Serivisi yo kubaka isomero rya Phage Display

Alpha Lifetech imaze imyaka myinshi ikora cyane mu ikoranabuhanga ryo kwerekana Phage. Yubatse urubuga rwiza rw'ikoranabuhanga ryo kwerekana Phage rutuma umuntu atakaza umwanya wo gukora ubushakashatsi bwa siyansi cyangwa ubushakashatsi ku mishinga, kandi rukorohereza umusaruro ukurikiraho. Alpha Lifetech ishobora kandi guha abakiriya serivisi nko gukora antibody ya vhh, gukora antibody ya scFv, gukora antibody ya Fab.

Alpha Lifetech ifite urubuga rwa M13/T7 phage rwuzuye rwo kwerekana no gukora amasomero y’abarwanya indwara. Dushobora kandi gutanga serivisi zihariye, nko gukora antibody scFv no gusuzuma antibody nyinshi, kugira ngo duhuze n’ibyo ukeneye.

Intangiriro ku imurikagurisha rya Phage

Ikoranabuhanga ryo kwerekana uduce duto (Phage display) ni uburyo bukoresha phages (virusi itera bagiteri) kugira ngo ishakishe molekile zihuza poroteyine cyangwa peptides runaka. Uburyo bwo kubaka isomero rya phage antibody burimo kubaka isomero rya Fab antibody, kubaka isomero rya antibody scFv, kubaka isomero rya nanobody, nibindi. Dukurikije ubwoko bwa bacteriophages, zishobora kugabanywamo M13, T7, T4, λ, n'izindi sisitemu. Dukurikije ubwoko bw'isomero, ishobora kugabanywamo isomero rya peptide ridasanzwe, isomero rya cDNA, isomero rya antibody, n'isomero rya poroteyine. Ikoranabuhanga ryo kwerekana uduce duto (Phage display) ryoroshye gukoresha kandi rihendutse kurikoresha. Ariko, ubu buryo bugabanya ubudasa bw'uturemangingo tw'uturemangingo mu isomero ridashobora kugaragaza urukurikirane rurerure cyane,

Muri iki gihe, ikoranabuhanga ryo kwerekana phage rikoreshwa cyane. Muri byo, hari byinshi byagezweho mu bushakashatsi no guteza imbere inkingo nshya (inkingo zihendutse kandi zikora neza), iterambere ry'imiti irwanya indwara (ikingira enzymes zo gusuzuma), ikwirakwizwa ry'ibimenyetso by'uturemangingo (gusuzuma epitopes zakozwe), n'ubushakashatsi ku epitopes za antigen (gutegura antibody za monoclonal).

Intangiriro kuri bacteriophage ya T7

Bacteriophage ya T7 ni ADN ifite imigozi ibiri, uburebure bwa ADN ni 40kb kandi ipfunyitse muri capsid ifite umurambararo wa 60nm. Ihuza hagati y'umutwe n'umurizo ni imiterere y'impeta igizwe na kopi nyinshi za gp8. Umutwe n'imbere bya bacteriophage ya t7 bigize imiterere y'uruziga ifatana na gp8 kandi ishobora guhuza umutwe n'umurizo.

Igishushanyo cya 1 Ishusho ya bacteriophage ya T7.(Icyitonderwa: Iterambere muri sisitemu yo kwerekana T7 phage (Isuzuma))

Intangiriro kuri Bacteriophage ya M13

Bacteriophage M13 ibarizwa mu itsinda ry’udusimba twitwa Ff phages. Ifite uburebure bwa nm 900 n’ubugari bwa nm 6.5. Irimo genome ya ADN imwe ifite umugozi umwe (ssDNA) ifite uburebure bwa bp 6407 igizwe na za jeni icyenda zigizwe na poroteyine 11 zitandukanye. Muri zo, poroteyine eshanu ni poroteyine zikozwe mu mavuta, kandi poroteyine esheshatu zigize uruhare mu kongera no guteranya poroteyine. Ingano nini ya poroteyine zikozwe mu mavuta ni poroteyine ya capsid G8P, igizwe na poroteyine zigera ku 2,700 kandi ikora agace gakikije kromosomu.

Igishushanyo cya 2 Ishusho ya bagiteriofage M13.(Icyitonderwa: Iby'ibanze ku ikoranabuhanga ryo kwerekana antibody phage)

Intangiriro ku isomero rya Phage Display Antibody

Kuvumbura uturemangingo tw’umubiri byarushijeho kuba ingenzi mu buvuzi bwa none. Hari uburyo butandukanye bwo kuvumbura uturemangingo tw’umubiri, ariko isomero ry’uturemangingo tw’uturemangingo tw’umubiri tw’ubwoko bwa phage display rikoreshwa cyane mu buvuzi.

Kuva mu 1990, imiterere itandukanye y’uturemangingo tw’umubiri yagiye ikoreshwa mu kubaka amasomero ya phage, harimo VH, VHH, scFvs, diabodies, na antibody za Fab. ScFv igizwe n’imiterere ya VH na VL ni antibody z’uruhererekane rumwe zikoreshwa mu kubaka amasomero ya antibody scFv. scFv irangwa no kubaho igihe gito kandi ikaba idafite ubudahangarwa buhagije. Fab Antibody Library igizwe na VH, VL, CL, na CH1, ishobora gupima vuba antibody nziza zifite affinity nyinshi. Igice gito cyane gishobora guhuza antigen-VHH kigize isomero rya nanobody ubu, VHH ifite ibyiza by’imiterere yoroshye, ingano nto, gushonga cyane, gutuza neza, no gutegurwa no kwigaragaza byoroshye. Synthetic nanobody (Nb) isomero ririmo kugaragara nk'uburyo bwiza bwo gukingira inyamaswa kuri nanobody ihamye kandi ifite affinity nyinshi. Muri rusange, utu duce twa antibody duhuzwa na G3P ya M13 phage, kandi binyuze mu gukora umubare munini wa genes zikora agace ka antibody, amasomero manini ya antibody ya phage display arashobora gukorwamo antibody nyinshi zitandukanye zishobora gutoranywamo.

Inzira yo kubaka Isomero rya Phage

Uburyo bwo kubaka isomero rya phage bukurikira: primers zihariye zigenewe kongera imbaraga za PCR zigakuramo ibyo bicuruzwa, bihuzwa na enzyme-ligated na T7/M13 phage vector, maze plasmid ya phage recombinant ikorwa neza. Plasmid ya phage recombinant yahinduwemo uturemangingo twa TG1 receptor, hanyuma ishyirwa ku kintu kirimo antibiyotike zikwiye, maze culture yagutse ikorwa nyuma yo gusuzuma transformers. Nyuma yo gusesengura inshuro nyinshi, phage yiyongera inshuro zo gusesengura muri bagiteri kandi ikagaragaza neza poroteyine cyangwa polypeptide. Hanyuma isomero rya phage rirasukurwa kugira ngo rikureho imyanda imwe n'imwe na phage zifunguye.

Uduce duhinduka (VH na VL) dufitanye isano n’ubwinshi bw’uturemangingo tw’umubiri, kandi VH na VL byashyizwe muri vector ya phage hamwe n’uruhererekane rw’uturemangingo twa phage PIII. Nyuma yo guterana, agace ka phage karagaragara kagahuzwa na N-terminal ya poroteyine ya III y’agace gato kugira ngo habeho agace k’uturemangingo tw’umubiri, hagafatwa isomero ririmo uruhererekane rwa ADN y’uturemangingo tw’umubiri.

Nyuma yo kurangiza gukingirwa, titer yarabonetse, maze amaraso y'inyamaswa afatwa nyuma yuko titer igaragaye. Lymphocytes zikurwa mu maraso, RNA irakurwamo, RT-PCR yongera igice cy’ingingo, hanyuma agace k’uturemangingo tw’akarere ka V kakaboneka. Gene V yongera hakoreshejwe primer yihariye.

Amasomero karemano yashinzwe arimo utunyangingo duto tw’umubiri dutera indwara mu nyamaswa dushobora kwibasira ikintu icyo ari cyo cyose. Utunyangingo duto tw’umubiri dufata neza antigen dushobora gukusanywa hakoreshejwe uburyo bwo gusuzuma bukemura cyangwa bushyiraho ikimenyetso cy’uko antigen ari yo.

Antigen ishyirwa ku kintu gikomeye gitwara ibintu, nk'umwobo wa microplate, cyangwa igashyirwa ku gasaro ka rukuruzi. Hanyuma isomero rya antibody ya phage rishyirwamo kugira ngo rifatanye na antigen. Nyuma yo gukurwaho inshuro nyinshi, phage zoroshye cyangwa zidafite umwihariko ziragezwaho amazi hanyuma izigaragaza antibody zigasigara.

Imikoreshereze ya Phage Display

*Guvumbura antibody byarushijeho kuba ingenzi mu buvuzi bwa none. Hari uburyo butandukanye bwo kuvumbura antibody, ariko ikoranabuhanga ryo kwerekana phage rikoreshwa cyane mu buvuzi. Kuva mu 1990, imiterere itandukanye y’antibody yakoreshejwe mu kubaka amasomero ya phage, harimo VH, VHH, scFvs, diabodies, na Fab antibodies.
*Amasomero ya peptide ya Phage yemerera kumenya vuba urukurikirane rwa epitopes za poroteyine kandi yabaye igikoresho gikomeye cyo gushakisha imikoranire iri hagati ya epitopes na antigen receptors.
*Ibice by’uturemangingo tw’umubiri bihuzwa na G3P ya phage ya M13, kandi binyuze mu guhuza umubare munini w’uturemangingo tw’umubiri dufata igice cy’uturemangingo tw’umubiri, hashobora gukorwa ibitabo binini by’uturemangingo tw’umubiri dufatanyirizwa hamwe, aho uturemangingo twinshi tw’uturemangingo dushobora gutoranywamo.
*Sisitemu yo kwerekana T7 phage ifite ibyiza byinshi, birimo koroshya, umutekano mwinshi, ituze, kubika byoroshye, no gutwara, bityo iyi sisitemu ikoreshwa mu nkingo zo kwirinda no kuvura.
*Sisitemu yo kwerekana T7 phage ishobora kubona antigen zitandukanye, nka antigen zo hejuru z'udukoko dutera indwara na antigen za kanseri.

Phage