Phage zakoreshejwe mu kubaka isomero zirimo phage zifite filamentous (M13 phage), phage za T7, phage za T4, phage za λ, nibindi. Phage zitandukanye zigaragaza imibare ya kopi, uburebure bwa peptide, impera zerekana, n'ibyo aside amine akunda biratandukanye. Kuri ubu, phage zifite filamentous zigaragara ku mpera ya amino na phage za T7 zigaragara ku mpera ya carboxyl zirakoreshwa cyane. Zishobora kwerekana isomero rya aside amino peptide ridasanzwe no kubaka isomero ry'uturere tw'umubiri.

Sisitemu yo kwerekana phage ya M13 ni ikoranabuhanga rikoresha phage ya M13 kugira ngo rigaragaze peptide cyangwa poroteyine zo hanze. Bacteriophage M13 ni phage idakora filamentous igizwe n'igishishwa cya poroteyine na ADN ifite umugozi umwe. Ahantu hayo hashobora gukoreshwa poroteyine ya capsid (pIII, pVI, pVII, pVIII na pIX) mu gukora uburyo bwo kwerekana, aho poroteyine za pIII na pVIII ari zo zikunze kugaragara:

Poroteyine ya pIII iherereye ku mpera ya phage kandi ifite uburemere bunini bwa molekile. Ishobora gushyirwa muri peptides cyangwa poroteyine zo hanze kandi ikoreshwa cyane. Ariko, pIII-mediated display ni display ifite imvange nyinshi, idakwiye gukoreshwa mu gusuzuma phage zo guhuza cyane, kandi igipimo cyo kwerekana poroteyine zimwe na zimwe kiri hasi, ibi bishobora kugira ingaruka ku bushobozi bwa phage bwo kwandura. Kubwibyo, sisitemu yo kwerekana phage yakozwe kugira ngo yongere ubushobozi bwo gutuza no gukora neza.

Poroteyine ya pVIII niyo poroteyine nyamukuru ya capsid ya phage ya M13, iherereye ku mpande zombi za phage kandi ifite uburemere buto bwa molekile. Ishusho ya pVIII muri rusange ihuza urukurikirane rw'inyuma kuri N-terminus, kandi uburebure bwa peptide ya fusion ni buke, ariko umubare wa kopi ya poroteyine ya fusion ya hanze ni munini, ikaba ikwiriye gusuzuma peptide zifite affinity nke.

Sisitemu yo kwerekana T4 phage ikoresha poroteyine ya capsid ya phage ya T4 virulent kugira ngo igaragaze peptides cyangwa poroteyine zo hanze. Phage ya T4 igizwe n'umutwe wa icosahedral n'umurizo ushobora gusubira inyuma, kandi gene ni ADN ifite umurongo ugororotse w'imirongo ibiri. Poroteyine z'agakingirizo ka SOC na HOC ku buso bwa capsid zikunze gukoreshwa mu kwerekana, kandi kuba zihari cyangwa zitariho byombi ntibigira ingaruka ku mikorere ya phage. Phage ya T4 ishobora kugaragazwa ahantu hamwe ha SOC cyangwa HOC, kandi ishobora no kwerekana imiterere itandukanye y'amahanga ahantu habiri icyarimwe. Sisitemu iteranyirizwa mu turemangingo tw'imyuga kandi ishobora kwerekana poroteyine zitandukanye. Sisitemu yo kwerekana ifite ubushobozi bwo kugeza kuri 35 kb n'umubare munini wa kopi, ariko ntabwo ifasha mu gusuzuma ligande zifatanye cyane.

Sisitemu yo kwerekana T7 phage ikoresha poroteyine ya 10B capsid ya T7 lytic phage kugira ngo igaragaze peptides cyangwa poroteyine zo hanze. T7 phage ikura vuba kandi idahindagurika, kandi genome yayo ni ADN ifite imigozi ibiri. Poroteyine ya 10B iherereye ku buso bwa phage kandi ishobora guhuzwa kugira ngo igaragaze urukurikirane rwo hanze kuri C-terminal yayo. Sisitemu ntishobora kwerekana peptides ngufi zifite umubare munini wa kopi gusa, ahubwo inagaragaza urukurikirane rurerure rufite umubare muto wa kopi, kandi imiterere yo gusuzuma ishobora gutoranywa hakurikijwe ibisabwa. T7 phage irekura urubyaro binyuze mu gusya bagiteri zisanzwe, zifite imbogamizi nke ku bwoko bwa peptides zigaragara, kandi zikura vuba kandi zikagira igihe gito cyo gusuzuma. Byongeye kandi, T7 phage ishobora gukura neza mu bidukikije bikomeye kandi ikwiriye gupimwa mu bidukikije bigoye.

Sisitemu yo kwerekana λ phage ikoresha poroteyine ya D na poroteyine ya V ya λ phage kugira ngo igaragaze poroteyine zo mu mahanga. λ phage igizwe n'umutwe wa icosahedral n'umurizo muto, kandi genome ni ADN ifite imigozi ibiri. Poroteyine ya D ni poroteyine nyamukuru mu mutwe naho poroteyine ya V ni poroteyine nyamukuru mu murizo. Zombi zishobora guhuza poroteyine zo hanze ku mpera runaka. Bitewe n'uko hariho ahantu henshi hahurira, ntabwo byoroshye gupima peptides cyangwa poroteyine zifite affinity nyinshi. Ariko, λ phage irekurwa nyuma yo guterana mu karemangingo k'umuntu, ikagaragaza ubwoko bwinshi bwa peptides, zishobora kugaragaza macromolecules cyangwa poroteyine zikomeye zifite uburozi ku muntu.

Igishushanyo cya 2 Ubwoko bwa bacteriophages bukoreshwa mu kwerekana phages. (Inkomoko y'aho uherereye: Icyerekanwa cya Phage n'ubundi buryo bwo kwerekana peptide)

RNA yose yakuwe mu matsinda y’amafi ya lymphocyte (nk'amafi ya lymphocyte yo mu maraso y'umuntu, uturemangingo twa lymph node cyangwa uturemangingo twa spleen). MRNA yahinduwe mu buryo bwa cDNA hakoreshejwe uburyo bwa reverse transcription.

Udupira twihariye twakozwe kugira ngo twongere imbaraga z’uruhererekane rw’umubiri ruremereye (Fd) n’uturemangingo tw’akarere k’uruhererekane rw’ubugari (kuri Fab Library) cyangwa uduce twa VH na VL (kuri scFv Library) hakoreshejwe PCR hakoreshejwe cDNA nk'icyitegererezo. Ibicuruzwa bya PCR byatandukanyijwe na agarose gel electrophoresis maze uduce twa gene twibasiwe turaboneka.

Agace k’uturemangingo tw’ingirabuzimafatizo kashyizwe muri vekteri ya phagemid, kandi agace k’uturemangingo tw’ingirabuzimafatizo gakunze guhuzwa na vekteri binyuze mu igogora rya enzyme no guhuza. Ku bubiko bwa Fab, ni ngombwa guhuza igice cya Fd na gene y’uruhererekane rw’urumuri hanyuma bikavangwa mu buryo bwuzuye bwa Fab. Ku bubiko bwa scFv, uduce twa VH na VL dushobora guhuzwa hamwe mu buryo butaziguye hakoreshejwe uburyo bwa PCR.

Umusaruro wo guterwa washyizwe mu tunyangingo twa E.coli dufite ubushobozi bwo gukora isomero rya E.coli ririmo agace k’utunyangingo tw’umubiri. Utunyangingo twahinduwe twasizwe ku masahani yo guhingwa ya agarose kugira ngo dukure mu ijoro rimwe, hanyuma utwo tunyangingo twarakurwaho maze tubikwa muri glycerol kugira ngo habe isomero rya bagiteri.

Uturemangingo two mu bubiko bwa bagiteri twongerewe kandi dushyirwamo agace k’ubufasha kugira ngo hagaragazwe agace k’ubudahangarwa bw’umubiri ku buso bwa phage. Agace k’ubudahangarwa karakusanyijwe, ari na ko kari bubiko bw’ubudahangarwa bwa phage.

Guhuza udusaro twa magnetic. Nyuma yo kozwa na PBST, hongewemo udusaro twa magnetic 50 uL hamwe n'umuti wa phage kugira ngo hasuzumwe neza, kandi imiterere yo gucukura yari ihuye n'isuzuma rya positive. Nyuma y'ibyo, supernatant yakusanyijwe kugira ngo isuzumwe neza. Phage supernatant nyuma yo gucukura nabi yavuzwe haruguru yongewemo udusaro twa magnetic twateguwe kugira ngo tucuzumwe neza. Amazi ya phage yajugunywe hanyuma yozwa na PBST inshuro 3-5. Eluent yongewemo kugira ngo ihagarare, maze eleent ihita ihinduka pH = 7.0. Kumenya urugero rwa elelution phage: 10μL phage solution yavanzwe inshuro 10 za 2 × YT medium, kandi icyitegererezo cya 100μL hamwe n'ivumbikwa rikwiye ryongewemo uturemangingo twa 100μL TG1 mu cyiciro cyo gukura cya logarithmic, kivanze neza, maze cyandura kuri 37 °C mu gihe cy'iminota 15. Uturemangingo twa TG1 twanduye udusaro twa dilutions zitandukanye twakwirakwijwe ku isahani za LB / AMP-GLU kandi duhingwa kuri 37 °C mu gihe cy'amasaha 8. Amakoloni yari ku isahani yarabazwe maze hapimwa titer ya phage recombinant. Umuti wa phage wa eluted wakoreshejwe mu kwanduza TG1 mu cyiciro cyo gukura cya logarithmic hanyuma uhingwa kugeza ku cyiciro cyo gukura cya logarithmic. Phage helper M13K07 yongewe kuri icyo gice, yongera gushyirwaho muri 2 × YT / AMP-KAN medium, hanyuma ihingwa mu cyuma gishyushya amazi mu gihe cy'amasaha 14. Subiramo intambwe zavuzwe haruguru kugira ngo ukurikirane isuzuma.

Hafashwe microplate y'ibiziba 96, hashyirwaho umuti wa 100 μL wavanzwe (umuti wo gusiga) kuri buri riba kugira ngo utwikirize ijoro ryose kuri 4 °C. Reka amazi yo gusiga, ongeramo ifu y'amata yaciwe kuri buri mwobo, hanyuma ufunge ku bushyuhe bw'icyumba. Hakozwe PBST inshuro 4, umuti wa phage wateguwe wongewe kuri microplate hanyuma ushyirwa ku bushyuhe bw'icyumba. Ingero za phage zitari ziboshye zajugunywe, zogezwa na PBST hanyuma zogezwa na PBS, maze eluent ihita ihinduka pH = 7.0. Ingano ya phage ya recombinant muri eluent yaramenyekanye. Umuti wa phage wa eluted wakoreshejwe mu kwanduza TG1 mu cyiciro cyo gukura kwa logarithmic hanyuma ushyirwa ku cyiciro cyo gukura cya logarithmic. Umuti wa phage M13K07 wongewe ku gice cyo hagati, wongera gushyirwa mu gice cyo hagati cya 2 × YT / AMP-KAN, hanyuma ushyirwa mu cyuma gishyushya amasaha 14. Subiramo intambwe zavuzwe haruguru kugira ngo ukurikirane isuzuma.

Phage yongewe ku tunyangingo twapimwe nabi, kandi imiterere yo gupima ikwirakwira yari ijyanye n’ikwirakwira rya positive. Nyuma y’ibyo, isomero rya phage ridafite aho ribohewe ryakusanyijwe muri iki cyiciro cyo gupima positive. Phage supernatant yongewe ku tunyangingo twapimwe nabi hanyuma ishyirwa mu bushyuhe ijoro ryose kuri 4 °C. Nyuma yo gupima, utwo tunyangingo twogejwe maze utwo tunyangingo twa sieve positive turagaruka. Shyiramo eluent kugira ngo uhindure phage, kandi umenye titer ya phage. Umuti wa phage wa eluted wakoreshejwe mu kwanduza TG1 mu cyiciro cyo gukura kwa logarithmic hanyuma uhingwa mu cyiciro cyo gukura cya logarithmic. Phage helper M13K07 yongewe ku gice cyo hagati, yongera guhagarikwa mu gice cya 2 × YT / AMP-KAN, hanyuma ihingwa mu cyuma gishyushya amasaha 14. Subiramo intambwe zavuzwe haruguru kugira ngo uhindure ikwirakwira.

Binyuze mu ikoranabuhanga ryo kwerekana phage, gene y’agace gahinduka k’umubiri ishobora gushyirwa muri genome ya phage, kugira ngo umubiri ugaragara ku buso bwa phage, bityo hubakwe isomero ry’umubiri ugaragaza phage. Ibi bishobora kwigana inzira yo gukora antibody muri vitro no gusuzuma antibody iyo ari yo yose.

Ikoranabuhanga ryo kwerekana imiterere y’uturemangingo (Phage ecran) rishobora gukoreshwa mu gusuzuma molecule zifite ibikorwa byihariye bya biyoloji, zishobora gukoreshwa nk'imiti ikoreshwa mu bushakashatsi no mu iterambere ryinshi. Urugero, binyuze mu gusuzuma imiterere y’uturemangingo (phage ecran), birashoboka kuvumbura molecule zifatanya na poroteyine zijyanye n’indwara zishobora kuba zifite ubushobozi bwo kuvura indwara.

Hakoreshejwe ikoranabuhanga ryo kwerekana phage, hashobora gutegurwa molekile zigomba gukingirwa indwara ziterwa n’udukoko twihariye. Izi molekile zishobora kwigana udukoko twitwa antigenic, bityo zigatera umubiri imbaraga zo gukora ubudahangarwa bw’umubiri kugira ngo ugere ku ntego yo gukumira indwara.

Ikoranabuhanga ryo kwerekana uduce duto (Phage ecran) rishobora kandi gukoreshwa mu kubaka isomero ry’ibikoresho byo gupima indwara. Ibi bikoresho bishobora kumenya no guhambira indwara cyangwa molekile zifitanye isano nabyo kugira ngo bishobore gupima indwara vuba kandi neza.

Binyuze mu ikoranabuhanga ryo kwerekana phage, hashobora gupimwa uduce twa antigenic twifata ku tunyangingo twihariye. Ibi birafasha gusobanukirwa imiterere n'imikorere ya antigens, ndetse n'uburyo antibodies na antigens bikora.

Ikoranabuhanga ryo kwerekana phage rishobora kandi gukoreshwa mu kwiga imikoranire ya molekile mu guhindura ibimenyetso by'uturemangingo. Mu gusuzuma ibimenyetso by'urumogi, dushobora kubona molekile zifatanye na molekile zijyanye no guhindura ibimenyetso by'uturemangingo, kugira ngo tumenye neza uburyo urumogi ruhindura ibimenyetso by'uturemangingo.

Byongeye kandi, ikoranabuhanga ryo kwerekana phage ryakoreshejwe cyane mu gusuzuma ligand ya poroteyine, gutegura ibintu bivura no kuvumbura, gusuzuma no kuvura indwara z’ubudahangarwa bw’umubiri n’ibindi bikorwa by’ubushakashatsi bwa siyansi.

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