Chiyambi cha Utumiki Wowonetsera Ma Phage

Kuwonetsera kwa phage, monga njira ya mamolekyu yozikidwa pa kusintha kwa majini a phage DNA, kwakhala chida champhamvu kwambiri cha mamolekyulu popanga ma probe a mamolekyu motsutsana ndi zolinga zinazake mwa kuwonetsa zidutswa izi pamwamba pa phage motero kusankha zidutswa za peptide/antibody zokhala ndi katundu womangirira kuchokera ku mitundu yambiri (malaibulale).

Alpha Lifetech ali ndi zaka khumi akugwira ntchito yopanga ma antibodies a phage display ndipo amatha kupereka ntchito yomanga ndi kuyesa ma antibodies aukadaulo kwa makasitomala athu. Malinga ndi zosowa za makasitomala, titha kupereka mitundu yosiyanasiyana ya ma antibodies, monga malaibulale oteteza chitetezo cha mthupi, malaibulale achilengedwe, malaibulale opangidwa ndi ma synthetic laibulale. Ukadaulo wa laibulale yowonetsa phage, womwe umagwirizanitsa majini a antibody ndi majini a mapuloteni a phage coat monga M13, ndiye ukadaulo wodziwika bwino wokonzekera ma antibodies ndipo umagwiritsidwa ntchito kwambiri popanga ma antibodies apadera kwambiri.

Alpha Lifetech idzapatsa katemera alpaca, llama, alpaca, ndi shaki, ndi zina zotero, kuti mphamvu zawo zifike pa 10^5. Tidzatumiza lipoti loyesa mphamvu kwa kasitomala kuti titsimikizire kuti detayo ndi yolondola komanso yolondola. Malaibulale achilengedwe ali ndi ubwino wa poizoni wochepa komanso chitetezo chochepa cha mthupi. Alpha Lifetech ili ndi malaibulale ambiri achilengedwe opangidwa kale, omwe angagwiritsidwe ntchito mwachindunji poyesa ma antibodies popanda katemera wa nyama, motero kuchepetsa nthawi ya polojekitiyi, kupititsa patsogolo kupita patsogolo kwa mayeso a kasitomala, ndikutsimikizira kuti antibody ikugwira ntchito bwino.

Chithunzi 1: Mfundo yowonetsera phazi

Dongosolo Lathu Lowonetsera Phage

Chidziwitso chachikulu cha dongosolo lowonetsera la M13/T4/T7 phage chawonetsedwa mu Gome 1.

Gome 1 Magalimoto osiyanasiyana owonetsera phage ndi mawonekedwe awo
	M13	T4	T7
Kukula kwa majini	6407 bp	168895 bp	39937 bp
Mapuloteni Owonetsa	pVI, pIII ndi pVIII	SOC ndi HOC	gp10B
Kukula kwa Chiwonetsero	>110 kDa ndi pIII
Kukula kwa Chiwonetsero
Kuchuluka kwa chiwonetsero		Makope	Makope
Kuchuluka kwa chiwonetsero	Makope okwana 2700 pa pVIII	Makope ochepera 155 pa HOC	Makope
Mayendedwe amoyo	Lysogeny	Lytic	Lytic

Alpha Lfetech Angapereke

Kuwonetsera kwa Phage kumagwirizanitsa majini ndi phenotype kukhala chimodzi, kuphatikiza kusankha ndi kukulitsa ndi mphamvu zamphamvu zowunikira. Alpha Lifetech ikhoza kupereka ntchito zosiyanasiyana pakupanga ma antibodies a phage display.
Ntchito zazikulu zikuphatikizapo: nsanja ya laibulale ya ma antibodies a VHH, nsanja ya laibulale ya ma antibodies a scFv, nsanja ya laibulale ya ma antibodies a Fab, nsanja yomanga laibulale ya phage, nsanja yowunikira laibulale ya phage ndi ntchito yodziwira ma antibodies ndi ntchito zina.

chitukuko cha ma antibodies - Alpha Lifetech

Chithunzi 2: Njira yopangira ntchito yowonetsera mawonekedwe a phage

Njira Yopangira Ma Antibody Owonetsa Phage

Tikhoza kupereka katemera ku nyama ndi mapuloteni omwe amapezeka mu labotale yathu kapena operekedwa ndi makasitomala athu, kuphatikizapo mapuloteni osiyanasiyana a macromolecular, peptides ndi mapuloteni a membrane. Kudzera mu kuyesa kwa ELISA, ofufuza athu adachita PBMC isolation ndi RNA extractions kuchokera m'magazi a alpacas ndi zotsatira zabwino kwambiri za katemera. Mwa kubwerezabwereza transcription ndi gene amplification, tinapanga jini pa pMESC, pComb3XSS kapena pCANTAB 5E phage vectors. Mwa electrotransformation ya maselo oyenerera a TG1 E. coli, malaibulale a phage antibody okhala ndi mphamvu ya laibulale yoposa 10^9 angapezeke. Malaibulale a antibody okhala ndi mphamvu yayikulu ya laibulale ali ndi mitundu yosiyanasiyana ndipo amathandiza kupeza ma antibodies okhala ndi mphamvu. Kuti tiwonetsetse kuti laibulale ya antibody ili bwino, tidzafotokozera mphamvu ya laibulale ndi kuchuluka kwa laibulale. Pomaliza timatsata malaibulale kuti tipatse makasitomala athu ma sequences ofunikira 30-50. Kenako, timachita kafukufuku wa laibulale ya phage display, Choyamba, antigen imalephereka kuyenda pa ma polypropylene microplates, ndipo kudzera mu kafukufuku wa maulendo 3-5, ma phage-antibodies omwe ali ndi mphamvu yofooka yomangirira amachotsedwa, ndipo ma clones enieni omwe amamangirira ku antigen amasungidwa. Pa chiwonetsero cha phage, njira ya ELISA imagwiritsidwa ntchito kuti ikhale ndi clone yabwino, pomwe pa chiwonetsero cha yisiti, njira ya FACS imagwiritsidwa ntchito kuti maselo awonetse zidutswa za antibody zobwerezabwereza pogwiritsa ntchito ma antigen olembedwa. Kudzera mu kafukufuku, timakonzekera ma antibodies omwe ali ndi mphamvu yayikulu, yeniyeni komanso yogwirizana kwambiri.

Chithunzi 3 Njira yopangira ma antibodies a Phage display

UBWINO WA UTUMIKI
Ubwino wa UTUMIKI

Alpha Lifetech yadzipereka kwambiri pa ukadaulo wowonetsera ma phage, ndipo imatha kupatsa makasitomala ntchito zambiri komanso zabwino kwambiri zowonetsera ma phage.

Nthawi ndi magwiridwe antchito apamwamba

Kampani yathu ikhoza kufunafuna ma antibodies okwanira m'kanthawi kochepa; titha kuchita mayeso osiyanasiyana owunikira ma antibodies malinga ndi zosowa za makasitomala.

Ubwino wazinthu zapamwamba

Kampani yathu imathandiza makasitomala kukonzekera ma antibodies okhala ndi chidziwitso chapamwamba komanso kukhazikika, zomwe zimawonjezera zotsatira za kafukufuku wasayansi.

Perekani zosankha zosiyanasiyana

Kampani yathu ikhoza kupanga malaibulale a ma antibodies a mitundu yosiyanasiyana (a anthu, mbewa, kalulu, alpaca, ndi zina zotero) ndipo ikhoza kupanga mitundu yosiyanasiyana ya malaibulale a ma antibodies (scfv, Fab, VHH, ndi zina zotero).

Kutha Kwambiri

Laibulale yathu ya ma antibodies ili ndi mphamvu zambiri zokwana ma antibodies oposa 10^9

Mafunso Ofunsidwa Kawirikawiri - Mavuto Ambiri Okhudza Kuwonetsa Phage

Mafunso owonetsera phage ◢

Q.
Kodi ukadaulo wowonetsera phage ndi chiyani?

A.
Ukadaulo wowonetsa phage ndi kuyika jini lachilendo pamalo enieni a puloteni ya phage capsid, ndikuwonetsa jini lachilendo pamwamba pa phage popanda kusokoneza ntchito yachibadwa ya phage. Pambuyo poyesa kangapo, puloteni yolunjika yokhala ndi luso lapadera komanso ntchito yayikulu ya zamoyo pamapeto pake idapezeka.
Q.
Kodi ubwino wa ukadaulo wowonetsera phage ndi wotani?

A.
Malaibulale okhala ndi ma antibodies amphamvu kwambiri amatha kupangidwa pogwiritsa ntchito ukadaulo uwu. Nthawi yowunikira ndi yochepa, njira yogwirira ntchito ndi yosavuta, ndipo mitundu yosiyanasiyana ya ntchito ndi yayikulu, zomwe zimathandiza kuzindikira njira zabwino kwambiri za ma peptide kapena mapuloteni omwe akufuna. Mu ntchito zokhudzana ndi kachilomboka, ofufuza amatha kupeza ma antibodies omwe amamangirira ku ma epitopes ofunikira a kachilomboka powunika malaibulale akuluakulu a ma antibodies. Njirayi ingalepheretse kulowa, kubwerezabwereza, kapena njira zopewera chitetezo cha mthupi za ma virus, motero kukhazikitsa maziko ochizira matenda.
Q.
Kodi kusiyana pakati pa ukadaulo wa hybridoma ndi ukadaulo wa phage ndi kotani?

A.
Ukadaulo wowonetsa phage umagwiritsidwa ntchito kwambiri kuposa ukadaulo wa hybridoma, womwe umangokhala wopanga ma antibodies ochepa motsutsana ndi ma immunogen enaake. Mosiyana ndi zimenezi, ukadaulo wowonetsa phage ukhoza kupereka laibulale yonse ya ma antibodies ochokera ku mitundu yosiyanasiyana ya chitetezo chamthupi.
Q.
Kodi ukadaulo wowonetsera phage umagwiritsidwa ntchito bwanji?

A.
Ukadaulo wowonetsa phage uli ndi ntchito zosiyanasiyana, kuphatikizapo kufufuza ma receptors a tizilombo toyambitsa matenda, kuphunzira momwe mapuloteni amagwirira ntchito, kutsatira kufalikira kwa zizindikiro pakati pa maselo, kuzindikira matenda a nyama, ndikupanga katemera, pakati pa madera ena.
Q.
Kodi pali chiyembekezo cha ukadaulo wowonetsera phage?

A.
Ukadaulo wowonetsa phage umapereka zabwino zambiri ndipo ungagwiritsidwe ntchito m'magawo osiyanasiyana. Komabe, vuto lalikulu popanga ma antibodies ophatikizana pogwiritsa ntchito njira yowonetsera phage ndizovuta komanso mtengo wogwirizana ndi njirayi. Njirayi imafuna kuphatikiza njira za biology ya molecular, kuphatikizapo kusintha kwa majini, kupanga ma cloning, ndi kufotokozera, pamodzi ndi kusanthula kwa immunological, biochemical, ndi biophysical kuti tizindikire ndikuwonetsa ma antibodies omwe tikufuna.

Mafunso okhudzana ndi ma antibodies ◢

Q.
Kodi njira zowonetsera ma antigen ndi ziti?

A.
Kuwonetsedwa kwa antigen ndi mgwirizano wofunikira kwambiri. Njira zowonetsera antigen zimaphatikizapo makamaka kuwonetsedwa kwa antigen yophimbidwa mwachindunji, kuwonetsedwa kwa antigen yophimbidwa mwachindunji, kuwonetsedwa kwa antigen kudzera mu kuyesa kwa maselo onse, kuwonetsedwa kwa antigen kudzera mu liposomes, nano-phospholipid disks ndi VLPs.
Q.
Kodi pali kusiyana kotani pakati pa mitundu yosiyanasiyana ya ma antigen?

A.
Njira yophikira mwachindunji ndi yosavuta, koma mawonekedwe a antigen ndi osavuta kusintha, ndipo ntchito yophikira yosalunjika ndi yovuta, koma imatha kusunga mawonekedwe a antigen ndikuwonjezera magwiridwe antchito. Kuwunika maselo kumatha kutsanzira malo enieni mu vivo, koma pali vuto la kumangirira kosalunjika. Disiki ya Nano-phospholipid ili ndi kukula kochepa, magwiridwe antchito apamwamba komanso kukhazikika bwino; Ma VLP amaphatikizidwa ndi antigen kapena kukulungidwa ndi antigen, yomwe ili ndi chitetezo chamthupi chambiri komanso chitetezo chabwino, ndipo ingayambitse chitetezo chamthupi champhamvu.
Q.
Kodi kuchuluka kwa ma antigen kumakhudza bwanji zotsatira za ukadaulo wowonetsa phage?

A.
Ngati kuchuluka kwa madzi kuli kochepa kwambiri, mwayi wolumikizana ndi phage udzachepa, zomwe zimapangitsa kuti ntchito yowunikira ichepe. Mosiyana ndi zimenezi, kuchuluka kwa madzi ambiri kungawonjezere mwayi wolumikizana ndi madzi osadziwika bwino, kukweza chizindikiro chakumbuyo, ndikulepheretsa kuzindikira ma clones abwino.
Q.
Kodi tingatsimikizire bwanji kuti antigen imasunga mawonekedwe ake achilengedwe panthawi yowunika?

A.
Njira yochepetsera yofewa ingagwiritsidwe ntchito popewa kutentha kwambiri, asidi wamphamvu ndi alkali. Njira yophikira yosalunjika komanso dongosolo la biotin-streptavidin zinagwiritsidwa ntchito kuchepetsa kuwonongeka kwa kapangidwe ka antigen. Ngati antigen ndi puloteni ya nembanemba, imatha kuwonetsedwa pamwamba pa maselo osakhudzidwa kapena kugwiritsidwa ntchito kutsanzira chilengedwe cha nembanemba, monga nano-disk, kuti isunge mawonekedwe ake achilengedwe a nembanemba.
Q.
Kodi mungatsimikizire bwanji kutsimikizika kwa ma antibodies omwe amawunikidwa ndi ukadaulo wowonetsa phage ku ma antigen?

A.
Kuwonjezera pa zoyeserera zachizolowezi monga ELISA, Western Blotting (WB), ndi Immunofluorescence (IF), njira zodziwira Surface Plasmon Resonance (SPR) ndi Bio-Layer Interferometry (BLI) zingagwiritsidwe ntchito poyang'anira njira zomangira ndi kugawa ma antibodies ndi ma antigen nthawi yeniyeni, zomwe zimathandiza kudziwa momwe ma affinity ndi kinetic parameters amagwirira ntchito.

Mafunso Okhudzana ndi Chitetezo cha Mthupi ◢

Q.
Kodi chitetezo cha mthupi cha nyama chimayankha bwanji mu ukadaulo wowonetsa phage?

A.
Kuwonetsa kwa phage kungagwiritsidwe ntchito kupanga ma antibodies a monoclonal okhala ndi mgwirizano wapamwamba kwambiri pamitundu yosiyanasiyana yopanga ma antibodies, kuphatikiza koma osati anthu okha, mbewa, makoswe, akalulu, nkhuku, ngamila, alpaca, ng'ombe, agalu, nkhosa, anyani, ndi shaki.
Q.
Kodi ndi makhalidwe ndi zochitika ziti zomwe zimagwiritsidwa ntchito m'malaibulale a antibody ochokera ku mitundu yosiyanasiyana ya nyama mu ukadaulo wowonetsa phage?

A.
Ukadaulo womanga malaibulale a ma antibodies a mbewa ndi wodziwika bwino, ndi wotsika mtengo, ndipo uli ndi nthawi yochepa yoyesera, zomwe zimapangitsa kuti ikhale yoyenera kuyesa ma antibodies oyambirira komanso kafukufuku woyambira. Ma antibodies a kalulu amasonyeza kuyanjana kwakukulu komanso kudziwika bwino, ndi kuchuluka kwa kuzindikira kwa epitope poyerekeza ndi ma antibodies a mbewa. Ma antibodies a anthu amatha kupezeka mwachindunji kuchokera ku malaibulale a ma antibodies a anthu, motero kupewa zotsatira za chitetezo chamthupi zomwe zingachitike pamene ma antibodies a heterologous aperekedwa m'thupi la munthu. Njirayi ili ndi phindu lalikulu pakufufuza ndi kupanga mankhwala, makamaka popanga ma antibodies ochiritsira.
Q.
Kodi chitetezo chamthupi cha nyama chomwe chimagwiritsidwa ntchito muukadaulo wowonetsa phage ndi chiyani?

A.
Ma immunogen a ziweto nthawi zambiri amagawidwa m'magulu awiri: protein antigen, polypeptide antigen, nucleic acid antigen ndi pathogen-related antigen, pakati pawo pali pathogen-related antigen yomwe imaphatikizapo virus antigen (tinthu tathunthu ta virus, virus protein, virus non-structural protein, etc.), bacterial antigen (complete bacterial extract, cell wall component, secreted protein, toxin, etc.) ndi parasitic antigen.
Q.
Kodi tingathetse bwanji vuto la kulekerera kwa chitetezo chamthupi mwa nyama panthawi ya chitetezo chamthupi?

A.
Sinthani ma immunogens mwa kusintha ma antigen a chitetezo chamthupi ndi mapuloteni ophatikizana ndikuyika tinthu tating'onoting'ono ta ma virus ozimitsa moto m'malo mwa mapuloteni opangidwa ndi ma virus. Sinthani kapangidwe kake ka kuyesera ndikusintha mlingo wa chitetezo chamthupi.
Q.
Kodi mphamvu ya chitetezo cha m'thupi ya ma immunogens ingawongoleredwe bwanji mu ukadaulo wowonetsa phage?

A.
Kukhazikika kwa chitetezo chamthupi kumatha kukulitsidwa mwa kuphatikiza ma antigen ndi mapuloteni onyamula, monga bovine serum albumin. Kuphatikiza apo, zinthu zothandizira chitetezo chamthupi, kuphatikizapo Freund's adjuvant ndi aluminiyamu adjuvant, zitha kugwiritsidwa ntchito kuti ziwonjezere nthawi yokhala m'thupi ndikulimbikitsa kuyambitsa ndi kufalikira kwa maselo a chitetezo chamthupi, motero zimawonjezera mphamvu ya chitetezo chamthupi.

Mafunso Okhudza Kumanga Laibulale ◢

Q.
Kodi laibulale ya ma antibody a phage ndi chiyani?

A.
Laibulale ya ma antibodies a phage ndi gulu la ma antibodies osiyanasiyana a phage omwe amapangidwa mwa kusonkhanitsa majini onse a dera losiyanasiyana kuchokera ku ma antibodies kupita ku ma phage vectors ndikuwawonetsa pamwamba pa ma phage. Njirayi imapangitsa kuti pakhale laibulale ya ma antibodies a phage.
Q.
Njira yopangira laibulale ya antibody ya phage

A.
Njira yonse yowonetsera phage imakhudza masitepe angapo ofunikira: kuchotsa maselo a magazi a mononuclear (PBMC) kuchokera ku nyama zomwe zalandira katemera, kupatula RNA yonse ndikuisintha kukhala DNA yowonjezera (cDNA), kukulitsa jini yowunikira pogwiritsa ntchito ukadaulo wa polymerase chain reaction (PCR), kupanga jini yowunikira kukhala vekitala ya phage, ndikusintha vekitala kukhala selo lothandizira kuti liziwonekera. Njirayi imapangitsa kuti pakhale laibulale yowonetsera phage, yomwe imatha kuzindikira mwachangu ma antibodies apamwamba. Pambuyo pake, laibulale iyi ikhoza kukhala chida chofunikira pakupanga katemera, mankhwala ochiritsira, ndi ma reagents ozindikira.
Q.
Kodi mfundo zazikulu zomanga laibulale yapamwamba ndi ziti?

A.

Onetsetsani kuti chiwerengero chonse cha PBMC chili chokwanira. Onetsetsani kuti RNA ili bwino popanda kuwonongeka.

Kuti zitsimikizire kuti pali anthu onse osintha zinthu, laibulale iyenera kukhala ndi mphamvu zokwana 10 mpaka 100 kuposa kusiyana kwa mfundo.
Q.
Kodi magulu a laibulale ya ma antibody a phage ndi ati?

A.
Malinga ndi gwero la laibulale ya ma antibodies, malaibulale a ma antibodies a phage akhoza kugawidwa m'ma laibulale a ma antibodies a immune phage ndi malaibulale a ma antibodies a native phage. Kusiyana kwakukulu pakati pa mitundu iwiriyi kuli ngati nyamazo zalandira katemera. Kuphatikiza apo, malaibulale a ma antibodies a native angapangidwe kudzera mu semi-synthesis kapena synthesis.
Q.
Kodi munthu angayese bwanji ubwino wa laibulale ya ma antibody a phage?

A.
Pali zizindikiro ziwiri zazikulu zowunikira ubwino wa laibulale ya ma antibodies: mphamvu yosungira ndi kusiyanasiyana. Mphamvu yosungira yambiri, pamodzi ndi kusiyanasiyana kwakukulu, imatsimikizira kufufuza bwino ma antibodies ndi mphamvu yayikulu komanso yeniyeni.

Mafunso Okhudza Kufufuza Laibulale ◢

Q.
Kodi njira yowunikira laibulale ya ma antibodies a phage ndi yotani?

A.
Pakufufuza kwa zamoyo, laibulale yowonetsera imayikidwa ndi zolinga zokhazikika kenako imatsukidwa kwambiri kuti ichotse ma phage osagwira ntchito. Ma conjugates nthawi zambiri amachotsedwa pogwiritsa ntchito yankho la acidic kapena mchere wambiri kenako amawonjezeredwa m'maselo oyenera kuti awonjezere mphamvu. Pambuyo poyesa ma antibodies atatu mpaka asanu, amapezeka ma antibodies okhala ndi mphamvu yayikulu.
Q.
Kodi ndi njira ziti zomwe zimagwiritsidwa ntchito poyesa ma antibodies a phage?

A.
Njira zoyezera phage makamaka zimaphatikizapo kuyika panning yolimba, kuyezera gawo lamadzimadzi, ndi kuyezera maselo. Kusankha njira yoyenera yoyezera kuyenera kuganizira makhalidwe a mamolekyu omwe akufunidwa, zolinga za njira yoyezera, ndi malo ochitira kafukufuku.
Q.
Kodi mayeso abwino ndi oipa a phages ndi ati?

A.
Kuyezetsa kwabwino ndiyo njira yofunika kwambiri yoyezetsa ma antibodies a phage omwe amamangirira ku mapuloteni omwe akufuna. Kuyezetsa kwa negative kumathandiza kuchotsa ma antibodies osakhala enieni. Pa nthawi yoyezetsa ma antibodies a phage, ma antibodies osakhala enieni amatha kuzindikirika chifukwa cha kusokonezedwa ndi chizindikiro cha antigen, zinthu, kapena ma antigen ena omwe ali ndi kapangidwe kofanana ndi ka antigen yomwe akufuna. Pazochitika zotere, kuyezetsa kwa negative kungathandize kuti njira yoyezetsa igwire bwino ntchito.
Q.
Kodi mungapewe bwanji kuyesa ma antibodies osadziwika?

A.

Ma antigens olembedwa ndi mitundu yosiyanasiyana ya zizindikiro akhoza kuyikidwa patsogolo poyesa koyamba, kapena kuyesa koyipa kungagwiritsidwe ntchito kuchepetsa kuchuluka kwa ma antibodies osadziwika bwino.

Mungasankhe kusintha madzi otsekereza oyenera kapena kugwiritsa ntchito madzi otsekereza osiyanasiyana kuti mupewe vutoli. Ngati antibody yomwe imazindikira mzere wa maselo akumbuyo (CHO kapena HEK293) yafufuzidwa, mutha kusintha mizere yosiyanasiyana ya maselo akumbuyo kuti mufufuze kapena kuti mufufuze zolakwika.
Q.
Kodi munthu angatsimikizire bwanji ndikusanthula ma antibodies omwe apezeka?

A.
Njira zotsimikizira ma antibodies nthawi zambiri zimaphatikizapo ELISA, Western Blotting (WB), flow cytometry, immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry, ndi njira zina zosiyanasiyana zoyesera. Alpha Lifetech ali ndi luso lalikulu pakupeza ma antibodies ndipo ali ndi gulu la akatswiri oyesera, kuonetsetsa kuti ma antibodies akugwira ntchito bwino muzoyesera zotsatira.

Ngati muli ndi mafunso aliwonse, chonde musazengereze kulankhulana nafe nthawi iliyonse.

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