Basa rekuongorora raibhurari yeYeast Surface Display

Alpha Lifetech inonyanya kugadzira raibhurari yekuratidza yeast uye kuongorora maraibhurari ekudzivirira ma antibodies, ichipa kugona kugadzira mhando dzakasiyana dzemaraibhurari e yeast uye kuaongorora kuti aone kana aine hukama hwakakura uye ma antibodies chaiwo kune vatengi pasi rese. Nechikwata chevaongorori vane hunyanzvi, tekinoroji yepamusoro uye michina, tinogona kuvaka maraibhurari e yeast tichitarisa mapuroteni akasiyana-siyana ane chekuita nezvirwere. Tinogona kupa matekinoroji ekuratidza phage uye yeast, kusanganisira protein yeast display, small molecule yeast display, antibody yeast display, nezvimwewo.

Maraibhurari edu ekuratidza mbiriso ane huwandu hwakakura uye akasiyana-siyana, izvo zvinopa hwaro hwepamusoro hwekuongorora zvinobudirira maantibody sequences chaiwo. Tinogona kuvaka mhando dzakasiyana dzemaraibhurari ekuratidza mbiriso ye antibody (IgG, scFv, VHH, Fab antibody libraries), maraibhurari emapuroteni, maraibhurari epeptide, maraibhurari ecDNA, nezvimwewo zvichienderana nezvaunoda. Pamusoro pezvo, tinogonawo kugadzira maraibhurari ekudzivirira muviri ane hunhu hwakasiyana-siyana, kusanganisira maraibhurari ekudzivirira muviri, maraibhurari echisikigo, maraibhurari ekugadzira, maraibhurari e semi-synthetic, uye maraibhurari ekudzivirira chirwere.

Nhanganyaya yeSisitimu yeKuratidza Mbiriso

Tekinoroji yekuratidza pamusoro pembiriso inzira yekuratidza mapuroteni akasanganiswa pamusoro pembiriso kuburikidza nekubatanidzwa kwemajini. Sisitimu yekuratidza mbiriso inowanzo shandiswa inoshandisa protein yakasanganiswa neC-terminus yeAga2p subunit yealpha lectin mating protein, inonyanya kuve nema epitope tag kumativi ese eprotein yakatsanangurwa: N-terminal 9-amino acid hemagglutinin (HA) tag uye C-terminus 10 amino acid c-myc tag. 69 amino acid Aga2p subunit inosunga ku725 amino acid alpha lectin Aga1p subunit kuburikidza netwo disulfide bonds, uye Aga1p yakasungirirwa kumadziro esero kuburikidza ne β 1,6-glucan covalent bond. Nokudaro, protein yakatsanangurwa inoratidzwa pamusoro pemasero eyeast uye inozozivikanwa ne ligand inoenderana. Bvisa mapuroteni anoshanda kubva kumaraibhurari kuburikidza ne flow cytometry screening.

Mufananidzo 1: Nheyo yekuratidzwa kwembiriso pamusoro. (Mufananidzo wakabva: Mashandisirwo eYeast Surface Display yeProtein Engineering.)

Nhanganyaya yeRaibhurari yeYeast Surface Display

Kuronga mbiriso kunobva pakuratidzwa kwembiriso pamusoro pembiriso, kunonyanya kushandiswa kuongorora maraibhurari emasoja ekudzivirira muviri anotarisa mapuroteni pamusoro pemasero. Nekuda kwekusungwa kushoma kusingatsananguriki pakati pemasero embiriso nemamwe masero, kusarudzwa kwenyama yembiriso kunogona kuongorora maraibhurari makuru ekubatanidza kuti awane ma clones asingawanzoitiki.

Basa rekuongorora raibhurari yeYeast Surface Display

Gadzirira maplasmids nemasero embiriso yekurima, gadzira DNA inoisa mapuroteni anotarisirwa woibatanidza kuita yeast expression vector ine ma inducible promoters, signal peptides, uye ma target genes akabatanidzwa kuma protein ekuratidza pamusoro (akadai seAga2p). Jini rinoisa mapuroteni anotarisirwa rinogona kusanganiswa nejini rinoisa mapuroteni emadziro e yeast cell (kazhinji Aga2p), uye gene rakasanganiswa rinogona kushandurwa kuita ma yeast cells kuburikidza ne electroporation. Mushure mekushandurwa, ma yeast cells anoratidza ma antibody genes pamusoro pawo anogona kuedzwa antigen-specific screening tests: yeast library inoiswa mu incubated ne target antigen uye yeast cells dzinonyatsobatana nayo dzinosarudzwa. Uchishandisa fluorescence activated cell sorting (FACS) kupatsanura yeast cells dzinoratidza mapuroteni ane hunhu hwaunoda, yeast display library screening inogona kuitwa nehukuru hwe library hwe~10 ^ 8-10 ^ 9 yeast cells. Ma clones akanaka anogona kupatsanurwa kuti awedzere kuongororwa kana kushandiswa pasi. Kana ma clones chaiwo emasoja ekudzivirira chirwere awanikwa kubva muraibhurari yemasoja ekudzivirira chirwere, anogona kutsanangurwa zvakanyanya uye kugadzirwa zvakawanda, uye kucheneswa uchishandisa matekiniki akadai seprotein A/G affinity chromatography kuti apedzise maitiro ese ekuongorora yeast display uye kugadzirwa kwemasoja ekudzivirira chirwere.

Maitiro ekuongorora raibhurari yeYeast Display

Mufananidzo 2 Maitiro ekuongorora raibhurari yekuratidza mbiriso

Kufamba Kwebasa Rekuongorora Raibhurari Yembiriso

Matanho	Zviri Mukati meSevhisi	Nguva yekutevera nguva
Kugadzirira Antigen	Rudzi rweAntigen: Kana mutengi achigona kupa maantigen, sampuro dzinoenderana dzinofanira kuendeswa zvichienderana nerudzi: mapuroteni akasanganiswa anoda 3-3.5mg ane chinodiwa chekuchena chinodarika 85%, mamorekuru madiki anofanira kusanganiswa nechinodiwa chekuchena chinodarika 90%, kugadzirwa kwepeptide kunofanirwa kusanganiswa nechinodiwa chekuchena chinodarika 90%, mhando dzesample dzakadai semavhairasi dzinofanira kuvharwa, RNA inofanira kuongororwa kudzivirira kuora, uye mhando dzeantigen dziri pamusoro dzinogonawo kugadziriswa kuti dzigadzirwe.	Mavhiki maviri kusvika matatu
Kusadzivirirwa kweMhuka	Huwandu hwemajekiseni ekudzivirira mhuka hunosvika mashanu, uye zvakakosha kuona kana huwandu hwemajekiseni huchifanira kuwedzerwa zvichibva pakuongororwa kwe serum titer. Kudzivirira kweAntigen: simba reprotein/virus antigen>105; Peptide/small molecule antigen potency>10^4	Mavhiki mashanu kusvika matanhatu
Kugadzirira kweCDNA yeTemplate	Sarudza maPBMC eplasma, bvisa RNA yese (RNA extraction kit) wobva wadzosera ku cDNA.	Zuva rimwe chete
Kuvaka Raibhurari	Uchishandisa cDNA yeraibhurari se template, VHH gene yakawedzerwa nePCR kaviri, uye VHH gene splicing yeast display vector yakagadzirwa. Vector yakashandurwa kuita yeast cells ne electroporation kuti igadzire antibody library. Sarudza ma clones makumi mana nemasere uye ziva positive rate (>90%) uchishandisa nzira yePCR; Verenga kugona kweraibhurari (10^7-10^8), NGS sequencing kuti uone right insertion rate (>90%) uye library diversity.	Mavhiki maviri
Kuongorora Raibhurari	Madenderedzwa matatu ekuongorora: Kuongororwa kweprotein FACS kwakanyorwa kuti Fluorescence, kuchiteverwa neNGS sequencing mudenderedzwa rechitatu. Ma clones akanaka akasarudzwa kuti aonekwe sequencing ye single gram uye kuti aonekwe se ELISA. Ma clones ese akanaka akasarudzwa kuti aonekwe sequencing yemajini, uye maCDR region sequences akasiyana akasarudzwa.	Mavhiki maviri kusvika matatu
Kuongororwa kweAntibody	Kugadzira mavector akakodzera ekutaura kweantibody sequences kunogona kubatsira kutaura kweantibody, kucheneswa kweantibody, kusimbiswa kweELISA neBLI kweantibody antigen binding kuti ionekwe kuti antibody affinity iri sei, uye flow cytometry blockade kuti ione kuti masero anoshanda sei.	Vhiki imwe chete

Kuongororwa kweRaibhurari yeChiratidzo cheMbiriso pamusoro peRaibhurari

Muzvinyorwa zvepuratifomu yekuratidza yeast surface yekukurumidza kuwana ma nanobodies anosarudza conformationally, vanyori vakagadzira puratifomu yakazara ye in vitro nanobody discovery yakavakirwa pa yeast surface display. Kutanga, raibhurari ye synthetic nanobody yakagadzirwa kutanga kubva kumajini ekamera. Mufananidzo D unoratidza kuti nanobody ine HA tag kumagumo e carboxyl, uye ipapo nanobody inosungirirwa pamwe chete pamadziro eyeast cell. Mufananidzo E unoratidza maitiro ekuongorora nanobody. Mbiriso ine antigen affinity nanobodies yakaparadzaniswa, yakawedzerwa, uye yakasarudzwa kakawanda kuti ive antibodies neFACS. Vanyori vakawana conformational selective nanobodies yakanangana ne GPCRs mbiri dzakasiyana dzevanhu kuburikidza nepuratifomu iyi.