BAO CAOTdM TAT ' H O I N G H I KHOA HOC Kt N I C M 40 N A M (1975-2015) THANH IAP VI^N HAN LAM KHOA HOC VA CdNG NGHt VI$T NAM'
NGHIEN C ( h l T d i U U OlEU KI^N B\iu H l | N PROTEIN IL-3 D U ' 6 l D A N G LAI \lO\
PelB TRONG E. coli
Diromg Thu Hiro'ng, Nguyin Thj Qui, Dgng Thj Ngpc Hd, Ld Thj Thu HSng, Tnrcfng Nam Hai
van Cdng nghi sinh hoc, IS Hodng QuSc Vlit, do GiSy, Hi Nil, tnhaimbLacvn
Ttnh dp Ihiit. Interleukin-3 (IL-3) Id mpt cytokine cd bdn chit Id glycoprotein, dupe xem li cd phi tdc dvng rpng nhit ttong h$ tao miu nhu tham gia vdo qui tilnh tvr dli mdi, nhdn Ito vd biet hda cda cdc tl bdo nguln tgo mdu. Trto thi gidi, nhilu cdng ty lE tgo ra IL-3 theo con dudng tdi t l hpp cho myc dich nghito dm nhimg vdi gid thdnh tuong dli cao. Hau hit cic sdn phim niy dlu dupe bilu hipn td tl bdo E. coli do nhttng iru dilm nhu de nudi ciy, thdi gian sinh trudng nhanh, lupng protein tgo ra nhilu vd dl ddng thu nhgn protein muc tieu. Mdc dd bilu hipn cda protein ngogi lai trong tl bio £. coli phy thudc vio nhilu yen tl nhu die dilm cda gen, tinh chit cda protein, dilu kipn bilu hiptL
Trong nghito cdu trudc, ehting tdi dd thiit kl dupe gen il-3 gdn vdi tin hi$u tilt PelB dl djnh hudng protein ra khoang chu chit vi bilu hipn thdnh cdng protein IL-3 ttong tl bdo E. coli BL21. Tuy nhita IL-3 dupe ting hpp d mdc dO thip vd chl mdt phln d dgng don, phin cdn Igi vln cdn gdn vdi tin hi^u tilt Do dd, dl thu dupe protem IL-3 sau khi bilu hipn dupe cdt hit khdi PelB, ding thdi cdi thi^n sinh khii t l bdo, tdng khi ndng tong hpp protein, ehdng tdi tiln hdnh khdo sdt cdc ylu t l dnh hudng din khd ndng bilu hi§n cda gen il-3 bao gim chung E. coli bilu hita, cdc dilu ki§n Ito men nhu thinh phin mdi trudng, nlng dd chit cim dng, nhi^t dp bilu hi§n, mat d0 tl bdo ldc cdm dng.
Phutfngphip nghiin aim
Cic ehung E. colt BL21(DE3), JM109(DE3), Rosetta 2, BL21 Soluble mang vector bilu hidn pETpe[Bil-3 dupe nudi ciy ttong mdi tnrdng LB chda 100 |ig/ml ampicillin. Djch tl bdo dupe chuyin sang 9 blnh tam giic 100 ml chda 10 ml cic loai mdi trudng khdc nhau: LB, M9, M9 + 1% glucose, M9 +1% glycerol, M9 + 0,5%
yeast exttact, M9 + 1% glucose + 0,5% yeast extract, M9 + 1% glycerol + 0,5% yeast extract, TB, SB. Cdc mdu dupe nudi tilp din khi OD600 dat 0,4-0,6 thi cam dng sinh ting hpp protem bdng 0,5 mM IPTG d 30°C trong 4 gid. Protein ting so dupe dipn di biln tmh ttta gel SDS-PAGE dl xdc dinh mdi trudng phu hpp cho svt bilu hi$n protein IL-3.
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BAO CAOTdM TAT - H O I N S H I KHOA HQC K* N I $ M 40NAM (1975-2015) T H A N H LAP vt^N HAN I A M KHOA HQC V A CdNG N G H | V I ^ N A M "
Tuong tu, cdc thdng so vl nlng dO chit cdm m ^ nhiet dd bilu hi?n, mdt dO tl bdo li^ cam i3iig lin lugrt du^rc diay dli dl ddnh gid khd nat^ tong hop IL-3 vd sinh khii tl bao thu dugc dl tim ra dilm tii uru nhit Ndng dd IPTG dugc khdo sdt tir 0,05- 2 mM. Nhiet dO bilu hi$n dugc ddnh gid t^ 20°C, 25°C, SCC, STC, 40''C. Mdt d$ tl bdo liie cam iing (OD600) lin lugt Id 0,4; 0,8; 1,0; 1,5 vd 2,0.
Tdm tai kit qud vd thdo lu$n
Bieu hien gen il-3 trong cdc chiing E. coli tdi td hgp: Chiing E. coli BL2I vd Soluble t^ng hgp E.-3 k ^ trong khi chung JM109 vd Rosetta 2 ting hgp cd IL-3 d^ng don va d^ng gdn Ihem PelB. ChOng JMI09 dugc I^a chgn ldm chijng bilu hi$n do protein IL-3 ting hgp In djnh, khdng bi phdn cdt bdi protease vat chu theo thdi gian.
Anh hudng cua thdnh phdn mdi trudng din sir biiu hi?n protein IL-3: Mdi trudng nudi ciy Id ylu t l quan trgng dnh hudng din h i ^ suit bilu h i ^ protein vd mdt d$ tl bdo It^ thu mau. Chung tdi khdo sdt 9 cdng thiic mdi trudng vd mdi trudng LB dugc Ilia chgn do protein IL-3 dugc ting hgp nhilu nhdt vd mdt dg tl bdo thu dugc cao nhit, tuy nhien lugng IL-3 cdn gdn vdi tin hi^u tilt PelB tuong duong vdi IL-3 d^g don.
Anh hudng cda ndng dd IPTG din s{e biiu hi?n protein IL-3: Nlng dg IPTG cao cd thi gdy dOc cho tl bdo, lie chl vi khuin sinh trudng. Tuy nhign nlng dO IPTG qud thip 56 h£La chl toe d$ phi§n md, dnh hudng din ndng suit protein. Trong ddi nlng d$
IPTG khdo sdt, nlng dO 0,05 mM IPTG dugc lua chgn do m|t dO tl bdo tiiu mSu cao, lugng IL-3 dang don thu dugc nhilu, d^g gdn vdi PelB it hon hdn vd phii hgp vdi Igi ich kinh tl.
Anh hudng cua nhiet dg biiu hi$n din su biiu hi^n protein IL-3: Nhi$t d0 ciing Id ylu to dnh hudng din hi§u suit vd chit lugng protein tdi to hgp. NhiSt dg cao tiii khd nang ddo thdi plasmid Idn, mRNA dl bi phdn huy. Gidm nhi$t d$ cd thi han chl dugc s\i phdn cdt protein dich bdi protease n^i bdo, cdi thifn dg hda tan. Chung tdi danh gid mgt s6 dilm nhi^ dO vd nhdn tfaiy d 25°C, protein IL-3 bilu hi$n khd tit vd toan b$ dugc cdt khdi tin hi$u tilt PelB. Nguyen nhdn cd thi do d nhi$t d$ thip, cdc qud trmh trong tl bdo dien ra chdm hon, thudn Igi cho vigc cuOn xodn ddng ciu trtic, bOc lg viing nli giOa tin hi$u tilt vd protein 11^3 de enzyme nhdn biet vd cdt hi§u qud.
Anh hudng cda mgt dd ti bdo luc cdm img din sif hiiu hiin protein IL-3: Vi§c
BAO CAO T 6 M TAT ' ^ O I N G H I KHOA HQC K^ N | £ M 4 0 N A M (1975-2015) T H A N H LAP V 1 | N HAN L A M KHOA HQC V A C O N G N G H | V I | T NAM"
cam ling bilu hif n protein ngoai lai thudng thyc h i ^ 6 gi5a-cu6i pha sinh tnidi^ nam mu de ddm bdo hif u suit ting hgp protein ciing nhu hgn dil bilu hi$n cde protease khi te bdo budc vdo pha tihh. Cam iing d thdi dilm OD600=1 la tiilch hgp nhit vi m^t dO tl bdo culi cOng cao, lugng protem IL-3 ting hgp khd nhieu.
Sau khi khdo sdt cdc dieu kiSn bilu hi§n phii hgp, smh khii t l bdo tdng l&i khoang 2,3 lin, ddc bigt protem IL-3 sau Idii bilu hi|n khdng cdn gdn vdi PelB, thu|n Igi cho vifc tdch chilt vd tinh s ^ sau ndy.
Protein t6ng sfi bi^u hi§n trong E. coli mang gen il-3 gin tfn hi$u t i l t pelB tnrdrc (a) vi sau (b) khi toi lAj di^u ki#n \%n men. £>C: protein tir d6ng t l bdo khdng mang gen); M: thang protein chudn
(Fermentas).
Xdc dinh trgng thdi protein IL-3: Todn bO IL-3 ndm d pha tiia vd kit ty 6 dgng thi viii trong tl bdo chit mdc dii tiiilt kl bilu hi$n IL-3 djnh hudng tilt ra khoang chu chit nhd tfn hi$u tilt PelB, c6 thi do-protein dugc bilu hien vugt miic din din cu$n xodn sai l^ch, kit t^ hinh didnh dgng khdng tan hay vl m$t ca chl ndo dd md protein dfch khdng Iham gia vdo con dudng tilt Uu diem ciia protein it dang till vid Id thudn Igi eho qud trinh tdch chilt, tinh sgeh khdi cdc protein tap ctia vdt chii. Ket qud ndy la tien dl cho vi^ thu hii lugng Idn IL-3 de nghiSn eutu tinh chit cua protein.
Tif khda: Escherichia coli, protein tdi to hgp, IL-3, pelB. biiu hi$n protein.
