Serivisi yo gusuzuma isomero ry'ibishushanyo mbonera by'umusemburo

Alpha Lifetech inzobere mu kubaka isomero ry’ibyerekanwa ry’umusemburo no gusuzuma amasomero y’ibyerekanwa by’abasilikari, itanga ubushobozi bwo gukora ubwoko butandukanye bw’amasomero y’umusemburo no kuyasuzuma kugira ngo hamenyekane ko hari ubudahangarwa bwinshi n’ubw’abasilikari bwihariye ku bakiriya ku isi yose. Dufatanyije n’itsinda ry’abashakashatsi b’inzobere, ikoranabuhanga rigezweho n’ibikoresho, dushobora kubaka amasomero y’umusemburo twibanda ku bwoko butandukanye bwa poroteyine ziterwa n’indwara. Dushobora gutanga ikoranabuhanga ryo kwerekana no kwerekana umusemburo, harimo kwerekana umusemburo wa poroteyine, kwerekana umusemburo muto w’umusemburo, kwerekana umusemburo w’abasilikari, nibindi.

Amasomero yacu yo kwerekana imisemburo afite ubushobozi bwinshi kandi atandukanye cyane, ibyo bikaba bitanga ishingiro rikomeye ryo gusuzuma neza urutonde runaka rw’uturemangingo tw’umubiri. Dushobora kubaka ubwoko butandukanye bw’amasomero yo kwerekana imisemburo y’umubiri (IgG, scFv, VHH, Fab antibody libraries), amasomero ya poroteyine, amasomero ya peptide, amasomero ya cDNA, nibindi bitewe n’ibyo ukeneye. Byongeye kandi, dushobora no guteza imbere amasomero y’uturemangingo dufite imiterere itandukanye, harimo amasomero y’ubudahangarwa bw’umubiri, amasomero karemano, amasomero y’ubukorano, amasomero y’uturemangingo tw’umubiri, n’amasomero y’uturemangingo tw’indwara.

Intangiriro kuri Sisitemu yo Kugaragaza Umusemburo

Ikoranabuhanga ryo kwerekana ubuso bw'umusemburo ni uburyo bwo kwerekana poroteyine zisubiramo ku buso bw'umusemburo binyuze mu guhuza gene. Sisitemu isanzwe yo kwerekana umusemburo ikoresha poroteyine igamije guhuzwa na C-terminus y'igice cya Aga2p cya poroteyine yo guhuza alpha lectin, igizwe ahanini n'amatagi ya epitope ku mpande zombi za poroteyine igamije: tagi ya N-terminal ya 9-amino acid hemagglutinin (HA) na tagi ya C-terminus 10 amino acid c-myc. Aside amino 69 Aga2p subunit ihuzwa na aside amino alpha lectin ya 725 Aga1p subunit binyuze mu migozi ibiri ya disulfide, naho Aga1p ifatanye n'urukuta rw'uturemangingo binyuze mu mugozi wa β 1,6-glucan covalent. Kubwibyo, poroteyine igamije kwerekanwa ku buso bw'uturemangingo tw'umusemburo hanyuma ikamenyekana na ligand ijyanye nayo. Gutandukanya poroteyine zikora n'amasomero binyuze mu gusuzuma flow cytometry.

Igishushanyo cya 1: Ihame ryo kwerekana ubuso bw'umusemburo. (Ishusho ikomokaho: Imikoreshereze y'Icyerekezo cy'Ubuso bw'Imyembe mu Buhanga bwa Poroteyine.)

Intangiriro ku isomero ry'ibyerekanwa ry'ubuso bw'umusemburo

Gutondekanya imisemburo bishingiye ku kwerekana ubuso bw'umusemburo, ahanini bikoreshwa mu gusuzuma amasomero y'abashinzwe kurwanya indwara zishingiye ku poroteyine zo ku buso bw'uturemangingo. Bitewe n'uko imiterere y'uturemangingo tw'umusemburo n'utundi duce tw'uturemangingo igabanuka, guhitamo imiterere y'umusemburo bishobora gusuzuma amasomero manini afatanya kugira ngo haboneke ubwoko budasanzwe bw'uturemangingo.

Serivisi yo gusuzuma isomero ry'ibishushanyo mbonera by'umusemburo

Gutegura plasmide na selile z’umusemburo w’imisemburo, gukora ADN ikora poroteyine y’ingenzi hanyuma uyishyire muri vector y’igaragaza ry’umusemburo irimo ibintu bifasha mu gutuma ibintu bihinduka, peptide z’ibimenyetso, na genes zihujwe na poroteyine zigaragaza ubuso (nka Aga2p). Gene ikora poroteyine y’ingenzi ishobora guhuzwa na gene ikora poroteyine y’urukuta rw’iselile y’umusemburo (ubusanzwe Aga2p), kandi gene ihujwe ishobora guhinduka selile z’umusemburo binyuze mu gukoresha electroporation. Nyuma yo guhinduka, selile z’umusemburo zigaragaza genes z’abarwanyi ku buso bwazo zishobora gukorerwa ibizamini byihariye byo gusuzuma antigen: isomero ry’umusemburo rishyirwamo antigen y’ingenzi kandi selile z’umusemburo zifatanya na zo by’umwihariko ziratoranywa. Hakoreshejwe uburyo bwo gutondeka selile z’umusemburo zikoresha fluorescence (FACS) kugira ngo hatoranywe selile z’umusemburo zigaragaza poroteyine zifite imiterere wifuza, isuzuma ry’isomero ry’umusemburo rishobora gukorwa ku ngano ntarengwa y’isomero rya ~ 10 ^ 8-10 ^ 9 selile z’umusemburo. Hanyuma selile nziza zishobora gukurwaho kugira ngo hakorwe isesengura ryinshi cyangwa hakoreshwe mu buryo bukurikira. Iyo hamaze kumenyekana udukoko twinshi tw’umubiri dukomoka ku mfuruka, dushobora kugaragazwa no gukorwa ku bwinshi, no gutunganywa hakoreshejwe uburyo nka poroteyine A/G affinity chromatography kugira ngo harebwe inzira yose yo gusuzuma imisemburo no gukora antibody.

Uburyo bwo gusuzuma isomero ry'ibinyampeke

Igishushanyo cya 2 Uburyo bwo gusuzuma isomero ry'umusemburo

Imikorere ya serivisi yo gusuzuma isomero ry'umusemburo

Intambwe	Ibikubiye muri serivisi	Ingengabihe
Gutegura Antigen	Ubwoko bwa antigen: Niba umukiriya ashobora gutanga antigen, ingero zijyanye nazo zigomba gutangwa hakurikijwe ubwoko: poroteyine zisubiramo zikenera 3-3.5mg hamwe n’ubuziranenge burenze 85%, molekile nto zigomba guhuzwa hamwe n’ubuziranenge burenze 90%, peptide synthesis igomba guhuzwa hamwe n’ubuziranenge burenze 90%, ubwoko bw’ingero nka virusi zigomba kudakora, RNA igomba gusuzumwa kugira ngo hirindwe kwangirika, kandi ubwoko bwa antigen bwavuzwe haruguru bushobora no guhindurwa kugira ngo bukoreshwe.	Ibyumweru 2-3
Ubudahangarwa bw'inyamaswa	Umubare w’inkingo z’amatungo ni 5, kandi ni ngombwa kumenya niba umubare w’inkingo wongerwa hashingiwe ku isuzuma rya serum titer. Ubudahangarwa bw’ingufu zirwanya indwara: imbaraga za poroteyine/virusi za antigen>105; imbaraga za Peptide/antigen ntoya za antigen>10^4	Ibyumweru 5-6
Itegurwa rya cDNA y'icyitegererezo	Gutandukanya PBMC zo mu maraso, gukuramo RNA yose (RNA extraction kit) hanyuma uhindure kuri cDNA.	Umunsi umwe
Kubaka Isomero	Hakoreshejwe cDNA yo muri library nk'icyitegererezo, gene ya VHH yongerewe imbaraga hakoreshejwe PCR ebyiri, maze gene ya VHH ihuza gene na yeast display vector. Vector yahinduwemo uturemangingo tw'umusemburo hakoreshejwe electroporation kugira ngo hubakwe isomero rya antibody. Hitamo clones 48 mu buryo butaziguye hanyuma umenye igipimo cyiza (>90%) ukoresheje uburyo bwa PCR; Kubara ubushobozi bw'isomero (10^7-10^8), NGS sequencing kugira ngo hamenyekane igipimo gikwiye cyo gushyira isomero (>90%) n'ubusumbane bw'isomero.	Ibyumweru 2
Gusuzuma Isomero	Inshuro eshatu zisanzwe zo gusuzuma: Gupima poroteyine FACS byanditseho fluorescence, hagakurikiraho NGS sequencing mu cyiciro cya gatatu. Ingano nziza zatoranijwe kugira ngo hamenyekane uburyo bwo kugaragaza imiterere ya garama imwe no kubona ELISA. Ingano nziza zose zatoranijwe kugira ngo hamenyekane gene sequencing, kandi hatoranijwe imiterere itandukanye ya CDR region sequences.	Ibyumweru 2-3
Igenzura ry'umubiri urwanya indwara	Kubaka uburyo bukwiye bwo kugaragaza ibimenyetso bya antibody bishobora koroshya imikorere ya antibody, gutunganya antibody, kwemeza ELISA na BLI ko antibody ifatanye kugira ngo harebwe niba antibody ifatanye, no gukumira flow cytometry kugira ngo byemezwe imikorere y’uturemangingo.	Icyumweru kimwe

Isuzuma ry'isomero ry'ikirahure cy'umusemburo

Mu nyandiko z’urubuga rwo kwerekana ubuso bw’umusemburo kugira ngo havumburwe vuba udukoko tw’ingirabuzimafatizo twatoranijwe mu buryo bw’imiterere y’umubiri, abanditsi bashyizeho urubuga rwuzuye rwo kuvumbura udukoko tw’ingirabuzimafatizo rwakozwe muri vitro rushingiye ku kwerekana ubuso bw’umusemburo. Ubwa mbere, hakozwe isomero rya nanobody ryakozwe rihereye kuri genes za ngamiya. Ishusho D igaragaza ko nanobody ifite ikimenyetso cya HA ku mpera ya carboxyl, hanyuma nanobody ishyirwa ku rukuta rw’uturemangingo tw’umusemburo. Ishusho E igaragaza uburyo bwo gusuzuma nanobody. Umusemburo ufite antigen affinity nanobodys waratoranijwe, wongerwa, kandi watoranijwe kenshi kugira ngo uhabwe antibodies na FACS. Abanditsi bavumbuye nanobodys zitoranijwe mu buryo bw’imiterere y’umubiri zigamije GPCR ebyiri zitandukanye z’abantu binyuze muri uru rubuga.

Igishushanyo cya 3: Igishushanyo n'iyubakwa ry'isomero rya nanobody ryakozwe mu buryo bwa sintetike. (Ishusho ikomokaho: Urubuga rwo kwerekana ubuso bw'umusemburo kugira ngo havumburwe vuba udukoko duto twatoranijwe mu buryo bw'imiterere.)

Ibyiza byo kwerekana umusemburo

Dufite ubushobozi bwo kubaka amasomero y’ibice by’ibice bifite ubushobozi bwo kubika amakuru kugeza kuri 10^9, hamwe n’uburyo bwo kuyashyiramo 100% n’uburyo butandukanye bwo kuyakoresha.