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Isithiyo segazi nobuchopho kanye nesithiyo segazi nobuchopho kuvimbela ama-biotherapeutic agents ukuthi afinyelele imigomo yawo ohlelweni lwezinzwa oluphakathi, ngaleyo ndlela kuvimbele ukwelashwa okuphumelelayo kwezifo zezinzwa. Ukuze sithole abathuthi bobuchopho abasha in vivo, sethule umtapo wezincwadi we-T7 phage peptide kanye negazi eliqoqwe ngokulandelana kanye noketshezi lwe-cerebrospinal (CSF) sisebenzisa imodeli ye-cannulated conscious large pool yamagundane. Ama-phage clones athile acebiswe kakhulu ku-CSF ngemuva kwemijikelezo emine yokukhetha. Ukuhlolwa kwama-peptide akhethiwe ngamanye kwembule ukucebisa okungaphezu kwe-1000-fold ku-CSF. Ukusebenza kwe-bioactivity kokulethwa okuqondiswa yi-peptide ebuchosheni kwaqinisekiswa ukwehla okungu-40% ezingeni le-amyloid-β ku-cerebrospinal fluid kusetshenziswa i-BACE1 peptide inhibitor exhunywe ne-transit peptide entsha ehlonziwe. Le miphumela iphakamisa ukuthi ama-peptide ahlonzwe ngezindlela zokukhetha i-phage in vivo angaba yizimoto eziwusizo zokulethwa kwe-macromolecule ebuchosheni ngomphumela wokwelapha.
Ucwaningo lokwelapha oluqondiswe ohlelweni lwezinzwa oluphakathi (CNS) lugxile kakhulu ekuboneni izidakamizwa nama-ejenti athuthukisiwe abonisa izakhiwo zokuqondisa i-CNS, ngomzamo omncane wokuthola izindlela eziqhuba ukulethwa kwezidakamizwa ezisebenzayo ebuchosheni. Lokhu sekuqala ukushintsha manje njengoba ukulethwa kwezidakamizwa, ikakhulukazi ama-molecule amakhulu, kuyingxenye ebalulekile yokuthuthukiswa kwemithi ye-neuroscience yesimanje. Indawo yesistimu yezinzwa ephakathi ivikelwe kahle uhlelo lokuvinjelwa kwemithambo yegazi, oluqukethe isithiyo segazi-ubuchopho (BBB) ​​​​kanye nesithiyo segazi-ubuchopho (BCBB)1, okwenza kube nzima ukuletha izidakamizwa ebuchosheni1,2. Kulinganiselwa ukuthi cishe yonke imithi yama-molecule amakhulu kanye nemithi engaphezu kuka-98% yama-molecule amancane isuswa ebuchosheni3. Yingakho kubaluleke kakhulu ukuhlonza izinhlelo ezintsha zokuthutha ubuchopho ezihlinzeka ngokulethwa okusebenzayo nokuqondile kwemithi yokwelapha ku-CNS 4,5. Kodwa-ke, i-BBB kanye ne-BCSFB nazo zinikeza ithuba elihle kakhulu lokulethwa kwezidakamizwa njengoba zingena futhi zingena kuzo zonke izakhiwo zobuchopho ngokusebenzisa imithambo yegazi ebanzi. Ngakho-ke, imizamo yamanje yokusebenzisa izindlela ezingangenisi ugesi zokulethwa ebuchosheni isekelwe kakhulu endleleni yokuthutha okuqondiswa yi-receptor (PMT) kusetshenziswa i-endogenous BBB6 receptor. Naphezu kwentuthuko ebalulekile yakamuva esebenzisa indlela ye-transferrin receptor7,8, kudingeka ukuthuthukiswa okuqhubekayo kwezinhlelo ezintsha zokulethwa ezinezakhiwo ezithuthukisiwe. Ukuze sifeze lokhu, umgomo wethu kwakuwukuthola ama-peptide akwazi ukuqondisa ukuthutha kwe-CSF, njengoba empeleni angasetshenziswa ukuletha ama-macromolecule ku-CNS noma ukuvula izindlela ezintsha ze-receptor. Ikakhulukazi, ama-receptor athile kanye nabathuthi besistimu ye-cerebrovascular (BBB kanye ne-BSCFB) angasebenza njengezinhloso ezingaba khona zokulethwa okusebenzayo nokuqondile kwemithi ye-biotherapeutic. Uketshezi lwe-cerebrospinal (CSF) luwumkhiqizo oyimfihlo we-choroid plexus (CS) futhi luxhumana ngqo noketshezi lwe-interstitial lobuchopho ngesikhala se-subarachnoid kanye nesikhala se-ventricular4. Muva nje kuye kwaboniswa ukuthi uketshezi lwe-subarachnoid cerebrospinal lusakazeka kakhulu lungene ngaphakathi kwe-interstitium yobuchopho9. Sithemba ukufinyelela isikhala se-parenchymal sisebenzisa le ndlela yokungena kwe-subarachnoid noma ngqo nge-BBB. Ukuze sifeze lokhu, sisebenzise isu eliqinile lokukhetha i-in vivo phage elihlonza kahle ama-peptide athuthwa yinoma iyiphi yalezi zindlela ezimbili ezihlukile.
Manje sesichaza indlela yokuhlola i-in vivo phage elandelanayo nge-CSF sampling ehambisana ne-high throughput sequencing (HTS) ukuqapha imijikelezo yokuqala yokukhetha enokwehlukahluka okuphezulu komtapo wolwazi. Ukuhlolwa kwenziwe kumagundane aqaphelayo nge-cannula enkulu ye-cisterna (CM) efakwe unomphela ukuze kugwenywe ukungcoliswa kwegazi. Okubalulekile, le ndlela ikhetha kokubili ukuqondiswa kobuchopho kanye nama-peptide anomsebenzi wokuthutha ngaphesheya kwesithiyo se-cerebrovascular. Sisebenzise ama-phage e-T7 ngenxa yobukhulu bawo obuncane (~60 nm)10 futhi saphakamisa ukuthi afanele ukuthuthwa kwama-vesicles avumela ukuwela kwe-transcellular kwesithiyo se-endothelial kanye/noma se-epithelial-medulla. Ngemva kwemijikelezo emine ye-panning, imiphakathi yama-phage yahlukaniswa ebonisa ukunotha kwe-CSF okunamandla kanye nokuhlangana kwemithambo emincane yobuchopho. Okubalulekile, sikwazile ukuqinisekisa okutholakele kwethu ngokubonisa ukuthi ama-peptide angcono kakhulu akhethwayo futhi akhiwe ngamakhemikhali ayakwazi ukuthutha impahla yamaprotheni aye ku-cerebrospinal fluid. Okokuqala, imiphumela ye-pharmacodynamic ye-CNS yasungulwa ngokuhlanganisa i-transit peptide ehamba phambili kanye ne-inhibitor ye-BACE1 peptide. Ngaphezu kokubonisa ukuthi amasu okuhlola ukusebenza kwe-in vivo angabona ama-peptide amasha okuthutha ubuchopho njengabathwali bemithwalo yeprotheni abaphumelelayo, silindele ukuthi izindlela ezifanayo zokukhetha ukusebenza nazo zibe ezibalulekile ekuboneni izindlela ezintsha zokuthutha ubuchopho.
Ngokusekelwe kumayunithi okwenza i-plaque (PFU), ngemva kwesinyathelo sokupakisha i-phage, kwaklanywa futhi kwadalwa umtapo wolwazi wama-peptide e-T7 phage angu-12-mer aqondile anokuhlukahluka okungaba ngu-109 (bheka Izinto Nezindlela). Kubalulekile ukuqaphela ukuthi sihlaziye ngokucophelela lo mtapo wolwazi ngaphambi kokusebenzisa i-in vivo panning. Ukwandiswa kwe-PCR kwamasampula omtapo wolwazi we-phage kusetshenziswa ama-primer aguquliwe kwakhiqiza ama-amplicons ayesebenza ngqo ku-HTS (Isithombe Esingeziwe 1a). Ngenxa a) amaphutha okulandelana kwe-HTS11, b) umthelela ekhwalithini yama-primer (NNK)1-12, kanye c) nokuba khona kwe-phage yohlobo lwe-wild-type (wt) (ama-skeleton inserts) kulabhulali yokulinda, inqubo yokuhlunga ukulandelana yaqaliswa ukukhipha ulwazi lokulandelana oluqinisekisiwe kuphela (Isithombe Esingeziwe 1b). Lezi zinyathelo zokuhlunga zisebenza kuwo wonke amalabhulali okulandelana kwe-HTS. Kulabhulali ejwayelekile, kutholakale inani lama-readings angu-233,868, lapho ama-39% awo aphumelele khona imigomo yokuhlunga futhi asetshenziselwa ukuhlaziya umtapo wolwazi kanye nokukhetha imijikelezo elandelayo (Isithombe Esingeziwe 1c–e). Ama-readings ayeyi-multiples yama-base pairs amathathu ubude ene-peak kuma-nucleotide angu-36 (Isithombe Esingeziwe 1c), okuqinisekisa ukwakheka komtapo wolwazi (NNK) 1-12. Okuphawulekayo ukuthi cishe ama-11% amalungu omtapo wolwazi aqukethe i-PAGISRELVDKL backbone engu-12-dimensional wild-type (wt) backbone, kanti cishe ingxenye yama-sequences (49%) yayiqukethe okufakiwe noma okususiwe. I-HTS yomtapo wolwazi iqinisekisile ukuhlukahluka okuphezulu kwama-peptide emtapo wolwazi: ama-peptide sequences angaphezu kwama-81% atholakale kanye kuphela futhi ama-1.5% kuphela avele kumakhophi angu-≥4 (Isithombe Esingeziwe 2a). Ama-frequency ama-amino acid (aa) kuzo zonke izikhundla ezingu-12 ku-repertoire ahlobene kahle nama-frequency alindelekile enanini lama-codon akhiqizwa yi-repertoire ye-NKK ewohlokile (Isithombe Esingeziwe 2b). Imvamisa ebonwe yezinsalela ze-aa ezifakwe yilezi zinto ezifakiwe ihlobene kahle nemvamisa ebalwe (r = 0.893) (Isithombe Esingeziwe 2c). Ukulungiswa kwama-library e-phage ukuze ajovwe kuhlanganisa izinyathelo zokukhulisa nokususa i-endotoxin. Lokhu kuye kwabonakala ngaphambilini ukuthi kunganciphisa ukuhlukahluka kwama-library e-phage12,13. Ngakho-ke, sihlele umtapo we-phage onama-plate-amplified owawususwe i-endotoxin futhi sawuqhathanisa nomtapo wezincwadi wokuqala ukuze silinganise imvamisa ye-AA. Ubudlelwano obuqinile (r = 0.995) bubonwe phakathi kwechibi lokuqala kanye nechibi elikhulisiwe nelihlanziwe (Isithombe Esingeziwe 2d), okubonisa ukuthi ukuncintisana phakathi kwama-clones akhulisiwe kuma-plate asebenzisa i-T7 phage akuzange kubangele ukucwaswa okukhulu. Lokhu kuqhathanisa kusekelwe ekuvameni kwezinhlobo ze-tripeptide emtatsheni ngamunye wezincwadi, njengoba ukuhlukahluka kwezincwadi (~109) kungenakuqoshwa ngokugcwele ngisho nange-HTS. Ukuhlaziywa kwemvamisa ye-aa endaweni ngayinye kwembule ukubandlulula okuncane okuncike endaweni ezindaweni ezintathu zokugcina ze-repertoire efakiwe (Isifanekiso Esingeziwe 2e). Ekuphetheni, siphethe ngokuthi ikhwalithi nokuhlukahluka komtapo wezincwadi kwakwamukelekile futhi izinguquko ezincane kuphela ekuhlukeni kwabonwa ngenxa yokwandiswa nokulungiswa kwezincwadi ze-phage phakathi kwemijikelezo eminingana yokukhetha.
Ukusampula uketshezi lwe-cerebrospinal serial kungenziwa ngokufaka i-cannula ngokuhlinzwa ku-CM yamagundane aqaphelayo ukuze kube lula ukuhlonza i-T7 phage efakwe nge-intravenously (iv) nge-BBB kanye/noma i-BCSFB (Isithombe 1a-b). Sisebenzise izingalo ezimbili zokukhetha ezizimele (izingalo A no-B) emizuliswaneni emithathu yokuqala yokukhetha kwe-in vivo (Isithombe 1c). Kancane kancane sandisa ukuqina kokukhetha ngokunciphisa inani eliphelele le-phage elethwe emizuliswaneni emithathu yokuqala yokukhetha. Emzuliswaneni wesine we-panning, sihlanganise amasampula avela emagatsheni A no-B futhi senza ukukhetha okuthathu okuzimele okwengeziwe. Ukuze sifunde izakhiwo ze-in vivo zezinhlayiya ze-T7 phage kulo modeli, i-wild-type phage (PAGISRELVDKL master insert) yafakwa emagundwini ngomthambo womsila. Ukubuyiselwa kwama-phage kusuka ku-cerebrospinal fluid kanye negazi ngezikhathi ezahlukene kubonise ukuthi ama-phage amancane e-T7 icosahedral ayenesigaba sokuqala sokususwa ngokushesha kusuka endaweni yegazi (Isithombe Esingeziwe 3). Ngokusekelwe kuma-titers anikezwe kanye nomthamo wegazi wamagundane, sibale ukuthi cishe i-phage engu-1% kuphela yesisindo esivela kumthamo onikezwe yatholakala egazini ngemuva kwemizuzu eyi-10 ngemuva kokujova nge-intravenous. Ngemva kwalokhu kwehla okusheshayo kokuqala, ukuhlanzwa okuhamba kancane kwe-primary kwalinganiswa nge-half-life yemizuzu engama-27.7. Okubalulekile, ama-phages ambalwa kakhulu athathwe endaweni ye-CSF, okubonisa isizinda esiphansi sokufuduka kwe-phage yohlobo lwe-wild-type kuya endaweni ye-CSF (Isithombe Esingeziwe 3). Ngokwesilinganiso, cishe ama-titers angu-1 x 10-3% kuphela e-T7 phage egazini kanye nama-4 x 10-8% e-phages afakwe ekuqaleni atholakala ku-cerebrospinal fluid phakathi nesikhathi sonke sokuthatha isampula (0-250 min). Okuphawulekayo ukuthi i-half-life (25.7 min) ye-phage yohlobo lwe-wild-type ku-cerebrospinal fluid yayifana naleyo ebonwe egazini. Le mininingwane ikhombisa ukuthi umgoqo ohlukanisa indawo ye-CSF egazini awuthintekile emagundwini afakwe i-CM, okuvumela ukukhethwa kwezincwadi ze-phage emzimbeni ukuze kutholakale ama-clone athuthwa kalula esuka egazini aye endaweni ye-CSF.
(a) Ukusetha indlela yokuphinda kuthathwe isampula yoketshezi lwe-cerebrospinal (CSF) echibini elikhulu. (b) Umdwebo okhombisa indawo yamaseli yesithiyo sesimiso sezinzwa esiyinhloko (CNS) kanye nesu lokukhetha elisetshenziswa ukuhlonza ama-peptide awela isithiyo segazi-ubuchopho (BBB) ​​​​kanye nesithiyo segazi-ubuchopho. (c) Ishadi lokugeleza kokuboniswa kwe-in vivo phage. Kumjikelezo ngamunye wokukhetha, ama-phage (izihlonzi zezilwane ngaphakathi kwemicibisholo) afakwa ngemithambo yegazi. Amagatsha amabili azimele ahlukile (A, B) agcinwa ngokwehlukana kuze kube umjikelezo wesine wokukhetha. Kumjikelezo wokukhetha 3 nowe-4, i-clone ngayinye ye-phage ekhishwe ku-CSF yahlelwa ngesandla. (d) I-Kinetics ye-phage ehlukaniswe egazini (imibuthano ebomvu) kanye noketshezi lwe-cerebrospinal (onxantathu abaluhlaza) ngesikhathi somjikelezo wokuqala wokukhetha emagundwini amabili afakwe i-cannulated ngemuva kokufakwa kwe-intravenous yelabhulali ye-T7 peptide (2 x 1012 phages/isilwane). Izikwele eziluhlaza okwesibhakabhaka zibonisa ukuhlushwa kokuqala okumaphakathi kwe-phage egazini, kubalwa kusukela enanini le-phage ejojiwe, kucatshangelwa inani legazi eliphelele. Izikwele ezimnyama zibonisa indawo lapho kuhlangane khona umugqa we-y othathwe ekuhlushweni kwe-phage yegazi. (e,f) Veza imvamisa ehlobene kanye nokusatshalaliswa kwazo zonke izinhlobo ze-tripeptide ezihlanganayo ezitholakala ku-peptide. Inani lezinhlobo ze-motif ezitholakala ekufundweni okungu-1000 liyaboniswa. Okuphawulekayo (p < 0.001) izinhlobo ze-motif ezicebile ziphawulwe ngamachashazi abomvu. (e) I-scatterplot yokuxhumana eqhathanisa imvamisa ehlobene ye-tripeptide motif yomtapo wolwazi ojojiwe ne-phage etholakala egazini evela ezilwaneni #1.1 kanye no-#1.2. (f) I-scatterplot yokuxhumana eqhathanisa imvamisa ehlobene yezinhlobo ze-tripeptide ze-phage yezilwane #1.1 kanye no-#1.2 ezihlukaniswe egazini kanye noketshezi lwe-cerebrospinal. (g, h) Ukumelwa kwe-Sequence ID ye-phage ecebile egazini (g) uma kuqhathaniswa nemitapo yolwazi ejojiwe kanye ne-phage ecebile ku-CSF (h) uma kuqhathaniswa negazi ngemva kokukhethwa kwe-in vivo kuzo zombili izilwane. Usayizi wekhodi enohlamvu olulodwa ubonisa ukuthi i-amino acid ivame kangakanani ukuvela kuleyo ndawo. Okuluhlaza = okune-polar, okunsomi = okungathathi hlangothi, okuluhlaza okwesibhakabhaka = okuyisisekelo, okubomvu = okune-acid kanye nokumnyama = ama-amino acid angaphoqelekile. Isithombe 1a, b saklanywa futhi sakhiqizwa ngu-Eduard Urich.
Sifake umtapo we-phage peptide emagundwini amabili e-CM instrument (clades A no-B) kanye ne-phage ehlukanisiwe kusuka ku-cerebrospinal fluid kanye negazi (Isithombe 1d). Ukususwa okusheshayo kokuqala komtapo wezincwadi bekungabonakali kakhulu uma kuqhathaniswa ne-wild-type phage. Isilinganiso sempilo yesigamu somtapo wezincwadi ofakwe kuzo zombili izilwane sasiyimizuzu engama-24.8 egazini, esifana ne-wild-type phage, kanye nemizuzu engama-38.5 ku-CSF. Amasampula e-phage egazi kanye ne-cerebrospinal fluid avela esilwaneni ngasinye afakwa ku-HTS futhi wonke ama-peptide ahlonziwe ahlaziywa ukuze kutholakale ukuthi kukhona i-tripeptide motif emfushane. Ama-Tripeptide motif akhethwe ngoba ahlinzeka ngesisekelo esincane sokwakheka kwesakhiwo kanye nokusebenzisana kwe-peptide-protein14,15. Sithole ubudlelwano obuhle ekusakazweni kwama-motif phakathi komtapo we-phage ofakwe kanye nama-clone akhishwe egazini lezilwane zombili (Isithombe 1e). Imininingwane ikhombisa ukuthi ukwakheka komtapo wezincwadi kucebiswe kancane kuphela endaweni yegazi. Ama-frequency e-amino acid kanye nokulandelana kokuvumelana kwahlaziywa kabanzi endaweni ngayinye kusetshenziswa i-adaptation yesofthiwe ye-Weblogo16. Ngokuthakazelisayo, sithole ukuceba okunamandla kuma-residues e-glycine egazini (Isithombe 1g). Lapho igazi liqhathaniswa nama-clone akhethwe ku-CSF, kwabonwa ukukhetha okunamandla kanye nokususwa kokukhethwa kwama-motif (Isithombe 1f), futhi ama-amino acid athile ayekhona ngokukhethekile ezindaweni ezinqunyiwe kumalungu ayi-12 (Isithombe 1h). Okuphawulekayo ukuthi izilwane ngazinye zazihluke kakhulu ku-cerebrospinal fluid, kanti ukuceba kwe-glycine egazini kwabonwa kuzo zombili izilwane (Isithombe Esingeziwe 4a-j). Ngemva kokuhlunga okuqinile kwedatha yokulandelana ku-cerebrospinal fluid yezilwane #1.1 kanye no-#1.2, kwatholakala ama-peptide ayi-12-mer angu-964 kanye no-420 ahlukile (Isithombe Esingeziwe 1d-e). Ama-clone e-phage ahlukanisiwe akhuliswa futhi afakwa emzuliswaneni wesibili wokukhetha kwe-in vivo. I-Phage ekhishwe emzuliswaneni wesibili wokukhetha yafakwa ku-HTS esilwaneni ngasinye futhi wonke ama-peptide atholakele asetshenziswa njengokufakwa ohlelweni lokuqashelwa kwe-motif ukuze kuhlaziywe ukuvela kwama-motif e-tripeptide (Isithombe 2a, b, ef). Uma kuqhathaniswa nomjikelezo wokuqala we-phage etholakale ku-CSF, sibone ukukhethwa okwengeziwe kanye nokususwa kokukhethwa kwama-motif amaningi ku-CSF emagatsheni A no-B (Isithombe 2). I-algorithm yokuhlonza inethiwekhi yasetshenziswa ukunquma ukuthi ngabe imele amaphethini ahlukene okulandelana okuhambisanayo. Ukufana okucacile kwabonwa phakathi kokulandelana okungu-12-dimensional okutholwe yi-CSF ku-alternative clade A (Isithombe 2c, d) kanye ne-clade B (Isithombe 2g, h). Ukuhlaziywa okuhlanganisiwe egatsheni ngalinye kwembule amaphrofayili ahlukene okukhetha ama-peptide angu-12-mer (Isithombe Esingeziwe 5c, d) kanye nokwanda kwesilinganiso se-CSF/blood titer ngokuhamba kwesikhathi kuma-clone ahlanganisiwe ngemuva komzuliswano wesibili wokukhetha uma kuqhathaniswa nomzuliswano wokuqala wokukhetha (Isithombe Esingeziwe 5e).).
Ukunotha kwama-motif nama-peptide ku-cerebrospinal fluid ngemijikelezo emibili elandelanayo yokukhethwa kokuboniswa kwe-phage okusebenzayo ngaphakathi kwe-vivo.
Zonke izinhlayiya ze-cerebrospinal fluid ezitholwe emzuliswaneni wokuqala wesilwane ngasinye (izilwane #1.1 kanye no #1.2) zahlanganiswa, zandiswa, zalandelaniswa nge-HT futhi zajovwa kabusha ndawonye (2 x 1010 phages/isilwane) amagundane amabili e-SM cannulated (#1.1 → #). 2.1 kanye no-2.2, 1.2 → 2.3 kanye no-2.4). (a,b,e,f) Ama-scatterplot okuhlangana aqhathanisa imvamisa ehlobene yezinhlayiya ze-tripeptide zazo zonke izinhlayiya ezithathwe yi-CSF emzuliswaneni wokuqala nowesibili wokukhetha. Imvamisa ehlobene kanye nokusatshalaliswa kwezinhlayiya ezimele zonke izinhlayiya ze-tripeptide ezinqwabelanayo ezitholakala kuma-peptide kuzo zombili izindlela. Inani lezinhlayiya ezitholakale ekufundweni okungu-1000 liyaboniswa. Izinhlayiya ezazikhethwe kakhulu (p < 0.001) noma zingafakwanga kwenye yezincwadi eziqhathaniswayo zigqanyiswe ngamachashazi abomvu. (c, d, g, h) Ukumelwa kwelogo yokulandelana kwazo zonke izilandelana ezinde ze-amino acid eziyi-12 ezicebile nge-CSF ngokusekelwe ezilandelaneni 2 no-1 zokukhetha kwe-in vivo. Usayizi wekhodi enohlamvu olulodwa ubonisa ukuthi leyo amino acid ivele kangaki kuleyo ndawo. Ukuze imele ilogo, imvamisa yezilandelana ze-CSF ezikhishwe ezilwaneni ngazinye phakathi kwezilandelana ezimbili zokukhetha iyaqhathaniswa futhi izilandelana ezicebile emzuliswaneni wesibili ziyaboniswa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 kanye (h) #1.2–#2.4. Ama-amino acid acebile kakhulu endaweni ethile ezilwaneni (c, d) inombolo 2.1 kanye nenombolo 2.2 noma (g, h) ezilwaneni inombolo 2.3 kanye nenombolo 2.4 aboniswa ngombala. Oluhlaza = oluyi-polar, olunsomi = olungathathi hlangothi, oluhlaza okwesibhakabhaka = oluyisisekelo, olubomvu = olune-acid kanye nomnyama = ama-amino acid angenawo umoya.
Ngemva komzuliswano wesithathu wokukhetha, sithole ukulandelana kwe-peptide okuhlukile okungu-124 (#3.1 kanye no-#3.2) okuvela kuma-clone e-phage akhiwe kabusha angu-332 e-CSF ahlukaniswe ezilwaneni ezimbili (Isithombe Esingeziwe 6a). I-sequence LGSVS (18.7%) yayinesilinganiso esiphezulu kakhulu, ilandelwa yi-wild-type inserts PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), kanye ne-SARGSWREIVSLS (2.2%). Emzuliswano wesine wokugcina, sihlanganise amagatsha amabili akhethwe ngokuzimela ezilwaneni ezintathu ezihlukene (Isithombe 1c). Kuma-clone e-phage ahleliwe angu-925 atholwe ku-CSF, emzuliswaneni wesine sithole ukulandelana kwe-peptide okuhlukile okungu-64 (Isithombe Esingeziwe 6b), phakathi kwawo isilinganiso esilinganiselwe se-wild-type phage sehle saya ku-0.8%. Ama-clone e-CSF avame kakhulu emzuliswaneni wesine kwakuyi-LYVLHSRGLWGFKLAAALE (18%), i-LGSVS (17%), i-GFVRFRLSNTR (14%), i-KVAWRVFSLFWK (7%), i-SVHGV (5%), i-GRPQKINGARVC (3.6%) kanye ne-RLSSVDSDLSGC (3, 2%). %)). Ububanzi bobude bama-peptide akhethiwe bubangelwa ukufakwa/ukususwa kwe-nucleotide noma ama-codon okuma ngaphambi kwesikhathi kuma-primer omtapo wolwazi lapho kusetshenziswa ama-codon agugile ekwakhiweni komtapo wolwazi we-NNK. Ama-codon okuma ngaphambi kwesikhathi akhiqiza ama-peptide amafushane futhi akhethwa ngoba aqukethe i-motif enhle ye-aa. Ama-peptide amade angase avele ekufakweni/ukususwa kuma-primer omtapo wolwazi wokwenziwa. Lokhu kubeka i-stop codon eklanywe ngaphandle kohlaka futhi kuyifunda kuze kube yilapho kuvela i-stop codon entsha ezansi. Ngokuvamile, sibale izici zokucebisa kuzo zonke izigaba ezine zokukhetha ngokuqhathanisa idatha yokufaka nedatha yokukhipha isampula. Emzuliswaneni wokuqala wokuhlola, sisebenzise ama-phage titer e-wild-type njengereferensi yangemuva engacacisiwe. Ngokuthakazelisayo, ukukhethwa kwe-phage okungekuhle kwakunamandla kakhulu emzuliswaneni wokuqala we-CSF, kodwa hhayi egazini (Isithombe 3a), okungase kube ngenxa yamathuba aphansi okusabalala okungenamsebenzi kwamalungu amaningi omtapo we-peptide engxenyeni ye-CSF noma ama-phage ahlobene avame ukugcinwa noma ukususwa egazini kangcono kunama-bacteriophages. Kodwa-ke, emzuliswaneni wesibili we-panning, ukukhethwa okunamandla kwama-phage ku-CSF kwabonwa kuzo zombili izigaba, okuphakamisa ukuthi umzuliswano wangaphambilini wawucebile kuma-phage abonisa ama-peptide akhuthaza ukuthathwa kwe-CSF (Isithombe 3a). Futhi, ngaphandle kokunotha kwegazi okuphawulekayo. Futhi emzuliswaneni wesithathu nowesine, ama-phage clones anothe kakhulu ku-CSF. Uma siqhathanisa imvamisa ehlobene yochungechunge ngalunye lwe-peptide oluhlukile phakathi kwemizuliswano emibili yokugcina yokukhetha, sithole ukuthi imizuliswano yayinothe kakhulu emzuliswaneni wesine wokukhetha (Isithombe 3b). Kukhishwe ama-motif e-tripeptide angu-931 kuzo zonke izilandelaniso ze-peptide ezihlukile ezingu-64 kusetshenziswa kokubili ukuqondiswa kwe-peptide. Ama-motif acebiswe kakhulu emzuliswaneni wesine ahlolwe ngokuseduze amaphrofayili awo okucebisa kuzo zonke izizungezo uma kuqhathaniswa nomtapo wolwazi ofakiwe (ukusika: ukucebisa okungu-10%) (Isithombe Esingeziwe 6c). Amaphethini ajwayelekile okukhetha abonise ukuthi iningi lezisusa ezifundwe licebiswe kuzo zonke izizungezo zangaphambilini zamagatsha womabili okukhetha. Kodwa-ke, amanye ama-motif (isb. SGL, VSG, LGS GSV) ayevela kakhulu ku-alternative clade A, kanti amanye (isb. FGW, RTN, WGF, NTR) acebiswe ku-alternative clade B.
Ukuqinisekiswa kokuthuthwa kwe-CSF kwama-peptide aboniswe nge-phage acebile nge-CSF kanye nama-peptide ahamba phambili e-biotinylated ahlanganiswe nemithwalo yokukhokha ye-streptavidin.
(a) Izilinganiso zokunotha ezibalwe kuzo zonke izilinganiso ezine (R1-R4) ngokusekelwe kuma-titer ajovwe (okufakwayo = I) phage (PFU) kanye nama-titer e-CSF phage anqunyiwe (okukhiphayo = O). Izici zokunotha zezilinganiso ezintathu zokugcina (R2-R4) zibalwe ngokuqhathaniswa nomjikelezo wangaphambilini kanye nomjikelezo wokuqala (R1) ngedatha yesisindo. Amabha avulekile ayi-cerebrospinal fluid, amabha anombala ayi-plasma. (***p<0.001, ngokusekelwe ku-t-test yoMfundi). (b) Uhlu lwama-peptide e-phage amaningi kakhulu, ahlelwe ngokwesilinganiso sawo esihlobene nawo wonke ama-phage aqoqwe ku-CSF ngemuva komjikelezo wesi-4 wokukhetha. Ama-clone e-phage ayisithupha avame kakhulu aqokonyiswe ngombala, anezinombolo kanye nezici zawo zokunotha phakathi kwezilinganiso zesi-3 nesesi-4 zokukhetha (ama-insets). (c,d) Ama-clone e-phage ayisithupha acebile kakhulu, ama-phage angenalutho kanye nemitapo yolwazi ye-phage peptide yabazali kusukela kumjikelezo wesi-4 ahlaziywe ngawodwana kumodeli yokusampula ye-CSF. Amasampula e-CSF kanye negazi aqoqwe ngesikhathi esibonisiwe. (c) Inani elilinganayo lama-clone angu-6 e-candidate phage (ama-phage/izilwane angu-2 x 1010), ama-phage angenalutho (#1779) (ama-phage/izilwane angu-2 x 1010) kanye nemitapo yolwazi ye-stock phage peptide (ama-phage/izilwane angu-2 x 1012) Faka okungenani ama-CM angu-3 esilwaneni esifakwe ku-cannulated ngokwehlukana ngomthambo womsila. I-CSF pharmacokinetics yomtapo wolwazi ngamunye we-phage clone kanye ne-phage peptide ofakwe ku-injection ngokuhamba kwesikhathi iyaboniswa. (d) ikhombisa isilinganiso se-CSF/igazi esimaphakathi sawo wonke ama-phage/mL atholakele ngesikhathi sokuthatha isampula. (e) Ama-peptide amane ahamba phambili okwenziwa kanye nokulawula okukodwa okuxutshwe kwaxhunyaniswa ne-biotin ne-streptavidin nge-N-terminus yawo (ukuboniswa kwe-tetramer) kulandelwe umjovo (umthambo womsila iv, 10 mg streptavidin/kg). Okungenani amagundane amathathu afakwe ku-intubation (N = 3).). Amasampula e-CSF aqoqwe ngesikhathi esibonisiwe kanti amazinga e-streptavidin alinganiswe yi-CSF anti-streptavidin ELISA (futhi = akutholakalanga). (*p<0.05, **p<0.01, ***p<0.001, ngokusekelwe ekuhlolweni kwe-ANOVA). (f) Ukuqhathaniswa kochungechunge lwe-amino acid lwe-clone ye-phage peptide ecebile kakhulu #2002 (onsomi) namanye ama-clone e-phage peptide akhethiwe kusukela emzuliswaneni wesine wokukhetha. Izingcezu ze-amino acid ezifanayo nezifanayo zinemibala.
Kuzo zonke izinhlayiya ezicebile emzuliswaneni wesine (Umfanekiso 3b), ama-clone ayisithupha akhethiwe akhethwe ukuze kuhlaziywe ngabanye kumodeli yokusampula ye-CSF. Inani elilinganayo le-phage eyisithupha ekhethiwe, i-phage engenalutho (engenayo insert) kanye nemitapo yolwazi ye-prophage peptide yafakwa ezilwaneni ezintathu ze-CM ezifakwe ku-cannulated, futhi i-pharmacokinetics yanqunywa ku-CSF (Umfanekiso 3c) kanye negazi (Umfanekiso Ongeziwe 7). Zonke izinhlayiya ze-phage ezihlolwe zihlose indawo ye-CSF ezingeni eliphakeme ngokuphindwe ka-10-1000 kunele-phage engenalutho (#1779). Isibonelo, ama-clone #2020 kanye no-#2077 ayeneziqu ze-CSF eziphakeme ngokuphindwe ka-1000 kune-phage yokulawula. Iphrofayili ye-pharmacokinetic ye-peptide ngayinye ekhethiwe ihlukile, kodwa zonke zinekhono eliphezulu lokufika kwe-CSF. Sibone ukwehla okuqhubekayo ngokuhamba kwesikhathi kwama-clone #1903 kanye no-#2011, kuyilapho kuma-clone #2077, #2002 kanye no-#2009 ukwanda phakathi nemizuzu yokuqala eyi-10 kungabonisa ukuthuthwa okusebenzayo kodwa kudinga ukuqinisekiswa. Ama-Clone #2020, #2002, kanye no-#2077 azinza emazingeni aphezulu, kuyilapho ukuhlushwa kwe-CSF kwe-clone #2009 kwehla kancane ngemva kokwanda kokuqala. Sabe sesiqhathanisa imvamisa ehlobene ye-CSF ngayinye kanye nokuhlushwa kwayo egazini (Isithombe 3d). Ukuxhumana kwe-titer ephakathi ye-CSF ngayinye kanye ne-titer yayo yegazi kuzo zonke izikhathi zokuthatha isampula kubonise ukuthi abathathu kwabayisithupha bacetshiswe kakhulu kwi-CSF yegazi. Ngokuthakazelisayo, i-clone #2077 ibonise ukuzinza kwegazi okuphezulu (Isithombe Esingeziwe 7). Ukuze siqinisekise ukuthi ama-peptide ngokwawo ayakwazi ukuthutha imithwalo ngenkuthalo ngaphandle kwezinhlayiya ze-phage endaweni ye-CSF, sakha ama-peptide amane ahamba phambili athathwe nge-biotin endaweni ye-N-terminus lapho ama-peptide enamathela khona ku-phage particle. Ama-peptide e-biotinylated (nos. 2002, 2009, 2020 kanye no-2077) ahlanganiswa ne-streptavidin (SA) ukuze kutholakale amafomu e-multimeric afana ne-geometry ye-phage. Le fomethi yasivumela futhi ukuthi silinganise ukuvezwa kwe-SA egazini kanye noketshezi lwe-cerebrospinal njengama-peptide amaprotheni athutha impahla. Okubalulekile, idatha ye-phage yayivame ukuphinda ikhiqizwe lapho ama-peptide okwenziwa enikezwa ngale fomethi ye-SA-conjugated (Isithombe 3e). Ama-peptide aqhekekile ayenokuvezwa okuncane kokuqala kanye nokucaciswa okusheshayo kwe-CSF ngamazinga angabonakali zingakapheli amahora angu-48. Ukuze sithole ukuqonda ngezindlela zokulethwa zala ma-peptide phage clones esikhaleni se-CSF, sihlaziye indawo yama-peptide e-phage peptide hits ngamanye sisebenzisa i-immunohistochemistry (IHC) ukuthola ngqo izinhlayiya ze-phage ihora eli-1 ngemuva kokujova nge-intravenous in vivo. Okuphawulekayo ukuthi ama-clone #2002, #2077, kanye no-#2009 angatholakala ngokudaya okunamandla kuma-capillary obuchopho, kuyilapho i-control phage (#1779) kanye ne-clone #2020 kungatholakalanga (Isithombe Esingeziwe 8). Lokhu kusikisela ukuthi la ma-peptide anegalelo emphumeleni ebuchosheni ngokuwela i-BBB ngqo. Kudingeka ukuhlaziywa okuningiliziwe okwengeziwe ukuze kuhlolwe lo mbono, njengoba umzila we-BSCFB nawo ungase uhileleke. Uma kuqhathaniswa ukulandelana kwama-amino acid kwe-clone ecebile kakhulu (#2002) namanye ama-peptide akhethiwe, kwaphawulwa ukuthi amanye awo anezandiso ezifanayo zama-amino acid, ezingase zibonise indlela efanayo yokuthutha (Isithombe 3f).
Ngenxa yephrofayili yayo ye-plasma ehlukile kanye nokwanda okukhulu kwe-CSF ngokuhamba kwesikhathi, i-phage display clone #2077 yahlolwa kabanzi esikhathini eside samahora angu-48 futhi yakwazi ukuphinda ikhiqize ukwanda okusheshayo kwe-CSF okubonwe ngokuhambisana namazinga e-SA aqhubekayo (Isithombe 4a). Ngokuphathelene namanye ama-phage clones atholakele, i-#2077 yangcola kakhulu ama-capillaries obuchopho futhi yabonisa i-colocalization ebalulekile nge-capillary marker lectin uma ibukwa ngesisombululo esiphezulu futhi mhlawumbe ingcola ethile esikhaleni se-parenchymal (Isithombe 4b). Ukuze sihlole ukuthi imiphumela ye-pharmacological eqondiswa yi-peptide ingatholakala yini ku-CNS, senze ucwaningo lapho izinguqulo ze-biotinylated ze-i) i-#2077 transit peptide kanye ne-ii) i-BACE1 inhibitor peptide zaxutshwa ne-SA ngezilinganiso ezimbili ezahlukene. Kokunye ukuhlanganiswa sisebenzise i-BACE1 peptide inhibitor kuphela kanti kwenye sisebenzise isilinganiso esingu-1:3 se-BACE1 peptide inhibitor ku-#2077 peptide. Womabili amasampula anikezwa ngemithambo yegazi kanye noketshezi lwe-cerebrospinal lwe-beta-amyloid peptide 40 (Abeta40) alinganiswa ngokuhamba kwesikhathi. I-Abeta40 yalinganiswa ku-CSF njengoba ibonisa ukuvinjelwa kwe-BACE1 ku-parenchyma yobuchopho. Njengoba bekulindelekile, zombili lezi zinhlanganisela zanciphisa kakhulu amazinga egazi le-Abeta40 (Umfanekiso 4c, d). Kodwa-ke, amasampula kuphela aqukethe ingxube ye-peptide no. 2077 kanye nesithiyo se-peptide ye-BACE1 ehlanganiswe ne-SA abangele ukwehla okukhulu kwe-Abeta40 kuketshezi lwe-cerebrospinal (Umfanekiso 4c). Idatha ikhombisa ukuthi i-peptide no. 2077 iyakwazi ukuthutha iphrotheni ye-60 kDa SA iye ku-CNS futhi ibangela nemiphumela yezokwelapha ngama-SA-conjugated inhibitors e-BACE1 peptide.
(a) Umjovo we-clonal (2 × 10 phages/isilwane) we-T7 phage obonisa amaphrofayili e-pharmacokinetic esikhathi eside we-CSF peptide #2077 (RLSSVDSDLSGC) kanye ne-control phage engajovwanga (#1779) okungenani kumagundane amathathu afakwe i-CM. (b) Isithombe esincane se-Confocal semithambo emincane ye-cortical emele kumagundane ajovwe nge-phage (2 × 10 10 phages/isilwane) esibonisa ukuphikisana kwe-peptide #2077 kanye nemithambo (i-lectin). Lawa ma-phage clones anikezwa amagundane ama-3 futhi avunyelwa ukuthi ajikeleze ihora eli-1 ngaphambi kokuphuma kwegazi. Ubuchopho bahlukaniswa futhi bagcotshwa ngama-antibodies ane-polyclonal FITC-labeled ngokumelene ne-T7 phage capsid. Imizuzu eyishumi ngaphambi kokuphuma kwegazi kanye nokufakwa okulandelayo, i-DyLight594-labeled lectin yanikezwa nge-intravenous. Izithombe ezikhanyayo ezibonisa ukudaya kwe-lectin (okubomvu) kohlangothi lwe-luminal lwemithambo emincane kanye nama-phage (oluhlaza) ku-lumen yama-capillary kanye nezicubu zobuchopho ezingaphakathi kwemithambo yegazi. Ibha yesikali ihambelana no-10 µm. (c, d) I-peptide yokuvimbela i-BACE1 ene-Biotinylated yodwa noma ihlanganiswe ne-biotinylated transit peptide #2077 ihlanganiswe ne-streptavidin kulandelwe ukujova nge-intravenous okungenani amagundane amathathu e-CM afakwe i-cannulated (10 mg streptavidin/kg). Ukunciphisa kwe-BACE1 peptide inhibitor-mediated ku-Aβ40 kulinganiswe nge-Aβ1-40 ELISA egazini (elibomvu) kanye noketshezi lwe-cerebrospinal (oluphuzi) ngezikhathi ezibonisiwe. Ukuze kucace kangcono, kudwetshwa umugqa onamachashazi kugrafu esikalini esingu-100%. (c) Ukwehla kwephesenti ku-Aβ40 egazini (onxantathu ababomvu) kanye noketshezi lwe-cerebrospinal (onxantathu abawolintshi) kumagundane aphathwe nge-streptavidin ehlanganiswe ne-transit peptide #2077 kanye ne-BACE1 inhibitory peptide ngesilinganiso esingu-3:1. (d) Ukwehla kwephesenti egazini i-Aβ40 (imibuthano ebomvu) kanye noketshezi lwe-cerebrospinal (imibuthano ye-orange) yamagundane aphathwe nge-streptavidin ehlanganiswe ne-BACE1 inhibitory peptide kuphela. Ukuhlushwa kwe-Aβ ekulawuleni kwakungu-420 pg/ml (ukuphambuka okujwayelekile = 101 pg/ml).
Ukuboniswa kwe-Phage kusetshenziswe ngempumelelo ezindaweni eziningana zocwaningo lwezokwelapha17. Le ndlela isetshenziswe ezifundweni zokuhlukahluka kwemithambo yegazi emzimbeni womuntu18,19 kanye nezifundo eziqondiswe emithanjeni yobuchopho20,21,22,23,24,25,26. Kulesi sifundo, sandise ukusetshenziswa kwale ndlela yokukhetha hhayi nje kuphela ekuboneni ngqo ama-peptide aqondiswe emithanjeni yobuchopho, kodwa nasekutholeni abantu abanezakhiwo zokuthutha ezisebenzayo ukuze bawele umgoqo wegazi nobuchopho. Manje sichaza ukuthuthukiswa kwenqubo yokukhetha umzimba womuntu emagundwini afakwe i-CM futhi sibonisa amandla ayo okuhlonza ama-peptide anezakhiwo ze-CSF homing. Sisebenzisa i-T7 phage ekhombisa umtapo wama-peptide angahleliwe angu-12-mer, sikwazile ukukhombisa ukuthi i-T7 phage incane ngokwanele (cishe ububanzi obungu-60 nm)10 ukuze ivumelane nomgoqo wegazi nobuchopho, ngaleyo ndlela iwele umgoqo wegazi nobuchopho noma i-choroid plexus. Sibonile ukuthi ukuvunwa kwe-CSF kumagundane e-CM afakwe ku-cannulated kwakuyindlela yokuhlola esebenza kahle elawulwa kahle emzimbeni, nokuthi i-phage ekhishwe ayibophanga nje kuphela emithanjeni yegazi kodwa futhi yayisebenza njengomthuthi ngaphesheya kwesithiyo segazi nobuchopho. Ngaphezu kwalokho, ngokuqoqa igazi kanyekanye nokusebenzisa i-HTS ku-CSF kanye nama-phage athathwe egazini, siqinisekisile ukuthi ukukhetha kwethu i-CSF akuzange kuthonywe ukucebisa igazi noma ukufaneleka kokukhula phakathi kwemijikelezo yokukhetha. Kodwa-ke, indawo yegazi iyingxenye yenqubo yokukhetha, njengoba ama-phage akwazi ukufinyelela indawo ye-CSF kumele asinde futhi ajikeleze egazini isikhathi eside ngokwanele ukuze azicebise ebuchosheni. Ukuze sikhiphe ulwazi lokulandelana oluthembekile kusuka kudatha ye-HTS eluhlaza, sisebenzise izihlungi ezivumelaniswe namaphutha okulandelana kweplatifomu ethile emsebenzini wokuhlaziya. Ngokufaka amapharamitha e-kinetic endleleni yokuhlola, siqinisekisile i-pharmacokinetics esheshayo yama-phage e-T7 e-wild-type (t½ ~ 28 min) egazini24, 27, 28 futhi sanquma nengxenye yokuphila kwawo ku-cerebrospinal fluid (t½ ~ 26 min) ngomzuzu). Naphezu kwamaphrofayili afanayo e-pharmacokinetic egazini kanye ne-CSF, yi-0.001% kuphela yokuhlushwa kwegazi kwe-phage okungatholakala ku-CSF, okubonisa ukuhamba okuphansi kwesizinda se-wild-type T7 phage ngaphesheya komgoqo wegazi nobuchopho. Lo msebenzi ugcizelela ukubaluleka komjikelezo wokuqala wokukhetha lapho kusetshenziswa amasu okuguqula i-in vivo, ikakhulukazi ezinhlelweni ze-phage ezisuswa ngokushesha ekujikelezeni kwegazi, njengoba ama-clone ambalwa akwazi ukufinyelela indawo ye-CNS. Ngakho-ke, emzuliswaneni wokuqala, ukwehla kokuhlukahluka komtapo wolwazi kwakukhulu kakhulu, njengoba inani elilinganiselwe lama-clone kuphela ekugcineni laqoqwa kule modeli ye-CSF eqinile kakhulu. Leli cebo lokuguqula i-in vivo lifaka izinyathelo eziningana zokukhetha njengokuqongelela okusebenzayo endaweni ye-CSF, ukusinda kwe-clone endaweni yegazi, kanye nokususwa ngokushesha kwama-clone e-T7 phage egazini phakathi nemizuzu yokuqala eyi-10 (Isithombe 1d kanye nesithombe esengeziwe 4M). ). Ngakho-ke, ngemva komjikelezo wokuqala, ama-clone e-phage ahlukene atholakala ku-CSF, yize ichibi lokuqala elifanayo lasetshenziswa ezilwaneni ngazinye. Lokhu kusikisela ukuthi izinyathelo eziningi eziqinile zokukhetha imitapo yolwazi yomthombo enamalungu amaningi omtapo wolwazi ziholela ekunciphiseni okukhulu kokwehlukahlukana. Ngakho-ke, izehlakalo ezingahleliwe zizoba yingxenye ebalulekile yenqubo yokuqala yokukhetha, zithonye kakhulu umphumela. Kungenzeka ukuthi ama-clone amaningi emtatsheni wolwazi wokuqala ayenokuthambekela okufanayo kakhulu kokunothisa i-CSF. Kodwa-ke, ngisho nangaphansi kwezimo ezifanayo zokuhlola, imiphumela yokukhetha ingahluka ngenxa yenani elincane le-clone ngayinye echibini lokuqala.
Ama-motif acebile ku-CSF ahlukile kulawo asegazini. Ngokuthakazelisayo, siphawule ushintsho lokuqala oluya kuma-peptide acebile nge-glycine egazini lezilwane ngazinye. (Isithombe 1g, Ama-Fig. 4e, 4f). Ama-peptide e-glycine aqukethe i-phage angase azinze kakhulu futhi angabi namathuba amaningi okukhishwa ekujikelezeni kwegazi. Kodwa-ke, la ma-peptide acebile nge-glycine awatholakalanga kumasampula oketshezi lwe-cerebrospinal, okuphakamisa ukuthi imitapo yolwazi ekhethiwe idlule ezinyathelweni ezimbili ezahlukene zokukhetha: eyodwa egazini kanti enye ivunyelwe ukuqongelela oketshezini lwe-cerebrospinal. Ama-clone acebile nge-CSF avela emzuliswaneni wesine wokukhetha ahlolwe kabanzi. Cishe wonke ama-clone ahlolwe ngawodwana aqinisekiswe ukuthi acebile ku-CSF uma kuqhathaniswa ne-blank control phage. I-peptide hit eyodwa (#2077) ihlolwe ngokuningiliziwe. Ibonise i-plasma half-life ende uma kuqhathaniswa nezinye izigameko (Isithombe 3d kanye nesithombe 7 esingeziwe), futhi ngokuthakazelisayo, le peptide yayiqukethe i-cysteine ​​​​residue ku-C-terminus. Muva nje kuye kwaboniswa ukuthi ukungezwa kwe-cysteine ​​​​kuma-peptide kungathuthukisa izakhiwo zawo ze-pharmacokinetic ngokubopha ku-albumin 29. Lokhu okwamanje akwaziwa nge-peptide #2077 futhi kudinga ucwaningo olwengeziwe. Amanye ama-peptide abonise ukuthembela kwe-valence ekuthuthukisweni kwe-CSF (idatha ayiboniswanga), okungase kuhlotshaniswe ne-geometry ebusweni ebonisiwe ye-T7 capsid. Uhlelo lwe-T7 esilusebenzisile lubonise amakhophi angu-5-15 e-peptide ngayinye nge-phage particle. I-IHC yenziwe kuma-clone e-lead phage afakwe nge-intravenously ku-cerebral cortex yamagundane (Isithombe Esingeziwe 8). Idatha ibonise ukuthi okungenani ama-clone amathathu (No. 2002, No. 2009 kanye noNo. 2077) asebenzisana ne-BBB. Kusazonqunywa ukuthi lokhu kusebenzisana kwe-BBB kuphumela ekuqongeleleni kwe-CSF noma ukunyakaza kwala ma-clone ngqo ku-BCSFB. Okubalulekile, sibonisa ukuthi ama-peptide akhethiwe agcina amandla awo okuthutha e-CSF lapho ehlanganiswa futhi eboshelwe ku-protein cargo. Ukubopha ama-peptide e-N-terminal biotinylated ku-SA empeleni kuphinda imiphumela etholwe ngama-clone awo e-phage egazini kanye noketshezi lwe-cerebrospinal (Isithombe 3e). Okokugcina, sibonisa ukuthi i-peptide eholayo #2077 iyakwazi ukukhuthaza isenzo sobuchopho se-peptide inhibitor ye-BACE1 ehlanganiswe ne-SA, okubangela imiphumela ye-pharmacodynamic evelele ku-CNS ngokunciphisa kakhulu amazinga e-Abeta40 ku-CSF (Isithombe 4). Asikwazanga ukuhlonza noma yimaphi ama-homologue ku-database ngokwenza usesho lwe-peptide sequence homology lwazo zonke izigameko. Kubalulekile ukuqaphela ukuthi usayizi womtapo wezincwadi we-T7 cishe u-109, kanti usayizi womtapo wezincwadi we-12-mers ungu-4 x 1015. Ngakho-ke, sikhethe ingxenye encane kuphela yesikhala sokwehlukahluka komtapo wezincwadi we-peptide we-12-mer, okungasho ukuthi ama-peptide alungiselelwe kakhulu angabonakala ngokuhlola isikhala sokulandelana esiseduze salezi zigameko ezitholiwe. Ngokucabangela, esinye sezizathu zokuthi kungani singakatholi noma yiziphi izinhlobo zemvelo zala ma-peptide kungaba ukungakhethwa ngesikhathi sokuziphendukela kwemvelo ukuze kuvinjelwe ukungena okungalawulwa kwezinhlobo ezithile zama-peptide ebuchosheni.
Uma sibheka ndawonye, ​​imiphumela yethu inikeza isisekelo somsebenzi wesikhathi esizayo wokuhlonza nokuchaza izinhlelo zokuthutha zesithiyo se-cerebrovascular in vivo ngokuningiliziwe. Ukusethwa okuyisisekelo kwale ndlela kusekelwe isu lokukhetha elisebenzayo elingagcini nje ngokuhlonza ama-clone anezakhiwo zokubopha imithambo yegazi yobuchopho, kodwa futhi kufaka phakathi isinyathelo esibalulekile lapho ama-clone aphumelelayo enomsebenzi wangaphakathi wokuwela izithiyo zebhayoloji in vivo aye engxenyeni ye-CNS. ukucacisa indlela yokuthutha la ma-peptide kanye nokukhetha kwawo ukubopha ku-microvasculature ethize esifundeni sobuchopho. Lokhu kungaholela ekutholakaleni kwezindlela ezintsha zokuthutha i-BBB nama-receptor. Silindele ukuthi ama-peptide atholakele angabopha ngqo kuma-receptor e-cerebrovascular noma kuma-ligand ajikelezayo athuthwa nge-BBB noma i-BCSFB. Ama-vector e-peptide anomsebenzi wokuthutha we-CSF atholakale kulo msebenzi azophenywa kabanzi. Njengamanje siphenya ukucaciswa kobuchopho kwala ma-peptide ngekhono lawo lokuwela i-BBB kanye/noma i-BCSFB. Lawa ma-peptide amasha azoba amathuluzi awusizo kakhulu ekutholakaleni okungenzeka kwama-receptor noma izindlela ezintsha kanye nokuthuthukiswa kwamapulatifomu amasha asebenza kahle kakhulu okulethwa kwama-macromolecule, njenge-biologics, ebuchosheni.
Hlanza i-cisterna enkulu (CM) usebenzisa ukuguqulwa kwendlela echazwe ngaphambilini. Amagundane e-Wistar afakwe i-anesthesia (200-350 g) afakwe kumshini we-stereotaxic futhi kwenziwa ukusika okuphakathi phezu kwekhanda eligundiwe nelilungisiwe ngendlela engenamagciwane ukuze kuvezwe ugebhezi. Bhoboza imigodi emibili endaweni ye-sash engenhla bese ubopha izikulufo zokulungisa emigodini. Kwabhobozwa imbobo eyengeziwe ku-lateral occipital crest ukuze kuqondiswe i-stereotaxic ye-cannula yensimbi engagqwali ku-CM. Faka usimende wamazinyo uzungeze i-cannula bese uqinisa ngezikulufo. Ngemva kokupholisa ngesithombe nokuqinisa usimende, inxeba lesikhumba lavalwa nge-supramid suture engu-4/0. Ukubekwa kahle kwe-cannula kuqinisekiswa ngokuvuza okuzenzakalelayo koketshezi lwe-cerebrospinal (CSF). Susa igundane kumshini we-stereotaxic, uthole ukunakekelwa okufanele ngemva kokuhlinzwa kanye nokuphathwa kobuhlungu, bese ulivumela ukuthi lilulame okungenani isonto elilodwa kuze kube yilapho izimpawu zegazi zibonakala kuketshezi lwe-cerebrospinal. Amagundane e-Wistar (Crl:WI/Han) atholakala eCharles River (France). Wonke amagundane agcinwe ngaphansi kwezimo ezithile ezingenazo izifo. Zonke izivivinyo zezilwane zavunyelwa yiHhovisi Lezilwane laseDolobheni laseBasel, eSwitzerland, futhi zenziwa ngokuhambisana neLayisensi Yezilwane No. 2474 (Ukuhlolwa Kokuthuthwa Kobuchopho Obusebenzayo Ngokulinganisa Amazinga Abantu Abazokwelashwa Ku-Cerebrospinal Fluid kanye Nobuchopho Begundane).
Gcina igundane liqaphile ngobumnene usebenzisa i-CM cannula esandleni. Susa i-Datura ku-cannula bese uqoqa u-10 µl woketshezi lwe-cerebrospinal olugeleza ngokuzenzakalelayo. Njengoba i-patency ye-cannula ekugcineni yaphazamiseka, amasampula oketshezi lwe-cerebrospinal acacile kuphela angenabo ubufakazi bokungcoliswa kwegazi noma ukushintsha kombala afakiwe kulolu cwaningo. Ngesikhathi esifanayo, cishe u-10-20 μl wegazi wathathwa ekusikeni okuncane esicongweni somsila wafakwa emapayipini ane-heparin (Sigma-Aldrich). I-CSF negazi kwaqoqwa ngezikhathi ezahlukene ngemva kokujova nge-intravenous ye-T7 phage. Cishe u-5-10 μl woketshezi walahlwa ngaphambi kokuba kuqoqwe isampula ngayinye ye-CSF, okuhambisana nomthamo ofile we-catheter.
Amalabhulali akhiqizwa kusetshenziswa i-vector ye-T7Select 10-3b njengoba kuchaziwe encwadini yesistimu ye-T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Kafushane, i-DNA insert engahleliwe engu-12-mer yaklanywa ngale ndlela elandelayo:
I-codon ye-NNK yasetshenziswa ukugwema ama-codon anqamulelayo kabili kanye nokwanda kwe-amino acid ku-insert. I-N iyisilinganiso se-equimolar esixutshwe ngesandla se-nucleotide ngayinye, kanti i-K iyisilinganiso se-equimolar esixutshwe ngesandla se-adenine kanye ne-cytosine nucleotides. Izindawo ezinqamulelayo ezi-single zaguqulwa zaba yi-DNA enqamulelayo kabili ngokuqhubeka nokufakwa kwe-dNTP (Novagen) kanye ne-enzyme ye-Klenow (New England Biolabs) ku-Klenow buffer (New England Biolabs) amahora ama-3 ku-37°C. Ngemva kokusabela, i-DNA enqamulelayo kabili yatholwa yi-EtOH precipitation. I-DNA eyavela yagaywa ngama-enzyme okukhawulela i-EcoRI kanye ne-HindIII (zombili ezivela ku-Roche). I-insert enqamulelayo nehlanziwe (QIAquick, Qiagen) (T4 ligase, New England Biolabs) yabe isifakwa ku-frame ibe yi-vector ye-T7 enqamulelayo ngaphambili ngemuva kwe-amino acid 348 ye-gene ye-10B capsid. Ukusabela kwe-ligation kwafakwa ku-16°C. amahora ayi-18 ngaphambi kokufakwa kwe-in vitro. Ukupakishwa kwe-Phage in vitro kwenziwa ngokwemiyalelo enikezwe nge-T7Select 10-3b cloning kit (Novagen) futhi isixazululo sokupakishwa sandiswa kanye ukuze sisetshenziswe kusetshenziswa i-Escherichia coli (BLT5615, Novagen). Ama-lysates afakwa ku-centrifuge, afakwa ku-tit futhi aqandisiwe ku--80° C. njengesisombululo se-glycerol esitokweni.
Ukwandiswa kwe-PCR okuqondile kwezifunda eziguquguqukayo ze-phage okukhuliswe kumhluzi noma epuletini kusetshenziswa ama-primer e-fusion angu-454/Roche-amplicon akhethekile. I-primer ye-fusion ehamba phambili iqukethe ama-sequence azungeze isifunda esiguquguqukayo (NNK) 12 (esiqondene nethempulethi), i-GS FLX Titanium Adapter A, kanye nochungechunge lwezihluthulelo zomtapo wolwazi olunezisekelo ezine (TCAG) (Isithombe Esingeziwe 1a):
I-reverse fusion primer iqukethe ne-biotin enamathiselwe kuma-capture beads kanye ne-GS FLX Titanium Adapter B edingekayo ekukhuliseni i-clonal ngesikhathi se-emulsion PCR:
Ama-amplicon abe esefakwa ku-454/Roche pyrosequencing ngokwephrothokholi ye-454 GS-FLX Titanium. Ukuze kwenziwe i-Sanger sequencing ngesandla (i-Applied Biosystems Hitachi 3730 xl DNA Analyzer), i-T7 phage DNA yandiswa yi-PCR futhi yahlelwa ngokulandelana ngama-primer pair alandelayo:
Ama-insert avela kuma-plaque ngamanye afakwe ku-PCR amplification kusetshenziswa i-Roche Fast Start DNA Polymerase Kit (ngokwemiyalelo yomenzi). Yenza i-hot start (imizuzu eyi-10 ku-95 °C) kanye nemijikelezo engu-35 yokukhulisa (imizuzwana engama-50 ku-95 °C, umzuzu o-1 ku-50 °C, kanye nomzuzu o-1 ku-72 °C).
I-Phage evela emitatsheni yezincwadi, i-phage yohlobo lwe-wild-type, i-phage esindiswe ku-CSF kanye negazi, noma ama-clone ngamanye akhuliswe ku-Escherichia coli BL5615 kumhluzi we-TB (Sigma Aldrich) noma ezitsheni ezingama-500 cm2 (Thermo Scientific) amahora ama-4 ku-37°C. I-Phage ikhishwe emapuletini ngokuhlanza amapuleti nge-Tris-EDTA buffer (Fluka Analytical) noma ngokuqoqa ama-plaque ngama-pipette angenawo amagciwane. I-Phage ihlukaniswe kusuka ku-culture supernatant noma ku-extraction buffer nge-round eyodwa ye-polyethylene glycol (PEG 8000) precipitation (Promega) futhi yaphinde yamiswa ku-Tris-EDTA buffer.
I-phage ekhulisiwe yafakwa imijikelezo engu-2-3 yokususwa kwe-endotoxin kusetshenziswa ubuhlalu bokususa i-endotoxin (i-Miltenyi Biotec) ngaphambi kokujova nge-intravenous (IV) (500 μl/isilwane). Emzuliswaneni wokuqala, kwafakwa ama-phage angu-2×1012; kwesibili, ama-phage angu-2×1010; emzuliswaneni wesithathu nowesine wokukhetha, ama-phage angu-2×109 ngesilwane ngasinye. Okuqukethwe kwama-phage ku-CSF namasampula egazi aqoqwe ngesikhathi esibonisiwe kwanqunywa ngokubalwa kwe-plaque ngokwemiyalelo yomenzi (incwadi yemiyalelo yesistimu ye-T7Select). Ukukhethwa kwama-phage kwenziwa ngokujova nge-intravenous imitapo yolwazi ehlanziwe emthanjeni womsila noma ngokujova kabusha i-phage ekhishwe ku-CSF emzuliswaneni wokukhetha wangaphambilini, futhi ukuvuna okulandelayo kwenziwa ngemizuzu eyi-10, imizuzu engama-30, imizuzu engama-60, imizuzu engama-90, imizuzu eyi-120, imizuzu eyi-180, kanye nemizuzu engama-240 ngokulandelana. Kwenziwe imijikelezo emine yokujikijela ngaphakathi emzimbeni lapho amagatsha amabili akhethiwe agcinwa futhi ahlaziywa ngokwehlukana phakathi kwemijikelezo emithathu yokuqala yokukhetha. Zonke izifakelo ze-phage ezikhishwe ku-CSF emijikelezweni emibili yokuqala yokukhetha zenziwe nge-454/Roche pyrosequencing, kuyilapho zonke izifakelo ze-clone ezikhishwe ku-CSF emijikelezweni emibili yokugcina yokukhetha zenziwe nge-454/Roche pyrosequencing. Zonke izifakelo zegazi ezivela emjikelezweni wokuqala wokukhetha nazo zenziwe nge-454/Roche pyrosequencing. Ukuze kujovwe ama-clone e-phage, izifakelo ezikhethiwe zandiswa ku-E. coli (BL5615) kumapuleti angu-500 cm2 ku-37°C amahora angu-4. Izifakelo ezikhethiwe ngazinye futhi ezenziwe nge-hand sequence zasakazeka endaweni ye-TB. Ngemva kokukhipha izifakelo, ukuhlanzwa nokususwa kwe-endotoxin (njengoba kuchaziwe ngenhla), izifakelo ze-2×1010/isilwane ku-300 μl zafakwa nge-intravenously emthanjeni owodwa womsila.
Ukucubungula kwangaphambili kanye nokuhlunga okunekhwalithi kwedatha yokulandelana. Idatha engahluziwe engu-454/Roche iguqulwe kusuka kufomethi yemephu yokusakaza ejwayelekile enama-binary (sff) yaya kufomethi efundekayo yomuntu ye-Pearson (fasta) kusetshenziswa isofthiwe yomthengisi. Ukucubungula okuqhubekayo kokulandelana kwe-nucleotide kwenziwa kusetshenziswa izinhlelo ze-C ezizimele kanye nezikripthi (iphakheji yesofthiwe engakhishwanga) njengoba kuchaziwe ngezansi. Ukuhlaziywa kwedatha eyinhloko kufaka phakathi izinqubo zokuhlunga eziqinile zezigaba eziningi. Ukuhlunga ukufundwa okwakungenalo uchungechunge lwe-DNA yokufaka olusebenzayo lwe-12mer, ukufundwa kwaqondaniswa ngokulandelana kwelebula yokuqala (GTGATGTCGGGGATCCGAATTCT), ilebula yokuma (TAAGCTTGCGGCCGCACTCGAGTA) kanye nokufakwa kwangemuva (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) kusetshenziswa ukuhlolwa kwe-Needleman-Wunsch global. ukulungiswa okuvumela ukungalingani okufika ku-2 ngokulungiswa ngakunye31. Ngakho-ke, ukufundwa ngaphandle kwamathegi okuqala nokuma kanye nokufundwa okuqukethe ukufakelwa kwangemuva, okungukuthi, ukuhambisana okudlula inani elivunyelwe lokungafani, kususiwe kulabhulali. Ngokuphathelene nokufundwa okusele, ukulandelana kwe-N-mer DNA okusukela kuphawu lokuqala kuya ekupheleni ngaphambi kokuba uphawu lokuma lukhishwe kuchungechunge lokuqala lokufunda futhi kuqhutshekwe kucutshungulwe (ngemuva kwalokhu okubizwa ngokuthi “faka”). Ngemva kokuhumusha i-insert, ingxenye ngemva kwe-codon yokuqala yokuma ekugcineni kwe-5′ ye-primer isuswa ku-insert. Ngaphezu kwalokho, ama-nucleotide aholela kuma-codon angaphelele ekugcineni kwe-3′ ye-primer nawo asusiwe. Ukuze kukhishwe ama-insert aqukethe ama-background sequences kuphela, ama-insert ahunyushwe aqala ngephethini ye-amino acid “PAG” nawo asusiwe. Ama-peptide anobude obungaphansi kwama-amino acid amathathu asusiwe kulabhulali. Ekugcineni, susa ukuphindaphinda echibini lokufaka bese unquma imvamisa ye-insert ngayinye eyingqayizivele. Imiphumela yalokhu kuhlaziywa ifaka uhlu lwe-nucleotide sequences (ifaka) kanye nama-frequency awo (okufunda) (Izibalo Ezingeziwe 1c no-2).
Ukufakwa kwe-DNA ye-Group N-mer ngokufana kokulandelana: Ukuze kususwe amaphutha okulandelana kwe-454/Roche (njengezinkinga zokwelulwa kwe-homopolymer kokulandelana) nokususa ama-redundancies angabalulekile kangako, ukufakwa kokulandelana kwe-DNA ye-N-mer okuhlungiwe ngaphambilini (okufakwayo) kuhlungwa ngokufana. ukufakwa (kuze kube yizisekelo ezi-2 ezingahambelani ezivunyelwe) kusetshenziswa i-algorithm ephindaphindayo echazwe kanje: ukufakwa kuhlungwa kuqala ngokuvama kwazo (okuphezulu kuya kokuphansi), futhi uma kufana, ngokuhlela kwazo kwesibili ngobude (okude kakhulu kuya kokufushane kakhulu) ). Ngakho-ke, ukufakwa okuvamile kakhulu nokude kakhulu kuchaza "iqembu" lokuqala. Imvamisa yeqembu isethwe kumvamisa yokhiye. Ngemuva kwalokho, ukufakwa ngakunye okusele ohlwini oluhleliwe kwazanywa ukungezwa eqenjini ngokulungiswa kwe-Needleman-Wunsch ngabubili. Uma inani lokungafani, ukufakwa, noma ukususwa ekulinganisweni lingadluli umkhawulo ongu-2, ukufakwa kwengezwa eqenjini, futhi imvamisa yeqembu iyonke iyanda ngokuthi ukufakwa kwengezwe kangaki. Ukufakwa kwengezwe eqenjini kumakwa njengokusetshenzisiwe futhi kungafakwa ekucutshungulweni okuqhubekayo. Uma ukulandelana kokufaka kungenakungezwa eqenjini elivele likhona, ukulandelana kokufaka kusetshenziselwa ukudala iqembu elisha elinemvamisa yokufaka efanele futhi kumakwe njengokusetshenziswayo. Ukuphindaphinda kuphela lapho ukulandelana ngakunye kokufaka sekusetshenziswe ukwakha iqembu elisha noma kungafakwa eqenjini elivele likhona. Phela, ukufakwa okuqoqwe ngamaqoqo okuhlanganisa ama-nucleotide ekugcineni kuhunyushwa kube ukulandelana kwama-peptide (imitapo yolwazi yama-peptide). Umphumela walokhu kuhlaziywa isethi yokufakwa kanye nemvamisa yazo ehambisanayo eyakha inani lokufunda okulandelanayo (Isithombe Esingeziwe 2).
Ukukhiqizwa Kwe-Motif: Ngokusekelwe ohlwini lwama-peptide ahlukile, kwadalwa umtapo wolwazi oqukethe wonke amaphethini e-amino acid (aa) angenzeka njengoba kuboniswe ngezansi. Iphethini ngayinye engaba khona yobude obungu-3 yakhishwa ku-peptide futhi iphethini yayo ephambene yanezelwa kanye nomtapo wolwazi ovamile we-motif oqukethe wonke amaphethini (ama-tripeptide). Imitapo yolwazi ye-motif ephindaphindwayo kakhulu yahlelwa ngokulandelana futhi kwasuswa ukuphindaphinda. Ngemuva kwalokho, ku-tripeptide ngayinye kumtapo wolwazi we-motif, sahlola ukuthi ikhona yini emtapweni wolwazi sisebenzisa amathuluzi okusebenzisa amakhompyutha. Kulesi simo, imvamisa ye-peptide equkethe i-tripeptide ye-motif etholakele iyangezwa futhi yabelwa ku-motif emtapweni wolwazi we-motif ("inani lama-motif"). Umphumela wokukhiqizwa kwe-motif uhlu olunezinhlangothi ezimbili oluqukethe konke ukuvela kwama-tripeptide (ama-motif) kanye namanani awo, okuyinani lokufunda ngokulandelana okuholela ku-motif ehambisanayo lapho ukufunda kuhlungwa, kuqoqwa, futhi kuhunyushwa. Ama-metric njengoba kuchaziwe ngemininingwane ngenhla.
Ukulungiswa kwenani lama-motif kanye nama-scatterplot ahambisanayo: Inani lama-motif esampula ngayinye lenziwe lajwayelekile kusetshenziswa
lapho u-ni kuyinani lezinto ezifundwayo eziqukethe isihloko u-i. Ngakho-ke, u-vi umele imvamisa yephesenti yezinto ezifundwayo (noma ama-peptide) aqukethe i-motif u-i kusampula. Amanani e-P enanini lezinto ezingajwayelekile ezifundwayo abalwe kusetshenziswa ukuhlolwa okuqondile kukaFisher. Ngokuphathelene nama-correlogram enani lezinto ezifundwayo, ukuhlangana kukaSpearman kubalwe kusetshenziswa inani lezinto ezifundwayo ezifundwayo no-R.
Ukuze kubonwe okuqukethwe kwama-amino acid endaweni ngayinye kulabhulali ye-peptide, kudalwe ama-logogram ewebhu 32, 33 (http://weblogo.threeplusone.com). Okokuqala, okuqukethwe kwama-amino acid endaweni ngayinye ye-peptide engu-12-mer kugcinwa ku-matrix engu-20×12. Ngemuva kwalokho, isethi yama-peptide ayi-1000 aqukethe okuqukethwe okufanayo kwe-amino acid endaweni ngayinye ikhiqizwa ngefomethi ye-fasta-sequence futhi inikezwe njengokufakwa ku-logo yewebhu 3, ekhiqiza ukumelwa kwesithombe kokuqukethwe kwe-amino acid ehlobene endaweni ngayinye. kulabhulali ye-peptide ethile. Ukuze kubonwe amasethi edatha amaningi, kwadalwa amamephu okushisa kusetshenziswa ithuluzi elithuthukiswe ngaphakathi ku-R (i-biosHeatmap, iphakheji ye-R engakakhishwa). Ama-dendrogram alethwe kumamephu okushisa abalwa kusetshenziswa indlela kaWard yokuhlanganisa ama-hierarchical nge-metric yebanga le-Euclidean. Ukuze kuhlaziywe izibalo zedatha yokulinganisa i-motif, amanani e-P okuthola amaphuzu angajwayelekile abalwa kusetshenziswa ukuhlolwa okuqondile kukaFisher. Amanani e-P kwamanye amasethi edatha abalwe ku-R kusetshenziswa i-t-test yomfundi noma i-ANOVA.
Ama-clone e-phage akhethiwe kanye nama-phage angenazo izifakelo ajovwe ngemithambo yomsila (ama-phage angu-2 × 1010/isilwane ku-300 μl PBS). Imizuzu eyishumi ngaphambi kokufakwa kwegazi kanye nokufakwa okulandelayo, izilwane ezifanayo zajovwe ngemithambo nge-100 μl ye-lectin ene-DyLight594 (i-Vector Laboratories Inc., DL-1177). Imizuzu engama-60 ngemva kokufakwa kwe-phage, amagundane ajovwe ngenhliziyo nge-50 ml PBS elandelwa yi-50 ml 4% PFA/PBS. Amasampula obuchopho aphinde alungiswa ubusuku bonke ku-4% PFA/PBS futhi acwiliswa ku-30% sucrose ubusuku bonke ku-4°C. Amasampula aqandisiwe nge-flash engxubeni ye-OCT. Ukuhlaziywa kwe-immunohistochemical kwamasampula aqandisiwe kwenziwe ekushiseni kwegumbi ku-cryosections ezingama-30 µm ezivinjiwe nge-1% BSA futhi kwafakwa ama-antibodies e-polyclonal FITC-labeled against T7 phage (Novus NB 600-376A) ku-4 °C. Kufakwe e-cubicle ubusuku bonke. Ekugcineni, izingxenye zagezwa izikhathi ezintathu nge-PBS futhi zahlolwa nge-confocal laser microscope (Leica TCS SP5).
Wonke ama-peptide anobumsulwa obuncane obungu-98% akhiwe yi-GenScript USA, afakwa i-biotinylated futhi afakwa i-lyophilized. I-Biotin iboshwe nge-triple glycine spacer eyengeziwe ku-N-terminus. Hlola wonke ama-peptide usebenzisa i-mass spectrometry.
I-Streptavidin (Sigma S0677) ixutshwe ne-peptide ye-biotinylated equimolar ephindwe kahlanu, i-peptide ye-BACE1 yokuvimbela i-biotinylated, noma inhlanganisela (isilinganiso esingu-3:1) ye-peptide ye-BACE1 yokuvimbela i-biotinylated kanye ne-peptide ye-BACE1 yokuvimbela ku-5–10% DMSO/efakwe ku-PBS. Ihora eli-1 ekushiseni kwegumbi ngaphambi komjovo. Ama-peptide ahlanganiswe ne-Streptavidin ajovwe nge-intravenously ngesilinganiso esingu-10 mg/kg komunye wemithambo yomsila yamagundane anomgodi wobuchopho.
Ukuhlushwa kwama-complexes e-streptavidin-peptide kuhlolwe yi-ELISA. Ama-plate e-Nunc Maxisorp microtiter (Sigma) ambozwe ubusuku bonke ku-4°C nge-antibody yegundane engu-1.5 μg/ml (Thermo, MA1-20011). Ngemva kokuvimba (i-blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) ekushiseni kwegumbi amahora ama-2, geza ipuleti nge-0.05% Tween-20/PBS (wash buffer) okwesibili, amasampula e-CSF kanye ne-plasma afakwe emithonjeni exutshwe ne-blocking buffer (plasma 1:10,000, CSF 1:115). Ipuleti labe selifakwa e-incubation ubusuku bonke ku-4°C nge-detection antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Ngemva kwezinyathelo ezintathu zokugeza, i-streptavidin yatholakala ngokufaka i-incubation kusisombululo se-TMB substrate (Roche) kuze kube yimizuzu engama-20. Ngemva kokumisa ukuthuthukiswa kombala nge-1M H2SO4, kala ukumuncwa ku-450 nm.
Umsebenzi we-streptavidin-peptide-BACE1 inhibitor complex uhlolwe yi-Aβ(1-40) ELISA ngokwephrothokholi yomenzi (Wako, 294-64701). Kafushane, amasampula e-CSF axutshwe ku-diluent ejwayelekile (1:23) futhi afakwa e-incuent ubusuku bonke ku-4°C kuma-plate angama-96-well ambozwe nge-antibody yokubamba i-BNT77. Ngemva kwezinyathelo ezinhlanu zokugeza, i-antibody ye-BA27 ehlanganiswe ne-HRP yanezelwa futhi yafakwa e-incubation amahora ama-2 ku-4°C., kwalandela izinyathelo ezinhlanu zokugeza. I-Aβ(1–40) yatholwa ngokufaka e-incubation kusisombululo se-TMB imizuzu engama-30 ekushiseni kwegumbi. Ngemva kokuthuthukiswa kombala kumiswe ngesisombululo sokumisa, linganisa ukumuncwa ku-450 nm. Amasampula e-plasma afakwa ekukhishweni kwesigaba esiqinile ngaphambi kwe-Aβ(1–40) ELISA. I-plasma yanezelwa ku-0.2% DEA (Sigma) kuma-plate angama-96-well futhi yafakwa e-incubation ekamelweni lokushisa imizuzu engama-30. Ngemva kokugeza amapuleti e-SPE ngokulandelana (Oasis, 186000679) ngamanzi kanye ne-100% methanol, amasampula e-plasma afakwa kumapuleti e-SPE futhi konke uketshezi kwasuswa. Amasampula ahlanzwa (kuqala nge-5% methanol bese kuba yi-30% methanol) futhi ahlanzwa nge-2% NH4OH/90% methanol. Ngemva kokomisa i-eluate ku-55°C imizuzu engama-99 ku-N2 current engaguquki, amasampula ancishiswa kuma-diluent ajwayelekile kwathi i-Aβ(1–40) yalinganiswa njengoba kuchaziwe ngenhla.
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