Yini i-Antibody Engineering?

Ngosizo lobunjiniyela be-antibody, kuye kwaba nokwenzeka ukuguqula usayizi wamangqamuzana, i-pharmacokinetics, i-immunogenicity, isibopho esibophezelayo, ukucaciswa kanye nomsebenzi womphumela wama-antibodies. Ngemva kokuhlanganiswa kwamasosha omzimba, ukubopha okukhethekile kwamasosha omzimba kuwenza abe yigugu kakhulu ekuxilongweni nasekwelashweni komtholampilo. Ngobunjiniyela bamasosha omzimba, bangahlangabezana nezidingo zokuthuthukiswa kwezidakamizwa nokuxilonga kusenesikhathi.

Inhloso yobunjiniyela bamasosha omzimba ukuklama nokukhiqiza imisebenzi eqondile, ezinzile amasosha omzimba emvelo angeke akwazi ukuyifeza, ukubeka isisekelo sokukhiqizwa kwamasosha omzimba okwelapha.

I-Alpha Lifetech, enokuhlangenwe nakho kwayo okubanzi kwephrojekthi kubunjiniyela bamasosha omzimba, ingahlinzeka ngezinsizakalo ezenziwe ngokwezifiso ze-monoclonal kanye ne-polyclonal antibody yezinhlobo eziningi, kanye nokwakhiwa komtapo wezincwadi we-phage kanye nezinsizakalo zokuhlola. I-Alpha Lifetech inganikeza amakhasimende amasosha omzimba ekhwalithi ye-biosimilar kanye nemikhiqizo yamaprotheni ehlanganiswa kabusha, kanye nezinsizakalo ezihambisanayo, ukuze kukhiqizwe amasosha omzimba asebenza kahle, acacile kakhulu, futhi azinzile. Ngokusebenzisa i-antibody ebanzi, izinkundla zamaprotheni kanye nezinhlelo zokubonisa i-fage, sihlinzeka ngamasevisi ahlanganisa umfula okhuphukayo nophansi wokukhiqizwa kwamasosha omzimba, okuhlanganisa nezinsizakalo zobuchwepheshe ezifana nokwenza i-antibody humanization, ukuhlanzwa kwe-antibody, ukulandelana kwe-antibody, nokuqinisekiswa kwe-antibody.

Isigaba sokuqala sobunjiniyela be-antibody sihlobene nobuchwepheshe obubili:

--Recombinant DNA technology

--Hybridoma technology

Ukuthuthukiswa okusheshayo kobunjiniyela be-antibody kuhlobene nobuchwepheshe obuthathu obubalulekile:

- Ubuchwepheshe be-Gene cloning kanye ne-polymerase chain reaction

--Inkulumo yamaprotheni: Amaprotheni aphinde ahlanganiswe akhiqizwa izinhlelo zokukhuluma ezifana nemvubelo, amagciwane amise okwenduku, nezitshalo.

--Umklamo wesakhiwo osizwa yikhompyutha

Isizukulwane sokuqala samasosha omzimba enziwe abantu ukuze kukhiqizwe amasosha omzimba e-chimeric, lapho isifunda esiguquguqukayo sama-antibody monoclonal egundane sasixhunywe endaweni engaguquki yama-molecule e-IgG yomuntu. Isifunda esibophezelayo se-antigen (CDR) segundane lesizukulwane sesibili se-monoclonal antibody safakwa ku-IgG yomuntu. Ngaphandle kwesifunda se-CDR, wonke amanye amasosha omzimba acishe abe ngamasosha omzimba womuntu, futhi kwenziwa imizamo yokugwema ukunxenxa izimpendulo ze-anti mouse antibody (HAMA) uma kusetshenziswa amasosha omzimba we-clone yegundane ekwelapheni abantu.

 

Ukwakha umtapo wezincwadi wokubonisa i-phage, isinyathelo sokuqala ukuthola izakhi zofuzo ezifaka amasosha omzimba, angahlukaniswa namaseli B ezilwane ezigonyiwe (ukwakhiwa komtapo wokuzivikela omzimba), akhishwe ngokuqondile ezilwaneni ezingagonyiwe (ukwakhiwa komtapo wezincwadi wemvelo), noma ahlanganiswe ku-vitro ngezingcezu zofuzo ze-antibody (ukwakhiwa komtapo wokwenziwa). Khona-ke, izakhi zofuzo zikhuliswa nge-PCR, zifakwe kuma-plasmid, futhi zivezwe ezinhlelweni zokubamba ezifanele (inkulumo yemvubelo (imvamisa i-Pichia pastoris), isisho se-prokaryotic (ngokuvamile i-E. coli), i-mammalian cell expression, i-plant cell expression, kanye ne-insect cell expression etheleleke ngamagciwane amise okwenduku). Okuvame kakhulu uhlelo lokukhuluma lwe-E. coli, oluhlanganisa ukulandelana kwe-antibody ebhala ngekhodi ku-phage futhi ihlanganise iphrotheni yegobolondo le-phage (pIII noma pVIII). Ukuhlanganiswa kwezakhi zofuzo, Futhi kuboniswa ebusweni be-bacteriophages. Umnyombo walobu buchwepheshe uwukwakha umtapo wolwazi wephage, onenzuzo ngaphezu kwemitapo yolwazi yemvelo ngoba ungaba nokubophezela okuthile. Kamuva, amasosha omzimba anemininingwane ye-antigen ayahlolwa ngenqubo yokukhetha yezinto eziphilayo, ama-antigen okuhlosiwe ayalungiswa, ama-phages angaboshiwe akhukhulwa ngokuphindaphindiwe, futhi ama-phages aboshiwe ayakhukhulwa ukuze athuthukiswe. Ngemuva kwemizuliswano emithathu noma ngaphezulu yokuphindaphinda, ukucaciswa okuphezulu kanye namasosha omzimba asondelene ayahlukaniswa.

Umfanekiso wesi-3: Ukwakhiwa Nokuhlolwa Kwelabhulali Ye-antibody

Ubuchwepheshe be-Recombinant DNA bungasetshenziswa ukukhiqiza izingcezu ze-antibody. Amasosha omzimba endwangu ekuqaleni angakhishwa kuphela nge-hydrolyzed nge-gastric protease ukuze akhiqize (Fab ') izingcezu ezi-2, abe esegaywa i-papain ukuze akhiqize izingcezu ze-Fab ngayinye. I-Fv fragment iqukethe i-VH ne-VL, ezinokuzinza okubi ngenxa yokungabikho kwamabhondi e-disulfide. Ngakho-ke, i-VH ne-VL zixhunywe ndawonye nge-peptide emfushane yama-amino acid angu-15-20 ukuze kwakheke i-antibody chain variable fragment (scFv) enesisindo se-molecular cishe esingama-25KDa.

Ucwaningo lwesakhiwo se-antibody e-Camelidae (Ikamela, i-LIama, ne-Alpaca) luye lwacacisa ukuthi amasosha omzimba anamaketanga asindayo kuphela futhi awekho amaketanga alula, yingakho ebizwa ngokuthi amasosha omzimba asindayo (hcAb). Isizinda esiguquguqukayo samasosha omzimba esindayo sibizwa ngokuthi amasosha omzimba wesizinda esisodwa noma ama-nanobodies noma i-VHH, enosayizi we-12-15 kDa. Njengama-monomers, awanawo amabhondi e-disulfide futhi azinze kakhulu, anokuhlobana okuphezulu kakhulu kwama-antigen.

Umfanekiso wesi-5: I-Heavy Chain Antibody kanye ne-VHH/Nanobody

Ukukhululeka kweseli kusebenzisa ukuvezwa kwe-DNA yemvelo noma yokwenziwa ukuze kuzuzwe ukwakheka kwamaprotheni e-in vitro, ngokuvamile kusetshenziswa isistimu yokusho ye-E. coli. Ikhiqiza amaprotheni ngokushesha futhi igweme umthwalo we-metabolic kanye ne-cytotoxic emangqamuzaneni lapho ikhiqiza inani elikhulu lamaprotheni ahlanganisiwe ku-vivo. Ingase futhi ikhiqize amaprotheni okunzima ukuwahlanganisa, njengalawo okunzima ukuwashintsha ngemva kokuhumusha noma ahlanganise amaprotheni olwelwesi.