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Ihe mgbochi ọbara-ụbụrụ na ihe mgbochi ọbara-ụbụrụ na-egbochi ndị na-agwọ ọrịa biotherapy iru ihe mgbaru ọsọ ha na sistemụ akwara etiti, si otú a na-egbochi ọgwụgwọ dị irè nke ọrịa akwara ozi. Iji chọpụta ndị na-ebuga ụbụrụ ọhụrụ na vivo, anyị webatara ọbá akwụkwọ peptide phage T7 ma chịkọta ọbara na mmiri cerebrospinal (CSF) site na iji ụdị oke oke. E mepụtara klọn phage kpọmkwem na CSF nke ukwuu mgbe agba anọ nke nhọrọ gasịrị. Nnwale nke peptides nke onye ọ bụla gosiri mmụba karịrị okpukpu 1000 na CSF. E gosipụtara bioactivity nke nnyefe peptide na-aga n'ụbụrụ site na mbelata 40% na ọkwa amyloid-β na mmiri cerebrospinal site na iji BACE1 peptide inhibitor jikọtara na peptide transit ọhụrụ achọpụtara. Nsonaazụ ndị a na-egosi na peptides achọpụtara site na ụzọ nhọrọ phage in vivo nwere ike ịbụ ụzọ bara uru maka nnyefe sistemụ nke macromolecules na ụbụrụ yana mmetụta ọgwụgwọ.
Nnyocha ọgwụgwọ lekwasịrị anya na sistemụ akwara etiti (CNS) lekwasịrị anya nke ukwuu n'ịchọpụta ọgwụ na ihe ndị e ji eme ka mmadụ nwee ike ibuga ọgwụ n'ụbụrụ, na-enweghị mgbalị dị ukwuu n'ịchọpụta usoro ndị na-akwalite ibuga ọgwụ n'ụbụrụ. Nke a amalitela ịgbanwe ugbu a ka ibuga ọgwụ, ọkachasị nnukwu molekul, bụ akụkụ dị mkpa nke mmepe ọgwụ neuroscience nke oge a. Gburugburu ebe obibi nke sistemụ akwara etiti nwere nchekwa nke ọma site na sistemụ mgbochi cerebrovascular, nke gụnyere ihe mgbochi ọbara-ụbụrụ (BBB) ​​​​na ihe mgbochi ọbara-ụbụrụ (BCBB)1, na-eme ka ọ sie ike ibuga ọgwụ n'ụbụrụ1,2. A na-eme atụmatụ na ihe fọrọ nke nta ka ọ bụrụ ọgwụ molekul buru ibu niile na ihe karịrị 98% nke obere molekul ọgwụ na-apụ n'ụbụrụ3. Ọ bụ ya mere o ji dị oke mkpa ịchọpụta usoro mbugharị ụbụrụ ọhụrụ nke na-enye nnyefe ọgwụ ọgwụgwọ dị irè na nke a kapịrị ọnụ na CNS 4,5. Agbanyeghị, BBB na BCSFB na-enyekwa ohere dị mma maka ibuga ọgwụ ka ha na-abanye ma banye n'ime usoro niile nke ụbụrụ site na nnukwu vasculature ya. Ya mere, mbọ ndị a na-agba ugbu a iji ụzọ ndị na-abụghị ịwakpo nke nnyefe na ụbụrụ dabere na usoro nke nnyefe receptor-mediated (PMT) site na iji ihe nnata BBB6 endogenous. N'agbanyeghị ọganihu dị mkpa na-adịbeghị anya site na iji ụzọ transferrin receptor7,8, mmepe ọzọ nke sistemụ nnyefe ọhụrụ nwere njirimara ka mma dị mkpa. Iji mezuo nke a, ebumnuche anyị bụ ịchọpụta peptides nwere ike imezi njem CSF, ebe ọ bụ na enwere ike iji ha mee ihe iji nyefee macromolecules na CNS ma ọ bụ mepee ụzọ nnata ọhụrụ. Karịsịa, ndị nnata na ndị na-ebuga sistemu cerebrovascular (BBB na BSCFB) nwere ike ịbụ ihe mgbaru ọsọ nwere ike maka nnyefe ọgwụ biotherapeutic na-arụ ọrụ na kpọmkwem. Mmiri Cerebrospinal (CSF) bụ ngwaahịa nzuzo nke choroid plexus (CS) ma na-akpakọrịta kpọmkwem na mmiri interstitial nke ụbụrụ site na oghere subarachnoid na oghere ventricular4. N'oge na-adịbeghị anya, egosiri na mmiri cerebrospinal subarachnoid na-agbasa nke ukwuu n'ime interstitium nke ụbụrụ9. Anyị nwere olileanya ịnweta oghere parenchymal site na iji usoro ntinye subarachnoid a ma ọ bụ ozugbo site na BBB. Iji mezuo nke a, anyị tinyere usoro nhọrọ phage siri ike nke na-achọpụta peptides nke otu n'ime ụzọ abụọ a dị iche na-ebuga.
Ugbu a, anyị na-akọwa ụzọ nyocha ngosipụta in vivo phage nke usoro ya na CSF sample yana usoro mmepụta dị elu (HTS) iji nyochaa agba nhọrọ mbụ yana ụdị ọbá akwụkwọ kachasị elu. Emere nyocha na oke ndị maara ihe na cannula cisterna (CM) nke etinyere n'ime ya ruo mgbe ebighị ebi iji zere mmetọ ọbara. Ihe dị mkpa bụ na ụzọ a na-ahọrọ ma ihe ndị na-elekwasị anya n'ụbụrụ na peptides nwere ọrụ njem gafee ihe mgbochi cerebrovascular. Anyị jiri phages T7 n'ihi obere nha ha (~ 60 nm) 10 ma tụọ aro na ha dabara adaba maka ibufe vesicles nke na-enye ohere ịgafe ihe mgbochi endothelial na/ma ọ bụ epithelial-medulla. Mgbe agba anọ nke panning gasịrị, e kewapụrụ ọnụọgụgụ phage na-egosi mmụba CSF ​​na njikọ microvessel nke ụbụrụ. Ihe dị mkpa bụ na anyị nwere ike ịkwado nchọpụta anyị site n'igosi na peptides kacha mma a họọrọ na nke ejiri kemịkalụ mepụta nwere ike ibufe ibu protein n'ime mmiri cerebrospinal. Nke mbụ, e guzobere mmetụta ọgwụ nke CNS site na ijikọta peptide transit kacha elu na ihe na-egbochi peptide BACE1. Na mgbakwunye na igosi na usoro nyocha ọrụ in vivo nwere ike ịchọpụta peptides transport ụbụrụ ọhụrụ dị ka ndị na-ebu ibu protein dị irè, anyị na-atụ anya na ụzọ nhọrọ ọrụ yiri nke ahụ ga-adịkwa mkpa n'ịchọpụta ụzọ njem ụbụrụ ọhụrụ.
Dabere na nkeji ndị na-akpụ plaque (PFU), mgbe usoro nkwakọ ngwaahịa phage gasịrị, e mepụtara ma mepụta ọbá akwụkwọ nke peptides phage T7 linear 12-mer nke nwere ụdị dị iche iche nke ihe dị ka 109 (lee Ihe na Ụzọ). Ọ dị mkpa ịmara na anyị nyochachara ọbá akwụkwọ a nke ọma tupu panning in vivo. Mmụba PCR nke ihe nlele ọbá akwụkwọ phage site na iji primers agbanwere emepụtara amplicons nke dabara kpọmkwem na HTS (Foto Mgbakwunye 1a). N'ihi a) njehie usoro HTS11, b) mmetụta na ịdị mma nke primers (NNK)1-12, na c) ọnụnọ nke phage ụdị ọhịa (wt) (ihe ntinye ọkpụkpụ) na ọbá akwụkwọ nchekwa, e tinyere usoro nzacha usoro iji wepụta naanị ozi usoro akwadoro (Foto Mgbakwunye 1b). Usoro nzacha ndị a metụtara ọbá akwụkwọ usoro HTS niile. Maka ọbá akwụkwọ ọkọlọtọ, enwetara ngụkọta ọgụgụ 233,868, nke 39% gafere ihe nzacha ahụ ma jiri ya mee ihe maka nyocha ọbá akwụkwọ na nhọrọ maka agba ndị sochirinụ (Foto mgbakwunye 1c–e). Ọnụọgụgụ ndị ahụ bụ ọtụtụ ọnụọgụ nke ụzọ atọ dị n'ogologo yana elu na nucleotides 36 (Foto mgbakwunye 1c), na-akwado nhazi ọbá akwụkwọ (NNK) 1-12. Ihe dị mkpa bụ na ihe dị ka 11% nke ndị otu ọbá akwụkwọ nwere ihe ntinye PAGISRELVDKL nke nwere ụdị ọhịa iri na abụọ (wt), ihe fọrọ nke nta ka ọ bụrụ ọkara nke usoro (49%) nwere ihe ntinye ma ọ bụ nhichapụ. HTS nke ọbá akwụkwọ ọbá akwụkwọ kwadoro nnukwu ụdị peptides na ọbá akwụkwọ: a chọtara ihe karịrị 81% nke usoro peptide naanị otu ugboro na naanị 1.5% mere na ≥4 mbipụta (Foto mgbakwunye 2a). Ugboro ole nke amino acid (aa) n'ọnọdụ iri na abụọ niile dị na repertoire ahụ jikọtara nke ọma na ugboro ole a tụrụ anya ya maka ọnụọgụ codons nke repertoire NKK mebiri emebi mepụtara (Foto mgbakwunye 2b). Ugboro ole a hụrụ nke residues aa nke ihe ndị a tinyere na ntinye ndị a jikọtara nke ọma na ugboro ole a gbakọrọ (r = 0.893) (Foto mgbakwunye 2c). Nkwadebe nke resịị phage maka ịgba ọgwụ gụnyere usoro nke mmụba na iwepụ endotoxin. Egosila na nke a nwere ike ibelata ụdịdị nke resịị phage12,13. Ya mere, anyị depụtara usoro resịị phage nke e wepụrụ endotoxin n'usoro ma tụnyere ya na resịị mbụ iji mee atụmatụ ugboro ole nke AA. A hụrụ njikọ siri ike (r = 0.995) n'etiti resịị mbụ na resịị a mụbara ma dị ọcha (Foto mgbakwunye 2d), na-egosi na asọmpi dị n'etiti resịị e mụbara na efere site na iji phage T7 emeghị ka nnukwu nsogbu pụta ìhè. Ntụnyere a dabere na ugboro ole nke ihe atụ tripeptide dị na ọbá akwụkwọ ọ bụla, ebe ọ bụ na enweghị ike ijide ụdị ọbá akwụkwọ dị iche iche (~109) ọbụlagodi na HTS. Nyocha ugboro ole nke aa na ọnọdụ ọ bụla gosiri obere echiche dabere na ọnọdụ n'ọnọdụ atọ ikpeazụ nke repertoire etinyere (Foto mgbakwunye 2e). Na mmechi, anyị kwubiri na ịdị mma na ụdị dị iche iche nke ọbá akwụkwọ ahụ bụ ihe a nabatara, naanị obere mgbanwe dị iche iche ka a hụrụ n'ihi mmụba na nkwadebe nke ọbá akwụkwọ phage n'etiti ọtụtụ agba nke nhọrọ.
Enwere ike ime nlele mmiri cerebrospinal serial site na itinye cannula n'ime CM nke oke ndị maara ihe iji mee ka njirimara nke T7 phage a gbara n'ime akwara (iv) site na BBB na/ma ọ bụ BCSFB (Fig. 1a-b). Anyị jiri ogwe aka nhọrọ abụọ nọọrọ onwe ha (ogwe aka A na B) na agba atọ mbụ nke nhọrọ in vivo (Fig. 1c). Anyị ji nwayọọ nwayọọ mụbaa oke nhọrọ ahụ site na ibelata mkpokọta phage ewebatara na agba atọ mbụ nke nhọrọ. Maka agba nke anọ nke panning, anyị jikọtara ihe nlele sitere na alaka A na B ma mee nhọrọ atọ ọzọ nwere onwe ha. Iji mụọ ihe onwunwe in vivo nke ihe T7 phage na ụdị a, a gbara phage ụdị ọhịa (PAGISRELVDKL master insert) n'ime oke site na akwara ọdụ. Iweghachite phages site na mmiri cerebrospinal na ọbara n'oge dị iche iche gosiri na obere phages T7 icosahedral nwere usoro mwepụ ngwa ngwa site na ngalaba ọbara (Fig. 3). Dabere na titers enyere na oke ọbara nke oke, anyị gbakọrọ na naanị ihe dị ka 1% wt. phage sitere na dose enyere ka achọpụtara n'ọbara nkeji iri mgbe a gbasịrị ya n'ime akwara. Mgbe mbelata ngwa ngwa a gasịrị, a tụrụ mwepụta mbụ nwayọ nwayọ na ọkara ndụ nke nkeji 27.7. Ihe dị mkpa bụ na naanị phages ole na ole ka ewepụtara na ngalaba CSF, nke na-egosi obere ndabere maka mbugharị phage ụdị ọhịa n'ime ngalaba CSF (Foto mgbakwunye 3). Na nkezi, naanị ihe dị ka 1 x 10-3% titers nke phage T7 n'ọbara na 4 x 10-8% nke phages e tinyere na mbụ ka achọpụtara na mmiri cerebrospinal n'oge nnwale niile (0-250 nkeji). N'ụzọ dị mkpa, ọkara ndụ (nkeji 25.7) nke phage ụdị ọhịa na mmiri cerebrospinal yiri nke a hụrụ n'ọbara. Ihe ndị a na-egosi na ihe mgbochi nke kewara ngalaba CSF na ọbara dị n'ime oke ndị nwere CM, na-enye ohere ịhọrọ n'ime ụlọ akwụkwọ phage iji chọpụta klọn ndị a na-ebufe ngwa ngwa site na ọbara gaa na ngalaba CSF.
(a) Ịtọlite ​​usoro maka ịnwalegharị mmiri cerebrospinal (CSF) site na nnukwu ọdọ mmiri. (b) Ihe osise na-egosi ebe ihe mgbochi sistemụ akwara etiti (CNS) dị na sel na usoro nhọrọ ejiri chọpụta peptides ndị gafere ihe mgbochi ọbara-ụbụrụ (BBB) ​​​​na ihe mgbochi ọbara-ụbụrụ. (c) Usoro nhazi ngosipụta phage In vivo. Na agba nke ọ bụla nke nhọrọ, a gbara phages (ihe njirimara anụmanụ dị n'ime akụ) n'ime akwara. A na-edobe alaka abụọ ọzọ nọọrọ onwe ha (A, B) iche iche ruo agba nke anọ nke nhọrọ. Maka agba nke 3 na nke 4 nke nhọrọ, ejiri aka hazie klọn phage ọ bụla ewepụtara na CSF. (d) Kinetics nke phage nke kewapụrụ site na ọbara (gburugburu uhie) na mmiri cerebrospinal (triangle akwụkwọ ndụ akwụkwọ ndụ) n'oge agba nke mbụ nke nhọrọ na oke abụọ a na-akpọ cannulated mgbe a gbasịrị ya n'ime akwara nke ọbá akwụkwọ peptide T7 (2 x 1012 phages/anụmanụ). Ákwà acha anụnụ anụnụ na-egosi nkezi mkpokọta mbụ nke phage n'ime ọbara, nke a gbakọrọ site na ọnụọgụ phage a gbara ọgwụ, na-eburu n'uche mkpokọta ọbara. Ákwà ojii ahụ na-egosi ebe njikọ nke ahịrị y nke ewepụtara site na njupụta phage ọbara. (e,f) Gosi ugboro ole na nkesa nke ihe niile nwere ike ịdị na peptide ahụ. Egosipụtara ọnụọgụ nke ihe ndị dị na ọgụgụ 1000. Ihe dị mkpa (p < 0.001) ihe ndị dị mma nwere ntụpọ uhie. (e) Ihe ndị dị mma nwere ntụpọ na-atụnyere ugboro ole nke ihe ndị dị mma nke tripeptide nke ọbá akwụkwọ a gbara ọgwụ na phage sitere na ọbara sitere na anụmanụ #1.1 na #1.2. (f) Ihe ndị dị mma nwere ntụpọ na-atụnyere ugboro ole nke ihe ndị dị mma nke phage anụmanụ #1.1 na #1.2 nke dịpụrụ adịpụ n'ọbara na mmiri cerebrospinal. (g, h) Ihe nnọchiteanya nke ihe ndị dị mma nke phage jupụtara na ọbara (g) megide ọbá akwụkwọ a gbara ọgwụ na phage jupụtara na CSF (h) megide ọbara mgbe agbachara nke nhọrọ in vivo na anụmanụ abụọ ahụ. Nha nke koodu otu mkpụrụedemede na-egosi ugboro ole amino acid ahụ na-apụta n'ọnọdụ ahụ. Akwụkwọ ndụ akwụkwọ ndụ = polar, odo odo = neutral, acha anụnụ anụnụ = basic, ọbara ọbara = acidic na oji = hydrophobic amino acid. Eduard Urich chepụtara ma mepụta Foto 1a, b.
Anyị gbara ọbá akwụkwọ phage peptide n'ime oke CM abụọ (clades A na B) ma kewapụ phage site na mmiri cerebrospinal na ọbara (Foto 1d). Mwepụ ngwa ngwa nke ọbá akwụkwọ ahụ adịchaghị mma ma e jiri ya tụnyere phage ụdị anụ ọhịa. Ọkara ndụ nke ọbá akwụkwọ a gbara n'ime anụmanụ abụọ ahụ bụ nkeji 24.8 n'ọbara, yiri phage ụdị anụ ọhịa, na nkeji 38.5 na CSF. E tinyere ihe nlele phage ọbara na mmiri cerebrospinal site na anụmanụ ọ bụla na HTS ma nyochaa peptides niile achọpụtara maka ọnụnọ nke obere tripeptide motif. A họọrọ ụdị tripeptide n'ihi na ha na-enye obere ntọala maka nhazi nhazi na mmekọrịta peptide-protein14,15. Anyị chọtara ezigbo njikọ na nkesa nke motifs n'etiti ọbá akwụkwọ phage a gbara na clones ewepụtara na ọbara anụmanụ abụọ ahụ (Foto 1e). Data ahụ na-egosi na ihe mejupụtara ọbá akwụkwọ ahụ bụ naanị obere ihe bara ụba na ngalaba ọbara. E nyochakwuru ugboro amino acid na usoro nkwekọrịta n'ọnọdụ ọ bụla site na iji mgbanwe nke ngwanrọ Weblogo16. N'ụzọ na-akpali mmasị, anyị hụrụ mmụba dị ukwuu na ihe fọdụrụ n'ọbara glycine (Foto 1g). Mgbe a tụnyere ọbara na klọn ndị a họọrọ site na CSF, a hụrụ nhọrọ siri ike na ụfọdụ nhọrọ nke motifs (Foto 1f), ụfọdụ amino acid dịkwa na ọnọdụ edobere na mmadụ iri na abụọ ahụ (Foto 1h). Ihe pụrụ iche bụ na anụmanụ dị iche iche nwere nnukwu ọdịiche na mmiri cerebrospinal, ebe a hụrụ mmụba glycine n'ọbara n'ime anụmanụ abụọ ahụ (Foto 4a–j). Mgbe a sachara data usoro dị na mmiri cerebrospinal nke anụmanụ #1.1 na #1.2 siri ike, enwetara mkpokọta peptides 12-mer pụrụ iche 964 na 420 (Foto 1d–e). E mekwuru klọn phage ndị dịpụrụ adịpụ ma tinye ha n'agba nke abụọ nke nhọrọ in vivo. E tinyere HTS n'ime anụmanụ ọ bụla, e jikwa peptide niile achọpụtara dị ka ntinye na mmemme njirimara motif iji nyochaa ihe ndị na-eme ka tripeptide pụta (Foto 2a, b, ef). Ma e jiri ya tụnyere okirikiri mbụ nke phage ahụ eweghachitere na CSF, anyị hụrụ nhọrọ ọzọ na iwepụ ọtụtụ motifs na CSF n'alaka A na B (Foto 2). E tinyere algọridim njirimara netwọk iji chọpụta ma ha nọchiri anya usoro dị iche iche nke usoro na-agbanwe agbanwe. A hụrụ myirịta doro anya n'etiti usoro 12-dimensional nke CSF weghachitere na clade ọzọ A (Foto 2c, d) na clade B (Foto 2g, h). Nnyocha a chịkọtara na alaka ọ bụla kpughere profaịlụ nhọrọ dị iche iche maka peptides 12-mer (Foto 5c, d) na mmụba na oke CSF/ọbara ka oge na-aga maka clones chịkọtara mgbe agba nke abụọ nke nhọrọ gasịrị ma e jiri ya tụnyere agba nke mbụ nke nhọrọ (Foto 5e).).
Ịbawanye ihe ndị e ji emepụta ihe na peptides n'ime mmiri cerebrospinal site na agba abụọ nke nhọrọ ngosipụta phage ọrụ in vivo.
E jikọtara phages mmiri cerebrospinal niile e nwetara site na agba mbụ nke anụmanụ ọ bụla (anụmanụ #1.1 na #1.2) ọnụ, gbasaa, tinye usoro HT ma gbanye ha ọzọ (phages 2 x 1010/anụmanụ) 2 SM cannulated oke (#1.1 → #). 2.1 na 2.2, 1.2 → 2.3 na 2.4). (a,b,e,f) Usoro mgbasa ozi mmekọrịta na-atụnyere ugboro ole nke motifs tripeptide nke phages niile sitere na CSF na agba nhọrọ mbụ na nke abụọ. Ugboro ole na nkesa nke motifs na-anọchite anya tripeptides niile nwere ike ịdị na peptides na ntụzịaka abụọ ahụ. Egosiputara ọnụọgụ motifs achọtara ma ọ bụ wepụ na otu n'ime ọbá akwụkwọ atụnyere ya na ntụpọ uhie. (c, d, g, h) Nnọchianya akara usoro nke usoro amino acid 12 niile bara ọgaranya na CSF dabere na agba nke 2 na 1 nke nhọrọ in vivo. Nha nke koodu mkpụrụedemede otu na-egosi ugboro ole amino acid ahụ na-eme n'ọnọdụ ahụ. Iji nọchite anya akara ngosi ahụ, a na-atụnyere ugboro ole usoro CSF ​​ewepụtara site na anụmanụ dị iche iche n'etiti agba nhọrọ abụọ ma gosipụta usoro ndị bara ọgaranya na agba nke abụọ: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 na (h) #1.2–#2.4. A na-egosi amino acid kachasị baa ọgaranya n'ọnọdụ enyere na (c, d) anụmanụ nọmba 2.1 na nọmba 2.2 ma ọ bụ (g, h) na anụmanụ nọmba 2.3 na nọmba 2.4 na agba. Akwụkwọ ndụ akwụkwọ ndụ = polar, odo odo = na-anọpụ iche, acha anụnụ anụnụ = isi, ọbara ọbara = acidic na oji = amino acid hydrophobic.
Mgbe agba nke atọ nke nhọrọ gasịrị, anyị chọpụtara usoro peptide pụrụ iche 124 (#3.1 na #3.2) site na klọn phage 332 nke CSF-reconstituted nke e kewapụrụ site na anụmanụ abụọ (Foto mgbakwunye 6a). Usoro LGSVS (18.7%) nwere oke kachasị elu, nke sochiri ntinye ụdị ọhịa PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), na SARGSWREIVSLS (2.2%). Na agba nke anọ ikpeazụ, anyị chịkọtara alaka abụọ ahọpụtara n'onwe ha site na anụmanụ atọ dị iche iche (Foto 1c). N'ime klọn phage 925 nke e weghachitere na CSF, na agba nke anọ anyị hụrụ usoro peptide pụrụ iche 64 (Foto mgbakwunye 6b), nke n'ime ha bụ oke nke phage ụdị ọhịa dara ruo 0.8%. Klọn CSF kachasị na agba nke anọ bụ LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) na RLSSVDSDLSGC (3, 2%). %). Ogologo peptides ahọpụtara bụ n'ihi ntinye/nhichapụ nucleotide ma ọ bụ codons stop stop na primers ọbá akwụkwọ mgbe ejiri codons degenerate maka imewe ọbá akwụkwọ NNK. Codons stop stop tupu oge eruo na-emepụta peptides dị mkpụmkpụ ma a na-ahọrọ ha n'ihi na ha nwere motif aa dị mma. Peptides ndị dị ogologo nwere ike ịpụta site na ntinye/nhichapụ na primers nke ọbá akwụkwọ sịntetik. Nke a na-etinye codon stop emebere n'èzí etiti ahụ ma na-agụ ya ruo mgbe codon stop ọhụrụ pụtara n'okpuru ala. N'ozuzu, anyị gbakọrọ ihe ndị na-eme ka agba nhọrọ anọ niile dị mma site na iji data ntinye tụnyere data mmepụta ihe atụ. Maka agba mbụ nke nyocha ahụ, anyị jiri ụdị phage nke ụdị ọhịa dị ka ntụaka ndabere na-abụghị nke a kapịrị ọnụ. N'ụzọ na-akpali mmasị, nhọrọ phage na-adịghị mma siri ike nke ukwuu na okirikiri CSF mbụ, mana ọ bụghị n'ọbara (Fig. 3a), nke nwere ike ịbụ n'ihi obere ohere nke mgbasa ozi na-enweghị isi nke ọtụtụ ndị otu peptide n'ime ngalaba CSF ma ọ bụ phages ndị metụtara ya na-adịkarị ka a na-ejide ma ọ bụ wepụ ya n'ọbara karịa bacteriophages. Agbanyeghị, na agba nke abụọ nke panning, a hụrụ nhọrọ siri ike nke phages na CSF na klades abụọ ahụ, na-atụ aro na agba gara aga bara ụba na phages na-egosipụta peptides nke na-akwalite nnabata CSF (Fig. 3a). Ọzọkwa, na-enweghị mmụba ọbara dị ukwuu. Ọzọkwa na agba nke atọ na nke anọ, clones phage bara ụba nke ukwuu na CSF. Na-atụnyere ugboro ole nke usoro peptide pụrụ iche ọ bụla n'etiti agba abụọ ikpeazụ nke nhọrọ, anyị chọpụtara na usoro ndị ahụ bara ụba karịa na agba nke anọ nke nhọrọ (Fig. 3b). E wepụtara mkpokọta ụdị tripeptide 931 site na usoro peptide pụrụ iche 64 niile site na iji ntụzịaka peptide abụọ ahụ. E lere ụdị tripeptide kachasị baa ọgaranya na agba nke anọ anya nke ọma maka profaịlụ mmụba ha n'ofe agba niile ma e jiri ya tụnyere ọbá akwụkwọ a gbara ọgwụ (mbepụ: 10% mmụba) (Foto mgbakwunye 6c). Usoro nhọrọ izugbe gosiri na ọtụtụ n'ime ebumnuche ndị a mụrụ bara ụba na agba niile gara aga nke alaka nhọrọ abụọ ahụ. Agbanyeghị, ụfọdụ ụdị (dịka SGL, VSG, LGS GSV) sitere na ụdị A ọzọ, ebe ndị ọzọ (dịka FGW, RTN, WGF, NTR) bara ọgaranya na ụdị B ọzọ.
Nnwale nke mbupu CSF nke peptides CSF-enriched phage-displayed na biotinylated leader peptides jikọtara na streptavidin payloads.
(a) Oke mmụba agbakọrọ na agba anọ niile (R1-R4) dabere na titers phage (PFU) a gbanyere (input = I) na titers phage (CSF phage titers) a chọpụtaworo (output = O). A gbakọrọ ihe ndị na-eme ka mmụba maka agba atọ ikpeazụ (R2-R4) site na iji ya tụnyere agba gara aga na agba nke mbụ (R1) yana data ibu. Ogwe ndị mepere emepe bụ mmiri cerebrospinal, ogwe ndị nwere onyinyo bụ plasma. (***p<0.001, dabere na ule t nke nwa akwụkwọ). (b) Ndepụta nke peptides phage kachasị baa ụba, nke a na-ahazi dịka nha ha si dị na phages niile anakọtara na CSF mgbe agba nke anọ nke nhọrọ gasịrị. A na-egosipụta clones phage isii kachasị na agba, ọnụọgụgụ na ihe ndị na-eme ka ha baa uru n'etiti agba nke 3 na nke 4 nke nhọrọ (insets). (c,d) E nyochachara clones phage isii kacha baa ọgaranya, phage efu na ọbá akwụkwọ peptide phage nne na nna site na agba nke anọ n'otu n'otu na ụdị nlele CSF. A na-anakọta ihe nlele CSF na ọbara na oge egosipụtara. (c) Nha nhata nke klọn phage isii (2 x 1010 phages/anụmanụ), phages efu (#1779) (2 x 1010 phages/anụmanụ) na stock phage peptide libraries (2 x 1012 phages/anụmanụ). A na-enye anụmanụ a na-akpọ cannulated ma ọ dịkarịa ala 3 CM n'ime ya iche iche site na ọdụ akwara. Egosiri pharmacokinetics nke ọ bụla a gbara ọgwụ phage clone na phage peptide library ka oge na-aga. (d) na-egosi nkezi oke CSF/ọbara maka phages/mL niile eweghachitere n'oge nlele. (e) E jikọtara peptides anọ e ji aka mee na otu njikwa a gbagọrọ agbagọ na biotin na streptavidin site na N-terminus ha (ngosipụta tetramer) wee nye ya ọgwụ (ọdụ akwara iv, 10 mg streptavidin/kg). Opekata mpe oke atọ a na-etinye n'ime mmiri (N = 3). A chịkọtara ihe nlele CSF n'oge a kapịrị ọnụ, e jikwa CSF anti-streptavidin ELISA tụọ njupụta streptavidin (nd = achọpụtaghị ya). (*p<0.05, **p<0.01, ***p<0.001, dabere na ule ANOVA). (f) Ntụnyere usoro amino acid nke clone peptide phage kachasị baa ọgaranya #2002 (odo odo) na clones peptide phage ndị ọzọ ahọpụtara site na agba nke anọ nke nhọrọ. E ji agba koodu agba mee iberibe amino acid ndị yiri ya na ndị yiri ya.
N'ime phages niile bara ọgaranya na agba nke anọ (Foto 3b), ahọpụtara klọn isii maka nyocha nkeonwe ọzọ na ụdị nlele CSF. E tinyere phage isii, phage efu (enweghị ntinye) na prophage peptide libraries n'ime anụmanụ CM atọ a na-akpọ cannulated, a chọpụtakwara pharmacokinetics na nnwale CSF (Foto 3c) na ọbara (Foto mgbakwunye 7). Klọn phage niile a nwalere lekwasịrị anya na ngalaba CSF na ọkwa 10-1000 ugboro karịa nke phage njikwa efu (#1779). Dịka ọmụmaatụ, klọn #2020 na #2077 nwere ihe dị ka ugboro 1000 karịa titers CSF karịa phage njikwa. Profaịlụ pharmacokinetic nke peptide ọ bụla ahọpụtara dị iche, mana ha niile nwere ikike ibugharị CSF dị elu. Anyị hụrụ mbelata na-adịgide adịgide maka klọn #1903 na #2011, ebe maka klọn #2077, #2002 na #2009 mmụba n'ime nkeji iri mbụ nwere ike igosi njem na-arụ ọrụ mana ọ dị mkpa ka a chọpụta ya. Klọn #2020, #2002, na #2077 kwụsiri ike na ọkwa dị elu, ebe CSF nke klọn #2009 belatara nwayọ nwayọ mgbe mmụba mbụ gasịrị. Anyị wee tụnyere ugboro ole nke onye CSF ọ bụla na ntinye ọbara ya (Fig. 3d). Njikọ nke nkezi titer nke onye CSF ọ bụla na titer ọbara ya n'oge nnwale niile gosiri na atọ n'ime ndị na-achọ CSF bara ụba nke ukwuu na CSF ọbara. N'ụzọ na-adọrọ mmasị, klọn #2077 gosipụtara nkwụsi ike ọbara dị elu (Foto Mgbakwunye 7). Iji gosi na peptides n'onwe ha nwere ike ibufe ibu ndị ọzọ na-abụghị mkpụrụ phage n'ime ngalaba CSF, anyị mepụtara peptides ndu anọ nke e si na biotin nweta na N-terminus ebe peptides na-ejikọ na mkpụrụ phage. E jikọtara peptides biotinylated (nọmba 2002, 2009, 2020 na 2077) na streptavidin (SA) iji nweta ụdị multimeric dị ka geometry phage. Usoro a nyekwara anyị ohere ịtụ mkpughe SA n'ọbara na mmiri cerebrospinal dị ka peptides protein na-ebu ibu. Ihe dị mkpa bụ na enwere ike ịmegharị data phage mgbe e nyere peptides sịntetik na usoro SA-conjugated a (Fig. 3e). Peptides ndị a gbagọrọ agbagọ enweghị mkpughe mbụ na mwepụ CSF ngwa ngwa yana ọkwa a na-apụghị ịchọpụta n'ime awa 48. Iji nweta nghọta n'ụzọ nnyefe nke clones phage peptide ndị a na oghere CSF, anyị nyochara ebe nke peptide phage nke ọ bụla na-abanye na oghere CSF site na iji immunohistochemistry (IHC) iji chọpụta kpọmkwem ihe ndị dị na phage otu awa mgbe a gbasịrị ya n'ime akwara na vivo. Ihe dị mkpa bụ na a pụrụ ịchọpụta klọn #2002, #2077, na #2009 site na ntụpọ siri ike na capillaries ụbụrụ, ebe achọpụtaghị phage njikwa (#1779) na klọn #2020 (Foto mgbakwunye 8). Nke a na-egosi na peptides ndị a na-enye aka na mmetụta na ụbụrụ kpọmkwem site na ịgafe BBB. A chọrọ nyocha zuru ezu iji nwalee echiche a, ebe ụzọ BSCFB nwekwara ike itinye aka. Mgbe a na-atụnyere usoro amino acid nke klọn kachasị baa ọgaranya (#2002) na peptides ndị ọzọ ahọpụtara, a chọpụtara na ụfọdụ n'ime ha nwere ndọtị amino acid yiri nke ahụ, nke nwere ike igosi usoro njem yiri nke ahụ (Foto 3f).
N'ihi profaịlụ plasma pụrụ iche ya na mmụba dị ukwuu na CSF ka oge na-aga, a nyochakwuru clone ngosi phage #2077 n'ime oge awa 48 dị ogologo ma nwee ike ịmụpụta mmụba ngwa ngwa na CSF a hụrụ na njikọ ya na ọkwa SA na-adịgide adịgide (Fig. 4a). Banyere clone phage ndị ọzọ achọpụtara, #2077 nwere agba siri ike maka capillaries ụbụrụ ma gosipụta njikọ dị mkpa na lectin akara capillary mgbe a lere ya na mkpebi dị elu yana ikekwe ụfọdụ ntụpọ na oghere parenchymal (Foto 4b). Iji nyochaa ma enwere ike nweta mmetụta ọgwụ peptide na CNS, anyị mere nnwale nke agwakọtara ụdị biotinylated nke i) peptide transit #2077 na ii) peptide inhibitor BACE1 na SA na oke abụọ dị iche iche. Maka otu njikọta anyị jiri naanị BACE1 peptide inhibitor na maka nke ọzọ anyị jiri oke 1:3 nke BACE1 peptide inhibitor na #2077 peptide. E tinyere ihe nlele abụọ a n'ime ọbara, a tụkwara ọkwa mmiri ọgwụ beta-amyloid peptide 40 (Abeta40) n'ọbara na cerebrospinal ka oge na-aga. A tụrụ Abeta40 na CSF ka ọ na-egosipụta mgbochi BACE1 na parenchyma ụbụrụ. Dịka a tụrụ anya ya, ihe mgbagwoju anya abụọ ahụ belatara ọkwa ọbara nke Abeta40 nke ukwuu (Foto 4c, d). Agbanyeghị, naanị ihe nlele nwere ngwakọta nke peptide nọmba 2077 na ihe na-egbochi peptide BACE1 nke ejikọtara na SA kpatara mbelata dị ukwuu na Abeta40 na mmiri cerebrospinal (Foto 4c). Data ahụ na-egosi na peptide nọmba 2077 nwere ike ibuga protein 60 kDa SA na CNS ma na-akpalikwa mmetụta ọgwụ site na ihe mgbochi SA nke BACE1 conjugated.
(a) Ntụtụ clonal (2 × 10 phages/anụmanụ) nke phage T7 nke na-egosi profaịlụ pharmacokinetic ogologo oge nke peptide CSF #2077 (RLSSVDSDLSGC) na phage njikwa na-enweghị ọgwụ (#1779) na opekata mpe oke atọ nke CM-intubated. (b) Foto mikroskopu nke confocal microvessels nke ndị nnọchi anya cortical na oke ndị a gbara phage (2 × 10 10 phages/anụmanụ) nke na-egosi mgbochi nke peptide #2077 na arịa (lectin). E nyere klọn phage ndị a na oke atọ ma hapụ ha ka ha gbasaa ruo otu awa tupu a gbaa ha. E kewara ụbụrụ ma tee ha ihe mgbochi polyclonal FITC nke a na-akpọ megide T7 phage capsid. Nkeji iri tupu a gbaa ha na mgbe e mesịrị, e nyere lectin DyLight594-labeled intravenously. Foto ndị na-enwu enwu na-egosi ntụpọ lectin (uhie) nke akụkụ luminal nke microvessels na phages (akwụkwọ ndụ akwụkwọ ndụ) na lumen nke capillaries na anụ ahụ ụbụrụ perivascular. Ogwe nha ahụ kwekọrọ na 10 µm. (c, d) E jikọtara biotinylated BACE1 inhibitory peptide naanị ya ma ọ bụ na njikọta ya na biotinylated transit peptide #2077 na streptavidin wee tinye ya n'ime akwara nke opekata mpe oke CM atọ a na-akpọ cannulated (10 mg streptavidin/kg). E jiri Aβ1-40 ELISA tụọ mbelata BACE1 peptide inhibitor na Aβ40 na ọbara (uhie) na cerebrospinal fluid (oroma) na oge egosiri. Maka nghọta ka mma, a na-ese ahịrị ntụpọ na eserese ahụ na nha nke 100%. (c) Mbelata pasentị na Aβ40 n'ọbara (triangle uhie) na mmiri cerebrospinal (triangle oroma) n'ime oke ejiri streptavidin jikọtara na transit peptide #2077 na peptide inhibitory BACE1 gwọọ na nha 3:1. (d) Mbelata pasentị na ọbara Aβ40 (gburugburu uhie) na mmiri cerebrospinal (gburugburu oroma) nke oke ejiri streptavidin gwọọ na peptide inhibitory BACE1 naanị. Ntinye Aβ na njikwa ahụ bụ 420 pg/ml (nhazi ọkọlọtọ = 101 pg/ml).
A na-eji ngosipụta phage eme ihe nke ọma n'ọtụtụ ebe nyocha ahụike17. A na-eji usoro a eme ihe maka ọmụmụ ihe gbasara ọdịiche dị n'ọbara in vivo18,19 yana ọmụmụ ihe gbasara arịa ụbụrụ20,21,22,23,24,25,26. N'ọmụmụ ihe a, anyị gbasaa ojiji nke usoro nhọrọ a ọ bụghị naanị na ịchọpụta kpọmkwem peptides na-elekwasị anya na arịa ụbụrụ, kamakwa na ịchọpụta ndị nwere ihe ndị na-eme ka mmadụ nwee ike ịgafe ihe mgbochi ọbara-ụbụrụ. Anyị na-akọwa ugbu a mmepe nke usoro nhọrọ in vivo na oke CM intubated ma gosipụta ikike ya ịchọpụta peptides nwere ihe ndị na-eme ka mmadụ nwee ike ịchọta CSF homing. Site na iji phage T7 nke na-egosipụta ọbá akwụkwọ nke peptides random 12-mer, anyị nwere ike igosi na phage T7 dị obere nke ukwuu (ihe dị ka 60 nm na dayameta)10 ka a gbanwee ya na ihe mgbochi ọbara-ụbụrụ, si otú a na-agafe ihe mgbochi ọbara-ụbụrụ ma ọ bụ choroid plexus ozugbo. Anyị hụrụ na owuwe ihe ubi CSF site na oke CM a kpọchiri akpọchi bụ usoro nyocha ọrụ in vivo nke a na-achịkwa nke ọma, nakwa na phage ewepụtara abụghị naanị nke ejikọtara na vasculature kamakwa ọ rụrụ ọrụ dị ka onye na-ebufe ihe mgbochi ọbara-ụbụrụ. Ọzọkwa, site n'ịchịkọta ọbara na itinye HTS na CSF ​​na phages sitere na ọbara, anyị kwadoro na nhọrọ anyị nke CSF abụghị nke mmụba ọbara ma ọ bụ ahụike maka mgbasawanye n'etiti agba nhọrọ. Agbanyeghị, ngalaba ọbara bụ akụkụ nke usoro nhọrọ, ebe ọ bụ na phages nwere ike iru ngalaba CSF ga-adị ndụ ma na-agbasa n'ọbara ruo ogologo oge iji mee ka onwe ha baa ọgaranya n'ụbụrụ. Iji wepụta ozi usoro a pụrụ ịdabere na ya site na data HTS raw, anyị tinyere nzacha ndị a haziri maka njehie usoro nhazi kpọmkwem na usoro nyocha. Site n'itinye paramita kinetic n'ime usoro nyocha, anyị kwadoro ngwa ngwa pharmacokinetics nke ụdị T7 phages (t½ ~ 28 nkeji) na ọbara24, 27, 28 ma chọpụtakwa ọkara ndụ ha na mmiri cerebrospinal (t½ ~ 26 nkeji) kwa nkeji). N'agbanyeghị ụdị profaịlụ ọgwụ pharmacokinetic dị otu a n'ọbara na CSF, naanị 0.001% nke mkpokọta ọbara nke phage ka achọpụtara na CSF, na-egosi obere ngagharị ndabere nke phage T7 ụdị ọhịa gafere ihe mgbochi ọbara-ụbụrụ. Ọrụ a na-egosi mkpa nke agba mbụ nke nhọrọ mgbe a na-eji usoro panning in vivo, karịsịa maka sistemụ phage nke a na-ewepụ ngwa ngwa site na mgbasa ozi, ebe ọ bụ na clones ole na ole nwere ike iru ngalaba CNS. Ya mere, na agba nke mbụ, mbelata na ụdị ọbá akwụkwọ dị oke ukwuu, ebe ọ bụ naanị ọnụọgụ clones ole na ole ka emechachara chịkọta na ụdị CSF siri ike a. Usoro panning in vivo a gụnyere ọtụtụ usoro nhọrọ dị ka mkpokọta na-arụ ọrụ na ngalaba CSF, ndụ clone na ngalaba ọbara, na mwepụ ngwa ngwa nke clones phage T7 site na ọbara n'ime nkeji iri mbụ (Fig. 1d na Fig. Mgbakwunye 4M). ). Ya mere, mgbe agba nke mbụ gasịrị, achọpụtara clones phage dị iche iche na CSF, ọ bụ ezie na ejiri otu ọdọ mmiri mbụ ahụ mee ihe maka anụmanụ nke ọ bụla. Nke a na-egosi na ọtụtụ usoro nhọrọ siri ike maka ọbá akwụkwọ isi mmalite nwere ọtụtụ ndị otu ọbá akwụkwọ na-ebute mbelata dị ukwuu na ụdịdị dị iche iche. Ya mere, ihe omume na-enweghị usoro ga-abụ akụkụ dị mkpa nke usoro nhọrọ mbụ, na-emetụta nke ukwuu nsonaazụ ya. O yikarịrị ka ọtụtụ n'ime klọn ndị dị na ọbá akwụkwọ mbụ ahụ nwere oke mmasị ịba ụba CSF. Agbanyeghị, ọbụlagodi n'okpuru otu ọnọdụ nnwale ahụ, nsonaazụ nhọrọ nwere ike ịdị iche n'ihi obere ọnụọgụ nke klọn ọ bụla dị na ọdọ mmiri mbụ.
Ihe ndị e ji CSF mee ka ha baa ọgaranya dị iche na nke dị n'ọbara. Ọ dị mma ịmara na anyị hụrụ mgbanwe mbụ e mere gaa na peptides bara ọgaranya na glycine n'ọbara anụmanụ dị iche iche. (Foto 1g, Foto Mgbakwunye 4e, 4f). Phage nwere peptides glycine nwere ike ịdịgide ma ghara ịdị mfe iwepụ ha n'ọbara. Agbanyeghị, achọpụtaghị peptides bara ọgaranya na glycine na ihe nlele mmiri cerebrospinal, na-egosi na ọbá akwụkwọ ndị a chịkọtara agafeela usoro nhọrọ abụọ dị iche iche: otu n'ime ọbara na nke ọzọ a na-ahapụ ka ọ gbakọta na mmiri cerebrospinal. A nwaleela clones bara ọgaranya nke CSF sitere na agba nke anọ nke nhọrọ nke ukwuu. E gosipụtara na ihe fọrọ nke nta ka ọ bụrụ clones niile a nwalere n'otu n'otu nwere CSF ma e jiri ya tụnyere phage njikwa efu. E lere otu peptide hit (#2077) anya nke ọma. O gosiri ọkara ndụ plasma dị ogologo ma e jiri ya tụnyere ndị ọzọ kụrụ (Foto 3d na Foto Mgbakwunye 7), ihe na-atọkwa ụtọ bụ na peptide a nwere ihe fọdụrụ na cysteine ​​​​na C-terminus. E gosila n'oge na-adịbeghị anya na mgbakwunye nke cysteine ​​​​na peptides nwere ike imeziwanye njirimara pharmacokinetic ha site na ijikọ na albumin 29. Nke a bụ ihe a na-amaghị ugbu a maka peptide #2077 ma chọọ ọmụmụ ihe ọzọ. Ụfọdụ peptides gosipụtara ịdabere na valence na mmụba CSF (egosighi data), nke nwere ike ịdị na geometry elu egosiri nke T7 capsid. Sistemụ T7 anyị jiri gosiri mbipụta 5-15 nke peptide ọ bụla kwa ihe phage. Emere IHC na clones lead phage ndị a gbanyere n'ime cerebral cortex nke oke (Foto mgbakwunye 8). Data ahụ gosiri na opekata mpe clones atọ (Nke 2002, Nke 2009 na Nke 2077) na BBB mekọrịtara. A ka ga-ekpebi ma mmekọrịta BBB a na-ebute mkpokọta CSF ma ọ bụ mmegharị nke clones ndị a ozugbo na BCSFB. Ihe dị mkpa bụ na anyị na-egosi na peptides ahọpụtara na-ejigide ikike njem CSF ha mgbe a na-emepụta ma jikọta ya na ibu protein. Njikọ nke peptides biotinylated N-terminal na SA na-emeghachi ihe ndị e nwetara site na klọn phage ha n'ọbara na mmiri cerebrospinal (Foto 3e). N'ikpeazụ, anyị na-egosi na peptide lead #2077 nwere ike ịkwalite ọrụ ụbụrụ nke biotinylated peptide inhibitor nke BACE1 jikọtara na SA, na-akpata mmetụta pharmacodynamic a ma ama na CNS site na ibelata ọkwa Abeta40 nke ukwuu na CSF (Foto 4). Anyị enweghị ike ịchọpụta homologues ọ bụla na nchekwa data site na ịme ọchụchọ homology usoro peptide nke ihe niile kụrụ. Ọ dị mkpa ịmara na nha nke ọbá akwụkwọ T7 dị ihe dị ka 109, ebe nha ọbá akwụkwọ echiche maka 12-mers bụ 4 x 1015. Ya mere, anyị họọrọ naanị obere akụkụ nke oghere dị iche iche nke ọbá akwụkwọ peptide 12-mer, nke nwere ike ịpụta na enwere ike ịchọpụta peptides kachasị mma site na ịtụle oghere usoro dị nso nke ihe ndị a chọpụtara. N'echiche, otu n'ime ihe kpatara na anyị ahụbeghị njikọ ọ bụla sitere n'okike nke peptides ndị a nwere ike ịbụ enweghị nhọrọ n'oge evolushọn iji gbochie ntinye nke ụfọdụ ihe peptide n'ụbụrụ na-enweghị njikwa.
N'ịchịkọta ihe anyị chọpụtara, ihe ndị a na-eme ka anyị mata ma kọwaa usoro njem nke ihe mgbochi cerebrovascular n'ime ahụ mmadụ n'ụzọ zuru ezu. Nhazi bụ isi nke usoro a dabere na atụmatụ nhọrọ ọrụ nke na-achọpụtaghị naanị clones nwere ihe jikọrọ akwara ụbụrụ, kamakwa ọ gụnyere usoro dị mkpa ebe clones ndị na-aga nke ọma nwere ọrụ dị n'ime ya iji gafee ihe mgbochi bayoloji na vivo n'ime ngalaba CNS. bụ ịkọwa usoro njem nke peptides ndị a na mmasị ha maka ijikọ na microvasculature kpọmkwem na mpaghara ụbụrụ. Nke a nwere ike iduga na nchọpụta nke ụzọ ọhụrụ maka mbugharị nke BBB na ndị nnata. Anyị na-atụ anya na peptides achọpụtara nwere ike ijikọ ozugbo na ndị nnata cerebrovascular ma ọ bụ na ligands na-agagharị agafe site na BBB ma ọ bụ BCSFB. A ga-enyocha vektọ peptide nwere ọrụ njem CSF nke a chọpụtara n'ọrụ a. Anyị na-enyocha ugbu a nkọwapụta ụbụrụ nke peptides ndị a maka ikike ha ịgafe BBB na/ma ọ bụ BCSFB. Peptides ọhụrụ ndị a ga-abụ ngwaọrụ bara uru nke ukwuu maka ịchọpụta ihe ndị na-anabata ma ọ bụ ụzọ ọhụrụ yana maka mmepe nke ikpo okwu ọhụrụ dị irè nke ukwuu maka nnyefe nke macromolecules, dị ka ihe ndị dị ndụ, na ụbụrụ.
Jiri mgbanwe nke usoro akọwapụtara na mbụ mee ka akụkụ ahụ buru ibu (CM) dị n'ime ya. E tinyere oke Wistar a na-eme ka ahụ ghara ịfụ (200-350 g) n'elu ihe eji eme stereotaxic, e mekwara mbepụ etiti n'elu isi ahụ a kpụchara akpụcha ma kwadebe ya nke ọma iji kpughee okpokoro isi ahụ. Gwuo oghere abụọ n'akụkụ nke eriri elu ahụ ma kechie skru ndị na-edozi ya n'ime oghere ndị ahụ. E tinyere oghere ọzọ n'akụkụ occipital crest maka nduzi stereotactic nke cannula ígwè anaghị agba nchara n'ime CM. Tinye simenti ezé gburugburu cannula ahụ ma jiri skru kpuchie ya. Mgbe e mechara foto ma sie ike na simenti, e ji suture supramid 4/0 mechie ọnya akpụkpọ ahụ. A na-akwado ntinye kwesịrị ekwesị nke cannula site na ntapu mmiri cerebrospinal (CSF) ozugbo. Wepụ oke ahụ na ngwaọrụ stereotaxic, nweta nlekọta kwesịrị ekwesị mgbe a wachara ahụ na njikwa ihe mgbu, ma kwe ka ọ gbakee ruo opekata mpe otu izu ruo mgbe a hụrụ ihe ịrịba ama nke ọbara na mmiri cerebrospinal. E si n'Osimiri Charles (France) nweta oke Wistar (Crl:WI/Han). E debere oke niile n'ọnọdụ pụrụ iche na-enweghị nje. Ụlọ Ọrụ Na-ahụ Maka Anụmanụ nke Obodo Basel, Switzerland kwadoro nnwale anụmanụ niile, e mekwara ha dịka Akwụkwọ ikike anụmanụ nke 2474 (Nyocha nke Njem Ụbụrụ Na-arụ Ọrụ site na Ịtụ Ọkwa Ndị Na-agwọ Ọrịa na Cerebrospinal Fluid na Brain of Rat) si dị.
Jiri nwayọ mee ka oke ahụ mara ihe ma jiri cannula CM n'aka. Wepụ Datura na cannula ahụ wee chịkọta 10 µl nke mmiri cerebrospinal na-asọpụta n'onwe ya. Ebe ọ bụ na ikike nke cannula ahụ mebiri emebi, naanị ihe nlele mmiri cerebrospinal doro anya na-enweghị ihe akaebe nke mmerụ ọbara ma ọ bụ mgbanwe agba ka etinyere na ọmụmụ ihe a. N'otu aka ahụ, e si na obere mbepụ dị n'ọnụ ọdụ were ihe dị ka 10-20 μl nke ọbara n'ime tubes nwere heparin (Sigma-Aldrich). E chịkọtara CSF na ọbara n'oge dị iche iche mgbe a gbasịrị T7 phage n'ime akwara. A tụfuru ihe dị ka 5-10 μl nke mmiri tupu a chịkọta ihe nlele CSF ọ bụla, nke kwekọrọ na olu nwụrụ anwụ nke catheter ahụ.
E ji T7Select 10-3b vektọ mepụta ọbá akwụkwọ dịka akọwara na ntuziaka sistemụ T7Select (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). N'ụzọ dị mkpirikpi, e tinyere DNA 12-mer na usoro a:
E jiri koodu NNK zere codon abụọ na-akwụsị akwụsị na oke mmetụta amino acid n'ime ihe ntinye ahụ. N bụ oke nha ... E mere nkwakọ ngwaahịa Phage in vitro dịka ntuziaka enyere na ngwa T7Select 10-3b cloning (Novagen) si dị, e wee mee ka ngwọta nkwakọ ngwaahịa ahụ dịkwuo elu otu ugboro iji lysis site na iji Escherichia coli (BLT5615, Novagen). E tinyere lysates ndị ahụ n'ime centrifuge, titrated ma kpọnwụọ na -80° C. dị ka ihe ngwọta glycerol.
Mmụba PCR ozugbo nke mpaghara mgbanwe phage nke e ji ofe ma ọ bụ efere mee ka ọ dịkwuo mma site na iji ihe nkwado 454/Roche-amplicon. Ihe nkwado 454/Roche-amplicon nke nwere njikọ n'ihu nwere usoro ndị gbara mpaghara mgbanwe ahụ (NNK) 12 (nke a kapịrị ọnụ), GS FLX Titanium Adapter A, na usoro igodo ọbá akwụkwọ anọ (TCAG) (Ihe mgbakwunye 1a):
Ihe mgbakwunye nke ngwakọta azụ ahụ nwekwara biotin nke ejikọtara na beads njide yana GS FLX Titanium Adapter B nke achọrọ maka mmụba clonal n'oge emulsion PCR:
E mechara jiri usoro 454/Roche pyrosequencing mee amplicon ndị a dịka usoro 454 GS-FLX Titanium si dị. Maka usoro Sanger aka (Applied Biosystems Hitachi 3730 xl DNA Analyzer), a mụbara T7 phage DNA site na PCR ma jiri usoro praịmarị ndị a mee ihe:
E jiri Roche Fast Start DNA Polymerase Kit (dịka ntuziaka onye nrụpụta si dị) mee ka ntinye sitere na plaques nke ọ bụla dịkwuo mma site na iji PCR. Mee mmalite ọkụ (nkeji 10 na 95 °C) na usoro mmụba 35 (s50 na 95 °C, nkeji 1 na 50 °C, na nkeji 1 na 72 °C).
E tinyere ọgwụ mgbochi nje sitere n'ọbá akwụkwọ, ọgwụ mgbochi nje nke ụdị ọhịa, ọgwụ mgbochi nje nke a napụtara site na CSF na ọbara, ma ọ bụ klọn nke ọ bụla n'ime Escherichia coli BL5615 na ofe TB (Sigma Aldrich) ma ọ bụ na efere 500 cm2 (Thermo Scientific) ruo awa anọ na okpomọkụ 37°C. E wepụtara ọgwụ mgbochi nje site na efere ndị ahụ site na iji Tris-EDTA buffer (Fluka Analytical) sachaa efere ndị ahụ ma ọ bụ site na ịchịkọta ihe mkpuchi ahụ na ntụ ntụ pipette na-adịghị ọcha. E wepụtara ọgwụ mgbochi nje site na ihe mkpuchi culture supernatant ma ọ bụ ihe nchekwa extraction site na iji otu gburugburu polyethylene glycol (PEG 8000) precipitation (Promega) ma tinyeghachi ya na ihe nchekwa Tris-EDTA.
E jiri mkpụrụ osisi endotoxin ewepụrụ (Miltenyi Biotec) mee ka phage a mụbara ugboro abụọ ruo atọ tupu e nye ya ọgwụ mgbochi n'ime ọbara (IV) (500 μl/anụmanụ). Na agba nke mbụ, e webatara phage 2×1012; na nke abụọ, phage 2×1010; na agba nke atọ na nke anọ, phage 2×109 kwa anụmanụ. A chọpụtara ọdịnaya phage na CSF na ihe nlele ọbara anakọtara n'oge egosiri site na ịgụta plaque dịka ntuziaka nke onye nrụpụta si dị (T7Select system manual). A na-eme nhọrọ phage site na ịgba ọbá akwụkwọ dị ọcha n'ime akwara ọdụ ma ọ bụ site na ịgba phage ewepụtara na CSF site na agba nke mbụ, a na-emekwa owuwe ihe ubi ndị sochirinụ na nkeji iri, nkeji iri atọ, nkeji iri isii, nkeji iri itoolu, nkeji iri abụọ, nkeji iri asatọ, na nkeji iri abụọ na anọ n'otu n'otu CSF na ihe nlele ọbara. E mere mkpokọta agba anọ nke in vivo panning ebe e debere ma nyochaa alaka abụọ ahọpụtara iche iche n'oge agba atọ mbụ nke nhọrọ. E tinyere phage niile ewepụtara na CSF site na agba abụọ mbụ nke nhọrọ ahụ na 454/Roche pyrosequencing, ebe e jiri aka hazie clones niile ewepụtara na CSF site na agba abụọ ikpeazụ nke nhọrọ ahụ. E tinyekwara 454/Roche pyrosequencing ọbara niile sitere na agba mbụ nke nhọrọ ahụ. Maka ịgba ọgwụ mgbochi phage, e tinyere phage ndị ahọpụtara na E. coli (BL5615) na efere 500 cm2 na 37°C ruo awa anọ. E tinyere clones ndị ahọpụtara ma jiri aka hazie n'otu n'otu na TB. Mgbe ewepụtara phage, sachaa ma wepụ endotoxin (dịka akọwara n'elu), a gbara phage 2×1010/anụmanụ na 300 μl n'ime otu akwara ọdụ.
Nhazi tupu oge eruo na nzacha nke data usoro. E si na usoro maapụ 454/Roche nke a na-akpọ Raw 454/Roche gbanwee data site na usoro maapụ iyi abụọ (sff) gaa na usoro Pearson nke a na-agụ mmadụ (fasta) site na iji ngwanrọ onye na-ere ahịa. Emere nhazi ọzọ nke usoro nucleotide site na iji mmemme na edemede C nkeonwe (ngwugwu ngwanrọ a na-ewepụtaghị) dịka akọwara n'okpuru. Nyocha nke data mbụ gụnyere usoro nzacha siri ike nke ọtụtụ ọkwa. Iji kpochapu agụ ndị na-enweghị usoro DNA ntinye 12mer dị irè, e doziri agụ ndị ahụ n'usoro ka ha bụrụ akara mmalite (GTGATGTCGGGGATCCGGAATTCT), akara nkwụsị (TAAGCTTGCGGCCGCACTCGAGTA) na ntinye ndabere (CCCTGCAGGGATATCCCCGGGAGCTCGTCGAC) site na iji ule Needleman-Wunsch zuru ụwa ọnụ. nhazi nke na-enye ohere ruo enweghị nkwekọrịta 2 kwa nhazi31. Ya mere, ewepụrụ agụ na-enweghị mmalite na nkwụsị tag na agụ nwere ntinye ndabere, ya bụ, nhazi ndị gafere ọnụọgụ ekwenyere nke enweghị ndakọrịta, na ọbá akwụkwọ. Maka agụmagụ ndị fọdụrụ, a chụpụla usoro DNA N-mer nke sitere na akara mmalite na njedebe tupu akara nkwụsị ahụ na usoro agụmagụ mbụ ma hazie ya ọzọ (nke a ga-akpọ "tinye"). Mgbe a tụgharịrị ihe ntinye ahụ, a ga-ewepụ akụkụ mgbe codon mbụ kwụsịrị na njedebe 5′ nke primer ahụ na ntinye ahụ. Na mgbakwunye, ewepụrụkwa nucleotides nke na-eduga na codons ezughị ezu na njedebe 3′ nke primer ahụ. Iji wepụ ihe ntinye nwere naanị usoro ndabere, ewepụrụ ihe ntinye a sụgharịrị nke na-amalite na ụkpụrụ amino acid "PAG". E wepụrụ Peptides nwere ogologo mgbe ntụgharị okwu nke na-erughị amino acid 3 na ọbá akwụkwọ. N'ikpeazụ, wepụ ihe mgbochi na ọdọ mmiri ntinye ahụ ma chọpụta ugboro ole ihe ntinye pụrụ iche ọ bụla dị. Nsonaazụ nke nyocha a gụnyere ndepụta nke usoro nucleotide (ihe ntinye) na ugboro ole ha (agụ) (Foto mgbakwunye 1c na 2).
Ntinye DNA nke otu N-mer site na myirịta usoro: Iji wepụ njehie nhazi usoro 454/Roche kpọmkwem (dịka nsogbu na ndọtị homopolymer nhazi) ma wepụ ihe ndị na-adịghị mkpa, a na-ahazi ntinye usoro DNA N-mer nke a kpụchara na mbụ (ihe ntinye) site na myirịta. ntinye (ruo ntọala abụọ na-adabaghị adaba kwere) site na iji algọridim ntụgharị akọwapụtara dị ka ndị a: a na-ahazi ntinye mbụ site na ugboro ole ha (kachasị elu ruo nke kacha ala), ọ bụrụkwa na ha bụ otu, site na ụdị nke abụọ ha site na ogologo (kachasị ogologo ruo nke kacha mkpụmkpụ)). Ya mere, ntinye kachasị na nke kachasị ogologo na-akọwapụta "otu" mbụ. A na-ahazi ugboro otu ahụ na ugboro igodo. Mgbe ahụ, a gbalịrị itinye ntinye ọ bụla fọdụrụ na ndepụta ahaziri ka etinye ya na otu ahụ site na nhazi Needleman-Wunsch abụọ. Ọ bụrụ na ọnụọgụ nke enweghị ndakọrịta, ntinye, ma ọ bụ nhichapụ na nhazi agaghị agafe oke nke 2, a na-agbakwunye ntinye na otu ahụ, a na-amụbakwa ugboro otu niile site na ugboro ole etinyere ntinye ahụ. A na-akara ntinye agbakwunyere na otu akara dị ka ejiri ma wepụ ya na nhazi ọzọ. Ọ bụrụ na enweghị ike itinye usoro ntinye na otu dị adị, a na-eji usoro ntinye emepụta otu ọhụrụ nwere ugboro ntinye kwesịrị ekwesị ma kaa akara dịka ejiri ya. Oge ntụgharị ahụ na-akwụsị mgbe ejiri usoro ntinye ọ bụla mepụta otu ọhụrụ ma ọ bụ enwere ike itinye ya na otu dị adị. E kwuwerị, a na-emesịa tụgharịa ntinye ndị nwere nucleotides n'ime usoro peptide (ọbá akwụkwọ peptide). Nsonaazụ nke nyocha a bụ otu ntinye na ugboro ole ha kwekọrọ nke mejupụtara ọnụọgụgụ nke ọgụgụ n'usoro (Foto mgbakwunye 2).
Ọgbọ Motif: Dabere na ndepụta nke peptides pụrụ iche, e mepụtara ọbá akwụkwọ nwere ụkpụrụ amino acid niile enwere ike (aa) dịka egosiri n'okpuru. E wepụtara usoro ọ bụla nwere ike ịdị ogologo 3 na peptide ahụ ma tinye usoro mgbanwe ya yana ọbá akwụkwọ motif nkịtị nwere ụkpụrụ niile (tripeptides). E tinyere usoro ọbá akwụkwọ nke motifs ugboro ugboro ma wepụ ihe ndị ọzọ. Mgbe ahụ, maka tripeptide ọ bụla dị na ọbá akwụkwọ motif, anyị lere anya maka ọnụnọ ya na ọbá akwụkwọ site na iji ngwaọrụ kọmputa. N'okwu a, a na-agbakwunye ugboro ole nke peptide nwere motif tripeptide achọtara ma kenye ya na motif dị na ọbá akwụkwọ motif ("ọnụọgụ motifs"). Nsonaazụ nke mmepụta motif bụ usoro akụkụ abụọ nwere ihe niile nke tripeptides (motifs) na ụkpụrụ ha, nke bụ ọnụọgụgụ nke ọgụgụ usoro nke na-ebute motif kwekọrọ mgbe a na-enyocha, chịkọta, ma sụgharịa ihe ndị a. Metrics dịka akọwara n'ụzọ zuru ezu n'elu.
Nhazi nke ọnụọgụ nke motifs na spatterplots kwekọrọ: E jiri usoro nhazi mee ka ọnụọgụ motifs maka ihe nlele ọ bụla dị mma.
ebe ni bụ ọnụọgụgụ agụmagụ nwere isiokwu i. Ya mere, vi na-anọchite anya pasentị ugboro nke agụmagụ (ma ọ bụ peptides) nwere motif i na ihe nlele ahụ. Ejiri ule Fisher kpọmkwem gbakọọ uru P maka ọnụọgụgụ motifs na-abụghị nke a na-ahazi. Banyere correlograms nke ọnụọgụgụ ebumnuche, ejiri ọnụọgụgụ ebumnuche ndị a na-ahazi na R gbakọọ njikọ Spearman.
Iji hụ ọdịnaya nke amino acid n'ọnọdụ ọ bụla n'ọbá akwụkwọ peptide, e mepụtara akara ngosi weebụ 32, 33 (http://weblogo.threeplusone.com). Nke mbụ, a na-echekwa ọdịnaya nke amino acid n'ọnọdụ ọ bụla nke peptide 12-mer na matrix 20 × 12. Mgbe ahụ, a na-emepụta otu peptides 1000 nwere otu ọdịnaya amino acid dị n'ọnọdụ ọ bụla na usoro ngwa ngwa ma nye ya dị ka ntinye na web-logo 3, nke na-emepụta ihe ngosi eserese nke ọdịnaya amino acid dị n'ọnọdụ ọ bụla. maka ọbá akwụkwọ peptide enyere. Iji hụ data dị iche iche, e mepụtara maapụ okpomọkụ site na iji ngwaọrụ emepụtara n'ime R (biosHeatmap, ngwugwu R nke a na-ewepụtabeghị). E jiri usoro nhazi ọkwa Ward na metric anya Euclidean gbakọọ dendrograms egosiri na maapụ okpomọkụ ahụ. Maka nyocha ọnụọgụgụ nke data akara motif, e jiri ule Fisher kpọmkwem gbakọọ ụkpụrụ P maka akara na-adịghị mma. E jiri ule t ma ọ bụ ANOVA nke ụmụ akwụkwọ gbakọọ uru P maka data ndị ọzọ na R.
A gbara ọgwụ phage clones na phages ahọpụtara na-enweghị ihe ntinye n'ime akwara site na akwara ọdụ (2 × 1010 phages/anụmanụ na 300 μl PBS). Nkeji iri tupu a gbanye ha ma tinye ha n'ime akpa, a gbara anụmanụ ndị ahụ 100 μl nke lectin DyLight594-labeled (Vector Laboratories Inc., DL-1177) n'ime akpa. Nkeji iri isii mgbe a gbasịrị ha phage, a gbara òké 50 ml PBS n'ime obi, wee tinye 50 ml 4% PFA/PBS. A na-edozikwa ihe nlele ụbụrụ n'ime 4% PFA/PBS n'abalị ma tinye ha na 30% sucrose n'ime otu abalị na 4°C. A na-akpọnwụ ihe nlele ndị ahụ ozugbo na ngwakọta OCT. E mere nyocha Immunohistochemical nke ihe nlele oyi kpọnwụrụ akpọnwụ na okpomọkụ ụlọ na 30 µm cryosections ejiri 1% BSA gbochie ma tinye ọgwụ mgbochi polyclonal FITC nke megidere T7 phage (Novus NB 600-376A) na 4 °C. Kpọọ ya n'abalị. N'ikpeazụ, e ji PBS saa akụkụ ndị ahụ ugboro atọ ma jiri igwe mikroskopu laser confocal (Leica TCS SP5) nyochaa ha.
GenScript USA mepụtara peptides niile nwere obere ọcha nke 98%, biotinylated ma lyophilized. A na-ejikọta biotin site na stripe glycine spacer ọzọ na N-terminus. Lelee peptides niile site na iji mass spectrometry.
A gwakọtara Streptavidin (Sigma S0677) na oke nha nha nha nke peptide biotinylated okpukpu ise, peptide inhibitory BACE1 biotinylated, ma ọ bụ ngwakọta (ratio 3:1) nke peptide BACE1 inhibitory biotinylated na peptide inhibitory BACE1 na 5–10% DMSO/etinyere na PBS. Otu awa n'ọnọdụ okpomọkụ ụlọ tupu a gbanye ya. A gbara peptides Streptavidin-conjugated n'ime akwara na dose nke 10 mg/kg n'ime otu n'ime akwara ọdụ nke oke nwere oghere ụbụrụ.
E jiri ELISA nyochaa oke mkpokọta nke ihe mgbagwoju anya streptavidin-peptide. E ji 1.5 μg/ml òké anti-streptavidin antibody (Thermo, MA1-20011) kpuchie efere Nunc Maxisorp microtiter (Sigma) n'abalị na 4°C. Mgbe ha gbachiri (ihe mgbochi nchekwa: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) na okpomọkụ ụlọ ruo awa abụọ, jiri 0.05% Tween-20/PBS (ihe nchekwa nchekwa) saa efere ahụ maka awa 3. Nkeji abụọ, etinyere ihe nlele CSF na plasma na olulu mmiri ejiri ihe nchekwa nchekwa (plasma 1:10,000, CSF 1:115) gwakọta efere ahụ n'abalị na 4°C na ihe mgbochi nchọpụta (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Mgbe e mechara usoro ịsa ahụ atọ, a chọpụtara streptavidin site na itinye ya n'ime mmiri TMB substrate (Roche) ruo nkeji iri abụọ. Mgbe ị kwụsịrị mmepe agba site na iji 1M H2SO4, tụọ mmịpụta ahụ na 450 nm.
Aβ(1-40) ELISA nyochachara ọrụ nke ihe mgbochi streptavidin-peptide-BACE1 dịka usoro nke onye nrụpụta si dị (Wako, 294-64701). N'ụzọ dị mkpirikpi, a gwakọtara ihe nlele CSF na ihe na-agbaze nkịtị (1:23) ma tinye ya n'ime oyi kpọnwụrụ akpọnwụ n'abalị na 4°C na efere olulu 96 nke ejiri ihe mgbochi njide BNT77 kpuchie. Mgbe usoro ịsa ahụ ise gasịrị, etinyere ihe mgbochi BA27 nke HRP jikọtara ma tinye ya na oyi kpọnwụrụ akpọnwụ ruo awa 2 na 4°C, wee soro usoro ịsa ahụ ise. A chọpụtara Aβ(1–40) site na itinye ya na mmiri TMB maka nkeji 30 na okpomọkụ ụlọ. Mgbe ejiri ihe ngwọta nkwụsị kwụsị, tụọ mmịpụta ahụ na 450 nm. E tinyere ihe nlele plasma siri ike tupu Aβ(1–40) ELISA. Agbakwunyere plasma na 0.2% DEA (Sigma) na efere olulu 96 ma tinye ya na okpomọkụ ụlọ maka nkeji 30. Mgbe e sachara efere SPE (Oasis, 186000679) na mmiri na 100% methanol, etinyere ihe nlele plasma na efere SPE ma wepụ mmiri mmiri niile. A sachara ihe nlele (na mbụ na 5% methanol wee 30% methanol) ma tinye 2% NH4OH/90% methanol. Mgbe e kpochara eluate ahụ na 55°C maka nkeji 99 na ọkụ N2 na-agbanwe agbanwe, e belatara ihe nlele ahụ na ihe ndị na-agbaze nkịtị ma tụọ Aβ(1–40) dịka akọwara n'elu.
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