyodwa noma ihlanganiswe ne-biotinylated transit peptide #2077 yahlanganiswa ne-streptavidin elandelwa umjovo ofakwa emthanjeni okungenani wamagundane e-CM e-cannulated (10 mg streptavidin/kg).Ukwehliswa kwe-BACE1 peptide inhibitor-mediated ku-Aβ40 kukalwe nge-Aβ1-40 ELISA egazini (bomvu) kanye noketshezi lwe-cerebrospinal (owolintshi) ngezikhathi ezibonisiwe.Ukuze kucace kangcono, umugqa wamachashazi udwetshwa kugrafu ngesilinganiso esingu-100%.(c) Ukuncipha kwamaphesenti ku-Aβ40 egazini (onxantathu ababomvu) noketshezi lwe-cerebrospinal (onxantathu abawolintshi) kumagundane aphathwa nge-streptavidin ehlanganiswe ukuze ihambe i-peptide #2077 kanye ne-BACE1 evimbela i-peptide ngesilinganiso esingu-3:1.(d) Ukuncipha kwamaphesenti egazi i-Aβ40 (imibuthano ebomvu) kanye noketshezi lwe-cerebrospinal (imibuthano ewolintshi) yamagundane aphathwa nge-streptavidin ehlanganiswe ne-BACE1 evimbela i-peptide kuphela.Ukugxila kwe-Aβ ekulawuleni kwakungu-420 pg/ml (ukuchezuka okujwayelekile = 101 pg/ml).
Isibonisi se-Phage sisetshenziswe ngempumelelo ezindaweni ezimbalwa zocwaningo lwe-biomedical17.Le ndlela isetshenziselwe izifundo ze-vivo vascular diversity18,19 kanye nezifundo eziqondise imikhumbi yobuchopho20,21,22,23,24,25,26.Kulolu cwaningo, sandise ukusetshenziswa kwale ndlela yokukhetha hhayi kuphela ekuhlonzweni okuqondile kwama-peptide aqondise imikhumbi yobuchopho, kodwa futhi ekutholakaleni kwamakhandidethi anezindawo zokuthutha ezisebenzayo ukuwela umgoqo wegazi-ubuchopho.Manje sichaza ukuthuthukiswa kwenqubo yokukhetha ku-vivo kumagundane afakwe ngaphakathi kwe-CM futhi sibonisa amandla ayo okuhlonza ama-peptide anezakhiwo ze-CSF homing.Sisebenzisa i-T7 phage ebonisa umtapo wezincwadi wama-peptide angahleliwe angu-12-mer, sikwazile ukukhombisa ukuthi i-T7 phage incane ngokwanele (cishe i-60 nm ububanzi) i-10 ukuze ijwayelane nomgoqo wegazi-yobuchopho, ngaleyo ndlela yeqe ngokuqondile umgoqo wegazi-ubuchopho noma i-choroid plexus.Siqaphele ukuthi ukuvunwa kwe-CSF kumagundane e-CM ekheniwe kwakuyindlela elawulwa kahle yokuhlola ukusebenza kwe-vivo, nokuthi i-phage ekhishiwe ayiboshiwe kuphela ku-vasculature kodwa futhi yayisebenza njengesithuthi ngaphesheya komgoqo wegazi-ubuchopho.Ngaphezu kwalokho, ngokuqoqa igazi ngasikhathi sinye nokusebenzisa i-HTS ku-CSF nasemaphashini athathwe egazini, saqinisekisa ukuthi ukukhetha kwethu i-CSF akuzange kuthonywe ukunothiswa kwegazi noma ukulungela ukwanda phakathi kwemizuliswano yokukhetha.Kodwa-ke, ingxenye yegazi iyingxenye yenqubo yokukhetha, njengoba ama-phage akwazi ukufinyelela ikhomishana ye-CSF kufanele aphile futhi ajikeleze egazini isikhathi eside ngokwanele ukuze azicebise ebuchosheni.Ukuze sikhiphe ulwazi lokulandelana olunokwethenjelwa kudatha ye-HTS eluhlaza, sisebenzise izihlungi eziguqulelwe kumaphutha okulandelanisa aqondene nenkundla ekugelezeni komsebenzi wokuhlaziya.Ngokuhlanganisa amapharamitha we-kinetic endleleni yokuhlola, siqinisekise i-pharmacokinetics esheshayo ye-wild-type T7 phages (t½ ~ 28 min) egazini24, 27, 28 futhi sanquma uhhafu wempilo yabo ku-cerebrospinal fluid (t½ ~ 26 min) ngomzuzu).Naphezu kwamaphrofayili afanayo e-pharmacokinetic egazini ne-CSF, kuphela i-0.001% ye-phage yegazi engatholakala ku-CSF, ekhombisa ukuhamba okuphansi kwesizinda se-wild-type T7 phage kuwo wonke umgoqo wegazi-ubuchopho.Lo msebenzi ugqamisa ukubaluleka komzuliswano wokuqala wokukhetha lapho usebenzisa amasu e-vivo panning, ikakhulukazi kumasistimu e-phage asulwa ngokushesha ekujikelezeni, njengoba ama-clone ambalwa akwazi ukufinyelela indawo ye-CNS.Ngakho, emzuliswaneni wokuqala, ukuncishiswa kokuhlukahluka komtapo wolwazi bekukukhulu kakhulu, njengoba inani elilinganiselwe kuphela lama-clones ekugcineni laqoqwa kule modeli ye-CSF eqinile.Lelisu le-vivo panning lalihlanganisa izinyathelo ezimbalwa zokukhetha ezifana nokuqoqwa okusebenzayo endaweni ye-CSF, ukusinda kwe-clone endaweni yegazi, nokususwa ngokushesha kwama-clone e-T7 phage egazini phakathi nemizuzu yokuqala ye-10 (Umfanekiso we-1d kanye ne-Supplementary Fig. 4M).).Ngakho-ke, ngemva komzuliswano wokuqala, ama-clone e-phage ahlukene ahlonzwa ku-CSF, nakuba ichibi elifanayo lokuqala lalisetshenziselwa izilwane ngazinye.Lokhu kuphakamisa ukuthi izinyathelo eziningi zokukhetha eziqinile zamalabhulali emithombo anezinombolo ezinkulu zamalungu omtapo wolwazi ziholela ekwehleni okukhulu kokuhlukahluka.Ngakho-ke, imicimbi engahleliwe izoba yingxenye ebalulekile yenqubo yokukhetha yokuqala, ibe nomthelela omkhulu kumphumela.Kungenzeka ukuthi ama-clones amaningi kumtapo wolwazi wokuqala abe nokuthambekela kokucebisa kwe-CSF okufanayo.Kodwa-ke, ngisho nangaphansi kwezimo ezifanayo zokuhlola, imiphumela yokukhetha ingase yehluke ngenxa yenani elincane le-clone ngayinye endaweni yokubhukuda yokuqala.
I-motifs enothisiwe ku-CSF ihlukile kulezo ezisegazini.Kuyathakazelisa ukuthi siphawule ushintsho lokuqala oluya kuma-peptide anothe nge-glycine egazini lesilwane ngasinye.(I-Fig. 1g, Amakhiwane engeziwe. 4e, 4f).I-Phage equkethe i-glycine peptides ingase izinze futhi mancane amathuba okuthi ikhishwe ekujikelezeni.Kodwa-ke, lawa ma-peptide anothe nge-glycine awazange abonwe kumasampula oketshezi lwe-cerebrospinal, okuphakamisa ukuthi imitapo yolwazi ekhethiwe idlule ezinyathelweni ezimbili ezihlukene zokukhetha: eyodwa esegazini kanti enye ivunyelwe ukunqwabelana oketshezini lwe-cerebrospinal.Ama-clones anothiswe yi-CSF ngenxa yomzuliswano wesine wokukhetha ahlolwe kakhulu.Cishe wonke ama-clone ahlolwe ngawodwana aqinisekiswa ukuthi anothisiwe ku-CSF uma kuqhathaniswa nephaji yokulawula engenalutho.Okukodwa kwe-peptide hit (#2077) kuhlolwe kabanzi.Ibonise uhhafu wempilo ende ye-plasma uma kuqhathaniswa namanye amahithi (Umfanekiso 3d kanye Nomfanekiso Owengeziwe 7), futhi ngokuthakazelisayo, le peptide iqukethe izinsalela ze-cysteine ​​​​ku-C-terminus.Kusanda kuboniswa ukuthi ukungezwa kwe-cysteine ​​​​kuma-peptide kungathuthukisa izakhiwo zabo ze-pharmacokinetic ngokubopha ku-albumin 29.Lokhu okwamanje akwaziwa nge-peptide #2077 futhi kudinga ucwaningo olwengeziwe.Amanye ama-peptide abonise ukuncika kwe-valence ekuthuthukisweni kwe-CSF (idatha engaboniswa), okungenzeka ihlobane nejometri engaphezulu ebonisiwe ye-T7 capsid.Uhlelo lwe-T7 esilusebenzisile lubonise amakhophi angu-5-15 we-peptide ngayinye ngezinhlayiyana zephage.I-IHC yenziwa kuma-clones e-phage ehola i-candidate ejovwe nge-intravenously ku-cortex ye-cerebral yamagundane (I-Supplementary Fig. 8).Idatha ibonise ukuthi okungenani ama-clone amathathu (No. 2002, No. 2009 kanye No. 2077) asebenzisana ne-BBB.Kusazonqunywa ukuthi ingabe lokhu kusebenzisana kwe-BBB kubangela ukunqwabelana kwe-CSF noma ukunyakaza kwalawa ma-clones ngqo ku-BCSFB.Okubalulekile, sibonisa ukuthi ama-peptide akhethiwe agcina umthamo wawo wezokuthutha we-CSF lapho ehlanganiswa futhi eboshelwe emthwalweni wamaprotheni.Ukubophezela kwe-N-terminal biotinylated peptides ku-SA ngokuyisisekelo kuphinda imiphumela etholwe ngama-phage clones awo afanele egazini kanye noketshezi lwe-cerebrospinal (Fig. 3e).Ekugcineni, sibonisa ukuthi i-peptide eholayo #2077 iyakwazi ukukhuthaza ukusebenza kobuchopho kwe-biotinylated peptide inhibitor ye-BACE1 ehlanganiswe ne-SA, okubangela imiphumela evelele ye-pharmacodynamic ku-CNS ngokunciphisa kakhulu amazinga e-Abeta40 ku-CSF (Fig. 4).Asikwazanga ukuhlonza noma yimaphi ama-homologue kusizindalwazi ngokwenza ukusesha kwe-homology yokulandelana kwe-peptide kukho konke okuphambili.Kubalulekile ukuqaphela ukuthi usayizi womtapo wezincwadi we-T7 ucishe ube ngu-109, kuyilapho usayizi womtapo wolwazi wama-12-mers ungu-4 x 1015. Ngakho-ke, sikhethe ingxenye encane yesikhala sokuhlukahluka komtapo wezincwadi we-peptide we-12-mer, okungase kusho ukuthi ama-peptide athuthukisiwe angabonakala ngokuhlola indawo eseduze yalezi zindawo ezilandelanayo.Ngokucatshangelwa, esinye sezizathu ezenza singatholi noma yimaphi ama-homologue emvelo alawa ma-peptide singase singakhethi ngesikhathi sokuziphendukela kwemvelo ukuze kuvinjelwe ukungena okungalawuleki kwe-peptide motifs ethile ebuchosheni.
Sekuhlangene, imiphumela yethu ihlinzeka ngesisekelo somsebenzi wesikhathi esizayo wokuhlonza kanye nokuveza izinqubo zokuthutha zesithiyo se-cerebrovascular in vivo ngokuningiliziwe.Ukusethwa okuyisisekelo kwale ndlela kusekelwe kuqhinga lokukhetha elisebenzayo elingagcini nje ngokuhlonza ama-clones anezakhiwo zokubopha imithambo yobuchopho, kodwa futhi kuhlanganisa nesinyathelo esibalulekile lapho ama-clone aphumelelayo anomsebenzi wangaphakathi wokuwela imigoqo yebhayoloji ku-vivo angene ekhomishini ye-CNS.iwukucacisa indlela yokuthutha lawa ma-peptide kanye nokukhetha kwawo ukubopha ku-microvasculature eqondene nendawo yobuchopho.Lokhu kungaholela ekutholakaleni kwezindlela ezintsha zokuthutha i-BBB nama-receptors.Silindele ukuthi ama-peptide akhonjiwe angabophezela ngokuqondile kuma-cerebrovascular receptors noma kuma-ligands ajikelezayo athuthwa nge-BBB noma i-BCSFB.Amavektha e-peptide anomsebenzi wezokuthutha we-CSF atholwe kulo msebenzi azophinde aphenywe.Okwamanje siphenya ukucaciswa kobuchopho balawa ma-peptide ngekhono lawo lokuwela i-BBB kanye/noma i-BCSFB.Lawa ma-peptide amasha azoba amathuluzi abaluleke kakhulu okutholakala okungenzeka kwama-receptors amasha noma izindlela kanye nokuthuthukiswa kwamapulatifomu amasha asebenza kahle kakhulu okulethwa kwama-macromolecules, njenge-biologics, ebuchosheni.
Cannulate i-cisterna enkulu (CM) usebenzisa ukuguqulwa kwendlela echazwe ngaphambilini.Amagundane e-Wistar anezinzwa (200-350 g) ayefakwe kumshini we-stereotaxic futhi ukusika okumaphakathi kwenziwa phezu kwekhanda elishefiwe futhi elilungiswe ngendlela engafanele ukuze kuvezwe ugebhezi.Gcoba izimbobo ezimbili endaweni ye-sash engenhla bese ufaka izikulufo zokulungisa emigodini.Imbobo eyengeziwe yabhojwa ku-lateral occipital crest ukuze kuqondiswe i-stereotactic ye-cannula yensimbi engagqwali ku-CM.Gcoba usimende wamazinyo eduze kwe-cannula futhi uvikeleke ngezikulufo.Ngemuva kokulungiswa kwesithombe nokuqina kukasimende, inxeba lesikhumba lalivalwa nge-4/0 supramid suture.Ukubekwa kahle kwe-cannula kuqinisekiswa ukuvuza okuzenzakalelayo kwe-cerebrospinal fluid (CSF).Susa igundane emshinini we-stereotaxic, thola ukunakekelwa okufanele kwangemva kokuhlinzwa kanye nokulawulwa kobuhlungu, futhi ulivumele ukuthi lilulame okungenani isonto elilodwa kuze kubonakale izimpawu zegazi oketshezini lwe-cerebrospinal.Amagundane e-Wistar (Crl:WI/Han) atholwe kwa-Charles River (France).Wonke amagundane agcinwa ngaphansi kwezimo ezithile ezingenalo i-pathogen.Konke ukuhlola kwezilwane kuvunywe Ihhovisi Lodokotela Wezilwane Ledolobha lase-Basel, e-Switzerland, futhi kwenziwa ngokuvumelana neLayisense Yezilwane No.
Gxila ngobumnene igundane linolwazi nge-CM cannula esandleni.Khipha i-Datura ku-cannula bese uqoqa u-10 µl woketshezi lwe-cerebrospinal olugeleza ngokuzenzekelayo.Njengoba amandla obunikazi be-cannula agcina esengozini, amasampula oketshezi lwe-cerebrospinal acacile kuphela angenabo ubufakazi bokungcoliswa kwegazi noma ukuguquguquka kombala afakiwe kulolu cwaningo.Ngokuhambisanayo, cishe u-10-20 μl wegazi wathathwa embotsheni encane ekugcineni komsila waba amashubhu ane-heparin (Sigma-Aldrich).I-CSF negazi kwaqoqwa ngezikhathi ezihlukahlukene ngemva kokujova ngomjovo we-T7 phage.Cishe i-5-10 μl yoketshezi yalahlwa ngaphambi kokuba isampula ngayinye ye-CSF iqoqwe, ehambisana nevolumu efile ye-catheter.
Amalabhulali akhiqizwa kusetshenziswa i-T7Select 10-3b vector njengoba kuchazwe kumanuwali wesistimu ye-T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996).Kafushane, ukufakwa okungahleliwe kwe-DNA ye-12-mer kwahlanganiswa ngale fomethi elandelayo:
I-NNK codon isetshenziselwe ukugwema ama-codons aphindwe kabili kanye ne-amino acid overexpression ekufakweni.I-N iyisilinganiso se-equimolar esixutshwe ngesandla senucleotide ngayinye, futhi i-K iyisilinganiso esilinganayo esixutshwe mathupha se-adenine ne-cytosine nucleotide.Izifunda ezinemicibisholo eyodwa zaguqulwa zaba yi-DNA ephindwe kabili ngokuqhubeka nokufukamela nge-dNTP (Novagen) kanye ne-Klenow enzyme (New England Biolabs) e-Klenow buffer (New England Biolabs) amahora angu-3 ku-37°C.Ngemuva kokusabela, i-DNA enemicu ekabili yatholwa yimvula ye-EtOH.I-DNA ewumphumela yagaywa ngama-enzyme avimbela i-EcoRI kanye ne-HindIII (womabili avela e-Roche).I-cleaved futhi yahlanzwa (QIAquick, Qiagen) insert (T4 ligase, New England Biolabs) yabe isixhunyaniswa kuhlaka lwaba yivektha ye-T7 eklanywe ngaphambili ngemva kwe-amino acid 348 yofuzo lwe-capsid engu-10B.Ukusabela kwe-ligation kwafakwa ku-16° C. amahora angu-18 ngaphambi kokupakishwa kwe-in vitro.Ukupakishwa kwe-Phage ku-vitro kwenziwa ngokuya ngemiyalo enikezwe i-T7Select 10-3b cloning kit (Novagen) futhi isixazululo sokupakisha sakhuliswa kanye ukuze kusetshenziswe i-Escherichia coli (BLT5615, Novagen).Ama-lysates aye-centrifuged, titrated futhi aqandiswe ku -80° C. njengesixazululo sesitoko se-glycerol.
I-PCR eqondile yokukhulisa izifunda eziguquguqukayo ze-phage ezikhuliswe kumhluzi noma ipuleti kusetshenziswa i-prietary 454/Roche-amplicon fusion primers.I-front fusion primer iqukethe ukulandelana okuzungeze isifunda esiguquguqukayo (NNK) 12 (i-template-specific), i-GS FLX Titanium Adapter A, kanye nokulandelana kokhiye belabhulali yesisekelo esine (TCAG) (Umfanekiso 1a Owengeziwe):
I-reverse fusion primer futhi iqukethe i-biotin enamathiselwe ukuthwebula ubuhlalu kanye ne-GS FLX Titanium Adapter B edingekayo ukuze kukhuliswe i-clonal ngesikhathi se-emulsion PCR:
Ama-amplicon abe esefakwa ngaphansi kwe-454/Roche pyrosequencing ngokusho kwe-454 GS-FLX Titanium protocol.Ngokulandelana kwemanuwali kwe-Sanger (Applied Biosystems Hitachi 3730 xl DNA Analyzer), i-T7 phage DNA yakhuliswa yi-PCR futhi yalandelwa ngamapheya okuqala alandelayo:
Okufakiwe okuvela kumaplakhu ngamanye kuye kwakhuliswa i-PCR kusetshenziswa i-Roche Fast Start DNA Polymerase Kit (ngokwemiyalo yomkhiqizi).Yenza isiqalo esishisayo (imizuzu eyi-10 ku-95 °C) kanye nemijikelezo ye-boost engu-35 (50 s ku-95 °C, iminithi elingu-1 ku-50 °C, kanye neminithi elingu-1 ku-72 °C).
I-Phage evela emitatsheni yolwazi, i-wild-type phage, i-phage esindiswe ku-CSF kanye negazi, noma ama-clones angawodwana akhuliswa ku-Escherichia coli BL5615 kumhluzi we-TB (Sigma Aldrich) noma ezitsheni ezingu-500 cm2 (Thermo Scientific) amahora angu-4 ku-37°C.I-Phage ikhishwe kumapuleti ngokuwasha amapuleti nge-Tris-EDTA buffer (Fluka Analytical) noma ngokuqoqa ama-plaque anamacebiso angenalutho.I-Phage yahlukaniswa ne-culture supernatant noma i-extraction buffer enomjikelezo owodwa we-polyethylene glycol (PEG 8000) precipitation (Promega) futhi yamiswa kabusha ku-Tris-EDTA buffer.
I-phage ekhulisiwe yayingaphansi kwemizuliswano engu-2-3 yokususwa kwe-endotoxin kusetshenziswa ubuhlalu bokususa i-endotoxin (i-Miltenyi Biotec) ngaphambi komjovo we-intravenous (IV) (500 μl/isilwane).Emzuliswaneni wokuqala, i-2 × 1012 phages yethulwa;kwesibili, 2×1010 izigaba;emzuliswaneni wesithathu nowesine wokukhetha, amaphaji angu-2×109 ngesilwane ngasinye.Okuqukethwe kwephage ku-CSF kanye namasampula egazi aqoqwe ngezikhathi ezikhonjisiwe kwanqunywa ngokubala koqwembe ngokwemiyalelo yomkhiqizi (i-T7Select system manual).Ukukhethwa kwe-Phage kwenziwa ngomjovo we-intravenous wemitapo yolwazi ehlanjululwe emthanjeni womsila noma ngokujova kabusha i-phage ekhishwe ku-CSF kusukela ekukhethweni kwangaphambilini, futhi ukuvuna okwalandela kwenziwa ku-10 min, 30 min, 60 min, 90 min, 120 min, 180 min, kanye ne-240 min kanye namasampula egazi ngokulandelana.Isamba semizuliswano emine ye-in vivo panning yenziwa lapho amagatsha amabili akhethiwe agcinwa ngokuhlukana futhi ahlaziywa phakathi nemizuliswano emithathu yokuqala yokukhetha.Konke ukufakwa kwe-phage okukhishwe ku-CSF emizuliswaneni emibili yokuqala yokukhetha kwakungaphansi kwe-454 / Roche pyrosequencing, kuyilapho wonke ama-clones akhishwe ku-CSF kusukela emizuliswaneni emibili yokugcina yokukhetha alandelwa ngesandla.Wonke ama-phages egazi kusukela emzuliswaneni wokuqala wokukhetha nawo afakwe ngaphansi kwe-454/Roche pyrosequencing.Ukuze uthole umjovo wama-clone we-phage, ama-phage akhethiwe akhuliswa ku-E. coli (BL5615) kumapuleti angu-500 cm2 ku-37 ° C amahora angu-4.Ama-clones akhethiwe futhi alandelaniswa ngesandla asakazwa ngendlela ye-TB.Ngemuva kokukhipha i-phage, ukuhlanzwa nokususwa kwe-endotoxin (njengoba kuchazwe ngenhla), i-2 × 1010 phages / isilwane ku-300 μl yajovwa nge-intravenously emthanjeni owodwa womsila.
Ukucubungula kwangaphambili kanye nokuhlunga kwekhwalithi yedatha yokulandelana.Idatha ye-Raw 454/Roche iye yaguqulwa isuka kufomethi yemephu yokusakaza evamile kanambambili (sff) yayiswa kufomethi ye-Pearson efundekayo yomuntu (i-fasta) kusetshenziswa isofthiwe yomthengisi.Ukucubungula okuqhubekayo kokulandelana kwe-nucleotide kwenziwa kusetshenziswa izinhlelo ze-C zobunikazi kanye nemibhalo (iphakheji yesofthiwe engakhishiwe) njengoba kuchazwe ngezansi.Ukuhlaziywa kwedatha eyinhloko kuhlanganisa izinqubo eziqinile zokuhlunga zezigaba eziningi.Ukuze uhlunge ukufundwa obekungaqukathi ukulandelana kwe-DNA ye-12mer evumelekile, ukufundwa kwaqondaniswa ngokulandelana ukuze kuqalwe ilebula (GTGATGTCGGGGATCCGAATTCT), ilebula yokumisa (TAAGCTTGCGGCCGCACTCGAGTA) kanye nokufakwa ngemuva (CCCTGCAGGGATATCCCGGAGCTCTCGTCGAC) kusetshenziswa i-Global Need Need.ukuqondanisa okuvumela okungafikela koku-2 ukungahambisani nokuqondanisa ngakunye31.Ngakho-ke, ukufunda ngaphandle kokuqala nokumisa amathegi nokufunda okuqukethe okufakwe ngemuva, okungukuthi, ukuqondanisa okudlula inombolo evunyelwe yokungafani, kukhishiwe kulabhulali.Ngokuqondene nokufundwa okusele, ukulandelana kwe-N-mer DNA esukela kuphawu lokuqala futhi iphele ngaphambi kokuba uphawu lokumisa lukhishwe ochungechungeni lokufunda lwangempela futhi luqhubeke lucutshungulwa (ngemuva kwalokhu olubizwa ngokuthi “faka”).Ngemva kokuhunyushwa kokufakwayo, ingxenye ngemva kwe-codon yokuqala yokumisa ekupheleni kuka-5′ we-primer iyasuswa ekufakweni.Ukwengeza, ama-nucleotide aholela kuma-codon angaphelele ekupheleni kwe-3 'ye-primer nawo asusiwe.Ukuze ukhiphe okufakiwe okuqukethe ukulandelana kwangemuva kuphela, okufakiwe okuhunyushiwe kuqala ngephethini ye-amino acid ethi “PAG” nakho kukhishiwe.Ama-Peptide anobude bangemva kokuhumusha obungaphansi kwama-amino acid angu-3 akhishiwe emtatsheni wezincwadi.Okokugcina, susa ukuphindaphindeka echibini lokufaka futhi unqume imvamisa yokufaka ngakunye okuhlukile.Imiphumela yalokhu kuhlaziywa yayihlanganisa uhlu lokulandelana kwe-nucleotide (ukufakwa) kanye nama-frequencies (okufundwayo) kwawo (Izibalo Ezingeziwe 1c no-2).
I-Group N-mer DNA ifaka ngokufana kokulandelana: Ukuqeda amaphutha wokulandelana we-454/Roche-specific (njengezinkinga zokulandelanisa izandiso ze-homopolymer) futhi kususwe ukuphindaphinda okungabalulekile kangako, okufakwe ngaphambilini okuhlungwayo kwe-N-mer DNA (okufakwayo) kuhlungwa ngokufana.okufakiwe (kufika kuzisekelo ezi-2 ezingahambelani ezivunyelwe) kusetshenziswa i-algorithm ephindaphindwayo echazwe ngale ndlela elandelayo: okufakiwe kuhlelwa kuqala ngokuvama kwakho (okuphezulu kuya kokuphansi kakhulu), futhi uma kufana, ngokuhlunga kwakho kwesibili ngobude (okude kakhulu kuye kokufushane kakhulu) ).Ngakho, ukufakwa okuvamile nokude kakhulu kuchaza "iqembu" lokuqala.Ifrikhwensi yeqembu isethwe kufrikhwensi yokhiye.Khona-ke, ukufakwa ngakunye okusele ohlwini oluhleliwe kuzanywe ukungezwa eqenjini ngokuhambisana nokuqondanisa okubili kwe-Needleman-Wunsch.Uma inani lokungafani, ukufakwa, noma ukususwa ekuqondaniseni lingeqi umkhawulo ongu-2, okufakiwe kuyengezwa eqenjini, futhi imvamisa yeqembu iyonke ikhuphuka ngokuthi ukufakwa kwengezwe kaningi kangakanani.Okufakwayo okwengezwe eqenjini kumakwa njengokusetshenzisiwe futhi akubandakanyiwe ekuqhutshweni okwengeziwe.Uma uchungechunge lokufaka lungakwazi ukwengezwa eqenjini eselivele likhona, ukulandelana kokufakwayo kusetshenziselwa ukwakha iqembu elisha elinefrikhwensi yokufaka efanele futhi imakwe njengesetshenzisiwe.Ukuphindaphinda kugcina lapho ukulandelana ngakunye kokufaka kusetshenziswe ukwakha iqembu elisha noma kungafakwa eqenjini eselivele likhona.Phela, ukufakwa kwamaqembu okuhlanganisa ama-nucleotide ekugcineni kuhunyushwa ngokulandelana kwe-peptide (imitapo yolwazi ye-peptide).Umphumela walokhu kuhlaziywa isethi yokufakwayo kanye nama-frequencies ahambisanayo enza inani lokufunda okulandelanayo (I-Supplementary Fig. 2).
Isizukulwane Se-Motif: Ngokusekelwe kuhlu lwama-peptide ahlukile, kwakhiwe umtapo wolwazi oqukethe wonke amaphethini e-amino acid angenzeka (aa) njengoba kukhonjisiwe ngezansi.Iphethini ngayinye engase ibe nobude obungu-3 yakhishwa ku-peptide futhi iphethini yayo ephambene yanezelwa kanye nomtapo wezincwadi ovamile oqukethe wonke amaphethini (ama-tripeptides).Imitapo yolwazi enezisusa eziphindaphinda kakhulu yalandelana futhi yasuswa ukungasasebenzi.Bese, ku-tripeptide ngayinye kumtapo wolwazi we-motif, sasihlola ukuthi ikhona yini emtatsheni wezincwadi sisebenzisa amathuluzi okubala.Kulokhu, imvamisa ye-peptide equkethe i-motif tripeptide etholakele iyengezwa futhi yabelwa i-motif kumtapo wezincwadi we-motif (“inombolo ye-motifs”).Umphumela wokwenziwa kwe-motif kuwuhlelo olunezinhlangothi ezimbili eziqukethe konke ukwenzeka kwe-tripeptides (ama-motif) kanye namanani awo ahlukahlukene, okuyinani lokufundwa okulandelanayo eliphumela ku-motif ehambisanayo lapho ukufundwa kuhlunga, kuqoqwa, futhi kuhunyushwa.Amamethrikhi njengoba kuchazwe ngokuningiliziwe ngenhla.
Ukujwayezwa kwenani lama-motifs kanye nama-scatterplots ahambisanayo: Inani le-motifs yesampula ngayinye lijwayele ukusetshenziswa
lapho i-ni inombolo yokufundwa okuqukethe isihloko i.Ngakho, i-vi imele imvamisa yephesenti yokufunda (noma ama-peptides) aqukethe i-motif i kusampula.Amanani we-P yenani elingajwayelekile lama-motif abalwe kusetshenziswa ukuhlolwa okuqondile kukaFisher.Ngokuphathelene nama-correlograms yenani lezisusa, ukuhlobana kuka-Spearman kubalwe kusetshenziswa inombolo evamile yezisusa ezino-R.
Ukuze ubone ngeso lengqondo okuqukethwe kwama-amino acid endaweni ngayinye kulabhulali ye-peptide, ama-logogram ewebhu 32, 33 (http://weblogo.threeplusone.com) adalwa.Okokuqala, okuqukethwe kwama-amino acid endaweni ngayinye ye-peptide engu-12-mer kugcinwa ku-matrix engu-20×12.Bese, isethi yama-peptide angu-1000 aqukethe okufanayo okuhlobene nokuqukethwe kwe-amino acid endaweni ngayinye ikhiqizwa ngefomethi ye-fasta-sequence futhi inikezwe njengokufakwayo kulogo 3 wewebhu, okhiqiza ukumelwa okuyisithombe kokuqukethwe kwe-amino acid ehlobene endaweni ngayinye.kumtapo wezincwadi we-peptide.Ukuze ubone ngeso lengqondo idathasethi ye-multidimensional, amamephu okushisa adalwe kusetshenziswa ithuluzi elithuthukiswe ngaphakathi ku-R (biosHeatmap, iphakheji engu-R esazokhishwa).Ama-dendrogram ethulwa kumamephu okushisa abalwe kusetshenziswa indlela yokuhlanganisa ye-Ward ye-hierarchical clustering nge-Euclidean distance metric.Ukuhlaziywa kwezibalo kwedatha ye-motif yamaphuzu, amanani we-P wokuthola amaphuzu angajwayelekile abalwe kusetshenziswa ukuhlolwa okuqondile kukaFisher.Amanani e-P amanye amasethi wedatha abalwe ngo-R kusetshenziswa i-t-test yoMfundi noma i-ANOVA.
Ama-phage clones akhethiwe kanye nama-phage ngaphandle kokufaka ajovwe nge-intravenously ngomthambo womsila (2×1010 phage/isilwane ku-300 μl PBS).Imizuzu eyishumi ngaphambi kokugcotshwa kanye nokulungiswa okulandelayo, izilwane ezifanayo zazijovwe ngomjovo nge-100 μl ye-DyLight594 ebhalwe ilectin (Vector Laboratories Inc., DL-1177).Imizuzu engama-60 ngemuva komjovo we-phage, amagundane agcotshwa ngenhliziyo nge-50 ml PBS elandelwa yi-50 ml 4% PFA/PBS.Amasampula obuchopho aphinde alungiswa ngobusuku obubodwa ku-4% PFA/PBS futhi acwiliswa ku-30% sucrose ngobusuku obungu-4°C.Amasampuli afriziwe afriziwe kungxube ye-OCT.Ukuhlaziywa kwe-Immunohistochemical kwamasampuli aqandisiwe kwenziwa kumazinga okushisa egumbi kuma-cryosection angu-30 µm avinjwe ngo-1% BSA futhi afakwa amasosha omzimba alebulwe nge-polyclonal FITC ngokumelene ne-T7 phage (Novus NB 600-376A) ku-4 °C.Fukamela ubusuku bonke.Ekugcineni, izigaba zagezwa izikhathi ezingu-3 nge-PBS futhi zahlolwa ngesibonakhulu se-laser confocal (Leica TCS SP5).
Wonke ama-peptide anobumsulwa obuncane obungu-98% ahlanganiswa yi-GenScript USA, i-biotinylated kanye ne-lyophilized.I-Biotin iboshwe nge-spacer ye-glycine ephindwe kathathu ku-N-terminus.Hlola wonke ama-peptide usebenzisa i-mass spectrometry.
I-Streptavidin (Sigma S0677) ixutshwe ne-5-fold equimolar excess of biotinylated peptide, biotinylated BACE1 inhibitory peptide, noma inhlanganisela (3:1 ratio) ye-biotinylated BACE1 inhibitory peptide kanye ne-BACE1 inhibitory peptide ku-5–10% ye-DMB ehlanganisiwe ye-DM.Ihora elingu-1 ekamelweni lokushisa ngaphambi kokujova.Ama-peptide e-Streptavidin-conjugated ajovwe nge-intravenously nge-dose ye-10 mg / kg komunye wemithanjeni yomsila wamagundane ane-cerebral cavity.
Ukuhlushwa kwe-streptavidin-peptide complexes kwahlolwa yi-ELISA.Amapuleti e-Nunc Maxisorp microtiter (Sigma) ahlanganiswe ubusuku bonke ku-4 ° C nge-1.5 μg/ml yegundane le-anti-streptavidin antibody (Thermo, MA1-20011).Ngemuva kokuvinjwa (i-blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ekamelweni lokushisa amahora angu-2, geza ipuleti ngo-0.05% Tween-20/PBS (ibhafa yokuwasha) ye-3 Second, CluSF100 isampula ye-plasma:0 i-plasma ehlukanisiwe 0, CSF 1:115).Ipuleti labe selifakwa ubusuku bonke ku-4 ° C nge-antibody yokuthola (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239).Ngemuva kwezinyathelo ezintathu zokugeza, i-streptavidin itholwe ngokufaka incubation ku-TMB substrate solution (Roche) kuze kufike kumaminithi angu-20.Ngemva kokumisa ukuthuthukiswa kombala nge-1M H2SO4, kala ukumunca ku-450 nm.
Umsebenzi we-streptavidin-peptide-BACE1 inhibitor complex uhlolwe yi-Aβ(1-40) ELISA ngokuya ngephrothokholi yomkhiqizi (Wako, 294-64701).Kafushane, amasampula e-CSF ahlanjululwe nge-diluent evamile (1:23) futhi afakwa ngobusuku obungu-4°C kumapuleti emithombo engu-96 ahlanganiswe ne-BNT77 yokubamba i-antibody.Ngemva kwezinyathelo ezinhlanu zokugeza, i-antibody ye-BA27 ehlanganisiwe ye-HRP yengezwa futhi yafukanyelwa amahora angu-2 ku-4° C., kulandelwa izinyathelo ezinhlanu zokugeza.I-Aβ(1–40) itholwe ngokufukamela kusixazululo se-TMB imizuzu engama-30 ekamelweni lokushisa.Ngemuva kokuthi ukuthuthukiswa kombala kumisiwe ngesisombululo sokumisa, linganisa ukumuncwa ku-450 nm.Amasampula e-Plasma angaphansi kokukhishwa kwesigaba esiqinile ngaphambi kwe-Aβ (1-40) ELISA.I-Plasma yengezwe ku-0.2% DEA (Sigma) kumapuleti anemithombo engu-96 futhi yafakwa endaweni yokushisa yegumbi imizuzu engu-30.Ngemva kokugeza ngokulandelana amapuleti e-SPE (Oasis, 186000679) ngamanzi kanye ne-methanol engu-100%, amasampula e-plasma engezwa kumapuleti e-SPE futhi lonke uketshezi lwakhishwa.Amasampuli ahlanzwa (okokuqala nge-5% methanol kwase kuba ngu-30% methanol) futhi akhishwa nge-methanol engu-2% NH4OH/90%.Ngemva kokumisa i-eluate ku-55 ° C ngamaminithi angu-99 ku-N2 yamanje, amasampuli ancishiswa kuma-diluent ajwayelekile futhi i-Aβ (1-40) ikalwa njengoba kuchazwe ngenhla.
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