Serivisi Yerekana Isomero Yerekana Isomero

Alpha Lifetech kabuhariwe mu kwerekana umusemburo werekana isomero ryubaka no kwerekana amasomero ya antibody, itanga ubushobozi bwo kubyara ubwoko butandukanye bwamasomero yimisemburo no kubisuzuma kugirango bihuze cyane na antibodi zihariye kubakiriya kwisi yose. Hamwe nitsinda ryabashakashatsi b'inzobere, ikoranabuhanga n'ibikoresho bigezweho, turashobora kubaka amasomero yimisemburo yibasira poroteyine zitandukanye ziterwa n'indwara. Turashobora gutanga fage yerekana na tekinoroji yerekana tekinoroji, harimo kwerekana umusemburo wa protein, kwerekana umusemburo muto wa molekile, kwerekana umusemburo wa antibody, nibindi

Amasomero yacu yerekana amasomero afite ubushobozi bunini kandi butandukanye, butanga umusingi muremure wo gusuzuma neza antibody yihariye. Turashobora kubaka uburyo butandukanye bwimisemburo ya antibody yerekana amasomero (IgG, scFv, VHH, amasomero ya Fab antibody), amasomero ya protein, amasomero ya peptide, amasomero ya cDNA, nibindi ukurikije ibyo ukeneye. Byongeye kandi, turashobora kandi guteza imbere amasomero ya antibody yibintu bitandukanye, harimo amasomero yumubiri, amasomero karemano, amasomero yubukorikori, amasomero yubukorikori, hamwe nububiko bwibitabo bya antibody.

Intangiriro kuri Sisitemu Yerekana Sisitemu

Ubuhanga bwo kwerekana umusemburo ni uburyo bwo kwerekana poroteyine za recombinant hejuru yumusemburo ukoresheje gene fusion. Sisitemu yerekana cyane imisemburo ikoresha poroteyine yagenewe guhuzwa na C-terminus ya Aga2p subunit ya alpha lectin mating protein, ahanini igizwe na tagito ya epitope kumpande zombi za poroteyine yagenewe: N-terminal 9-amino acide hemagglutinin (HA) hamwe na C-terminus 10 amino acide c-myc. Acide 69 amine aside Aga2p ihuza na 725 amino acide alpha lectin Aga1p subunit ikoresheje imigozi ibiri ya disulfide, naho Aga1p ikomekwa kurukuta rw'akagari ikoresheje β 1,6-glucan covalent. Kubwibyo, intego ya poroteyine igaragara hejuru yubusemburo bwimisemburo hanyuma ikamenyekana na ligand ihuye. Tandukanya poroteyine zikora mumasomero ukoresheje ecran ya cytometrie.

Igishushanyo 1: Ihame ryumusemburo werekana. (Inkomoko y'ishusho: Porogaramu Yumusemburo Ubuso bwerekanwe kuri Protein Engineering.)

Intangiriro Kumasomero Yerekana Isomero

Gutondekanya umusemburo gushingiye kubuso bwerekana hejuru, bikoreshwa cyane mugusuzuma amasomero ya antibody yibasira proteine zo hejuru. Bitewe no guhuza kwihariye kudasanzwe hagati yimisemburo nizindi selile, guhitamo ibinyabuzima byumusemburo birashobora kwerekana amasomero manini ahuza amasomero kugirango abone clone zidasanzwe.

Serivisi Yerekana Isomero Yerekana Isomero

Tegura plasmide hamwe numusemburo wimisemburo yumuco, ushushanya ADN ikubiyemo poroteyine yagenewe hanyuma uyishyire mubisemburo byerekana umusemburo urimo porotokoro idasobanutse, peptide yerekana ibimenyetso, hamwe na gen zigenewe guhuza poroteyine zigaragara (nka Aga2p). Gene ikubiyemo poroteyine yagenewe irashobora guhuzwa na gene ikubiyemo poroteyine yo mu rukuta rw'umusemburo (ubusanzwe Aga2p), kandi gene yahujwe ishobora guhinduka ingirabuzimafatizo binyuze mu mashanyarazi. Nyuma yo guhinduka, ingirabuzimafatizo zigaragaza ingirabuzimafatizo ya antibody hejuru yazo zishobora kwipimisha antigen yihariye: isomero ryimisemburo ryinjizwamo antigen hamwe na selile yimisemburo ihambiriye. Ukoresheje fluorescence ikora selile itondekanya (FACS) kugirango utandukane utugingo ngengabuzima twerekana poroteyine zifite ibimenyetso bifuza, kwerekana isomero ryerekana isomero rishobora gukorwa hamwe nubunini bwibitabo bunini bwa ~ 10 ^ 8-10 ^ 9. Clone nziza irashobora gutandukanywa kugirango irusheho gusesengura cyangwa porogaramu yo hasi. Iyo clone yihariye imaze kumenyekana mubitabo byibitabo bya antibody, birashobora kurushaho kurangwa no kubyara umusaruro, kandi bigasukurwa hakoreshejwe tekinoroji nka protein A / G affinity chromatography kugirango irangize inzira yose yerekana imisemburo yerekana no gukora antibody.

Inzira Yumusemburo Werekana Isomero

Igishushanyo.2 Umusemburo werekana uburyo bwo gusuzuma isomero

Umusemburo Werekana Isomero Kugenzura Serivise Yakazi

Intambwe	Ibirimo	Igihe ntarengwa
Gutegura Antigen	Ubwoko bwa Antigen: Niba umukiriya ashobora gutanga antigene, ingero zijyanye nazo zigomba gutangwa ukurikije ubwoko: proteine za recombinant zisaba 3-3.5mg zifite isuku irenga 85%, molekile nto zigomba guhuzwa nibisabwa byera hejuru ya 90%, synthesis ya peptide igomba guhuzwa nibisabwa byera hejuru ya 90%, ubwoko bwintangarugero nka virusi bigomba gukenerwa kugirango virusi ikorwe, RNA ikenera kwanduzwa na RNA; Guhindura.	Ibyumweru 2-3
Ubudahangarwa bw'inyamaswa	Umubare w’ikingira ry’inyamaswa ni 5, kandi ni ngombwa kumenya niba wongera umubare w’inkingo zishingiye ku gupima titer. Ubudahangarwa bwa antigen: imbaraga za poroteyine / virusi antigen> 105; Peptide / ntoya ya molekile antigen imbaraga> 10 ^ 4	Ibyumweru 5-6
Inyandikorugero cDNA Gutegura	Tandukanya plasma PBMCs, gukuramo RNA yose (ibikoresho byo gukuramo RNA) hanyuma usubire kuri cDNA.	Umunsi 1
Kubaka Isomero	Ukoresheje isomero cDNA nkicyitegererezo, gene ya VHH yongerewe ibyiciro bibiri bya PCR, hanyuma hubakwa gene ya VHH itanga imisemburo yerekana vector. Vctor yahinduwe mumasemburo na electroporation kugirango yubake isomero rya antibody. Bisanzwe hitamo clone 48 hanyuma umenye igipimo cyiza (> 90%) ukoresheje uburyo bwa PCR; Kubara ubushobozi bwibitabo (10 ^ 7-10 ^ 8), NGS ikurikirana kugirango umenye igipimo cyukuri cyo kwinjiza amasomero (> 90%) nuburyo butandukanye bwibitabo.	Ibyumweru 2
Kugaragaza Isomero	Mburabuzi ibyiciro bitatu byo gusuzuma: Fluorescence yanditseho protein FACS yerekana, ikurikirwa na NGS ikurikiranye mugice cya gatatu. Clone nziza zatoranijwe kugirango garama imwe yindobanure yerekanwe na ELISA. Clone nziza zose zatoranijwe kugirango zikurikirane gene, hanyuma hatoranijwe urutonde rwa CDR zitandukanye.	Ibyumweru 2-3
Kugenzura Antibody	Kubaka imvugo ikwiye kugirango ikurikirane antibody irashobora koroshya imvugo ya antibody, kweza antibody, ELISA na BLI kwemeza antibody antigen ihuza kugirango igenzure neza antibody, hamwe na cytometrie ikumira kugirango yemeze imikorere ya selile.	Icyumweru 1

Ikibazo cyumusemburo Ubuso bwerekana Isomero

Mu buvanganzo bwerekana umusemburo werekana urubuga rwo kuvumbura byihuse bya nanobodi byatoranijwe, abanditsi bashizeho uburyo bwuzuye bwo kuvumbura vitro nanobody bushingiye ku kwerekana umusemburo. Ubwa mbere, isomero rya nanobody isomero ryakozwe guhera kuri genes yingamiya. Igishushanyo D cyerekana ko nanobody ifite tag ya HA kumpera ya carboxyl, hanyuma nanobody igashyirwa hamwe kurukuta rwumusemburo. Igicapo E cerekana inzira ya nanobody. Umusemburo ufite antigen affinity nanobodies warahawe akato, wongerewe, kandi watoranijwe inshuro nyinshi kuri antibodies na FACS. Abanditsi bavumbuye uburyo bwo guhitamo nanobodies yibanda kuri GPCR ebyiri zitandukanye zabantu.