Tgp chi Cong light- Sinh hoc H((2), 31<>-325, 2(112
LEN M E N T i l N C ; M E I H N A N ( ; < A O .SAN L L O N C ; F L A < ; E I . L I N I I U H I ( | [ I I N ( ; V I K H U A N ESCIIERKHn (OU\\\ l ( > l E O I ' I R O N C . H E M I O N ( ; I I N M F N DA ItJMII G R E T V
O i r T h i l l u j e n ' , Le Q n j n h Ciani;'. .SNcn-OJofKiifois-. imntif. N;,,,, Hal' 'Vicn Cimg nglu; smh hoc, I 'n-ii Kluni hoc va Ciing nghe I id \,ini
" Vien Ce'mg nghe Hoang gia Thuy Dicn TOM TA 1
(.ten /hC mil IUKI cho lliigellin l l n cu.i \„lwonelU, cm hicn Iroiiii tc hao lis, luri, Ina enh Ml 21 dmVi d.iny duuy. Iio nguyen ticn gi'i do la ihioicdoMii (11\) nen dLr<,Tc yo' I'll !-i I nghien cim lam v.iccine pUowj. Salm,mella ho;ic l.iiii l.i cli.il len mon ^aii \ufu l i \ l li( iioni: liv lining Icn duong Irong mdi tnrouL! \,"i quy trinh len iiicii cung nhu sun lugiig piotein ii!io,u lai '1i\l IK sung gian doan ngudn dmh dirony ^ Ki (ca
•n,,(SCio\;ii lypliiiiiiiiiiiiiulildiiocnghiOiicim liiai ' ^ Ii'iii l<'' li'mi lang linh smh mien IIKII CU.I kli.ing i\l lit Dc CO duivc luoiiy Idn kli.iiiLi iiyiiycii cho cac .ho \accini.'. Uong cony Uinh ii.ij cliuiifitoi dii loi iru hinh (iKi.i (llHiy Dicn) liony hinh len men, chat diiili cv .mil hinrng Km deu smh kluH cua chung /: coh HI 21 Uii U< hyp
Kill nuoi i.u cliuiiL! Uii lo hi>]i o dicu kicn len men ll'd-biilch bo ol). .sinh klioi lc (\io tang nlianh, Tr\FliC duoc loni: hoii un \ oi ^an lu\mg dai lien 1.8 ij/l. urong dumig 0.14 y/g te kio khd Tuy nhien. cdng nghe len men led-bdlch ho SUIIL: guin diun cii kci hi.rp ho sung lien tyc nguon dinh duong Lim king dang ke san lircmg TrxFliC De tliu duoc luong hm TixlliC. \iec bo sung isopio|i>l-Kl.i-l)-ihiogiil;iclostde (IPTG) de cam img K^ng hgp prolein ngoai lai phai duoc uen hanh Irong giai doan len men Icd-halch co ho .sung gian doan nguon dinh duong khi lH),.i,i cua ic biio ikii klioang 24 Tiong ijuit Uinh lOn men limg me co bo sung lien luc nguon dinli duong, Ihanh phan dinh dirmig bo sung \ao binh len men co aiili huong (irn ddn mirc do on dmh cu.i TixMiC Ham luong Tr\l liC thu diroc lh;ip khi bo sung hen luc ngufiii glycerol hay Y'Ui, irong khi do, bn sung VG (glyceiol va ciionam men), lijni luong TrvFliC iluKiuoc<''iidinh him (tdicu kicn Icn men loi irii, lui,Tng'lrxlliC thu duoc dai 4.(tg/lii moi iiiiong. laiiggapkho.ing 9 l.iiiso\.Vi Icn men o hmh liim giac
TirUida E coli Bl.21, lliC. linig me. S Typliin <i. TixfliC.
MODAU
Flagellin FliC ma hoa btii gen fliC la mpt trong hai loai flagellin ctia le bao Salmonella enterica serovar Typhimurium (viel lal la S Typhi murium) da dugc chimg minh co linh sinh mien djch bao ho chong lai S Typhimurium. Flagellin noi chung va FliC noi rieng co kha nang kich thich ca the dong vat smh mien dich a ca hai nhanh la mien dich bam smh va mien dich tiep thu (qua mien dich dich the va mien dich trung gian te bao) giup ca the khang lai VI khuin (Cuadros et ai, 2004, Ramos el al., 2004).
U^u diem cua FliC la c6 kha nang kich thich tao lugng lan khang the tiet vao djch nhay ruot nham ngan chan sir xam nhiem Salmonella ngay a he tieu hoa cua dong vat (Strindelius et ai, 2004). Ngoai ra, khang nguyen nay con co the kich boat lam tang sinh so lugng te bao gi^t tir nhien (NK) tot hon khang nguyen O ciia t6 bao vi khuan (Tsujimoto et ai, 2005).
Tir nam 2006, flagellin da dugc nghien cim sir
dung do lam ta chat dimg cho phong va dieu trj ung thu (United States Patent Application Publication, so US 2006/0088555 AI), Trong \ai n.im Iro lai day. khang nguyen nay ngay cang thu hut dirge nhieu nhom tac gia tren the guVi quan tam nghien lam ta chat vaccine chong lai benh do virus. Dien hinh, nam 2010 mot nhom tac gia a My da thir nghiem tlianh cong flagellin eiia S. TyphimiiMum lam ta chat cho vaccine duai dan vj phoujj H5N1, Sau khi cong cuong dgc vm lieu cao gap 40 lan LDsn, 16 chuot duac gay mien dich van song 100%. Dieu dang chii y la khi sir dung llagellin lam ta chat, vaccine cum da dugc gay mien dich cho dgng vat qua phun .strong, nho mui nen rat thyan tien cho vi^c sir dung (Skountzou ei al. 2010).
Vol muc dich thu dugc luong Ion khang nguyen Flic phyc vy cho nghien ciru lam ta chat vaccine hoac lam vaccine phong S Typhimurium cho gia c3m, nhom nghien cmi da bieu h'lenJIiC duai d^ng rieng le trong E coli nhung khang nguyen tai to hgp sau khi sinh ra de bj phan cat bai protease ciia vi khuan chu (Tran Ngoc Tan el ai, 2003, Lopez-Boad 319
Do Thj Huyen etal el al. 2004), Khi dirge bicii liicii i.\uCi\ d^mg dung hgp
viVi thioredoxin 0\\). l i \ l l i C dugc bleu hi^n An dinh hon v^ san lirgng thu dugc cao lum. Trong nghien cCni n^y. chiing toi lya chgn dieu kivn len men. quy trinh len men di- Idni lang s.in lir(,Tiig TrxFIiC phyc vy cho nghien cim iieii dgng v(il
Mv thong len men (ireta dirge lya chgn de tin uu cac dicu kiC'ii len men do chiiiij: cii im diem s.iii (1) d;i\ lii In; llioiig li'ii men gom d binh len men giAng hC'l nhau. dirge ket noi cluing vin iiigt ni^y tinh nen h^n chi- dugc sg sai khac \ c cdc \eu lo ngo^i c^nh nhu sai khac \ e trying thai sinh ly cua inSu klu eliuven sang binh Icn men. c(> mau vA cAc yeu to khac; (2) co the thay doi mgl li.i> nhieu yen i6 tloi
\ a i mot hinh len men. (3) trying thai smh Irutnig. deu thy nguon dmh duiVng va linh triing Irao doi cliiit ciiii chijng dien ra trong tung binh len men dugc tich hgp chung nen de so s^nh nhgn biet anh liinnig ciia tirng
\eu to len sy smh Iruting, trao doi chat, tong hgp protein tii to hgp ciia chung E i oli lai to hgp,
VAT Uhl' VA PHU'ONCi PHAP
\ ' a t li^u Chiing vi .sinh vgt
Chung Escherichia coli BL21 [F amp hsd SB(rBmB)gal dem (DE3} plysSfCaml)] tai l6 hgp mang gen ma hoa flagellin FliC cua S. Typhimurium do phong Ky thuat di truyen thiet ke.
Thiet bj len men
H^ thong len men da binh Greta (Belanch Bioleknik, Thuy Dien).
.Mill trtrimg
• MVTGly: I lit moi trucmg chira 6,645 g Na2HP04 2 H ; 0 ; 3 g KH.P04: 0,5 g NaCI; 1 g NH4CI, 0,24 g M^SO.,; 0,024 g CaCl;.2H;0; 10 g tryptone; 5 g cao nam men va 10 g glycerol.
- YT- 5 g/l cao nam men va 10 g/l tryptone.
- YG, 5 g/l cao nam men va 10 g/I glycerol.
- YTG: 5 g/l cao nam men, 10 g/l tryptone va 10 g/l glycerol.
Dung dfch vi lupng
Mgt lit dung dich vi lugng bao g6m: 0,5 g CaCl2,2H20; 16,7 g FeCb.6H30; 0.18 g ZnS04,7H.O;
0,16 g CuS04.5H20; 0,11 g MnSO^.lHjO; 0,18 g CoCl;.6H20; 20,1 gNa-EDTA.
Phu'tfnj; phAp
I.en men sdn xu&l protein ldi lo h{rp
(^'himg /•,' (•(;// t^i to hgfp dugc toi uu len men trong hv ihi'iig len men da binh Greta gom 6 binh len men, dung lich I lit. Moi trudng MYTGly dugc chuan bi vino cic binh Icn men sau d6 khtj trung true Hep loan bO h? ihong, Ket thiic khir trimg, moi lrir(Tiig dugc lam ngugi nhanh ho$c de ngugi t^
nhien. sau do bo sung thfim dung djch vi lugng theo ly Iv 3 ml dung djch vi lugng cho I lit moi tnrong MYTCJly. C,ie dieu kivn nuoi cay chung tai lo hgp, iiliicl dg nuoi ban dau 37°C, nhivt dp cam img 2S-3()'C. pH 7.0 dugc dieu chinh bSng NH4OH 2.VI/C, vi IIJ'Oi 20% (Hedren d ai 2006); toe dg dong khi 0,5-1 vvm (the tich khi tren the tich moi irycmg trong I phiit). ap suat 0,1 bar; toe dp khuay 500-1500 vong/phul (v/p) de kiem soat gia trj nong dp oxy h6a tan (DOT) trong khoang 20-30%. Cir sau I h, tien hanh thu mau mpt Ian de phan tich loc dp sinh truang thong qua gia trj ODhoo va protein tai to hgp pha tan, pha khong t.m tren gel SDS-PAGE.
Cac quy trinh len men dugc khao sat (I) len men batch I: Ien men trong dieu ki^n thong thuong den khi nguon dinh dudng trong moi truang c p ki^t;
(2) len men limg me b6 sung gian do^n ngucn dinh dudng' sau khi ket thiic batch I. binh len men dugc bo sung 1 lan vai mgl lugng dinh dudng bang lupng ban dau nhung the tich b ^ g I/IO the tich moi truong ban dau; (3) Ien men timg me bo sung lien tyc nguon dinh dudng' sau khi ket thiic batch I. nguon dinh dudng vcri dp dgm d$c gap 10 Ian moi truong ban dau dugc bo sung lien tijc voi lupng nho dii de duy tri toe dp sinh truang 161 da ciia vi khuan (thong qua thong so DOT ho§c O2); (4) len men timg me bo sung gian d o ^ kel hpp voi b6 sung lien tuc nguon dinh dudng: sau khi ket thiic batch I. binh len tnen dugc b6 sung I lan \ a i mpt lugng dinh dudng bing lupng dinh dudng ban dau nhim^ vdi the tich bSng 1/10 the tich ban dau va nuoi cay tiep cho den khi nguon dinh dudng het. sau do tien hanh Ien men bo sung lien tyc nguon dinh dudng nhu quy trinh 3.
Danh gia khd ndng sinh tru&ng cda chiing E coU ldi to App
Kha nang sinh truong ctia te bao dupe danh gia thdng qua dg dye ciia mdi truong do d buoc song 600 nm (ODBOO). Trong qua trinh Ien men sinh khoi te bao tang nhanh va s6m vugt qua ngudng ciia may do quang pho, vi v^y mau te bao sau khi thu dugc hoa loang bang dung dich muoi sinh ly (NaCI 0,9%) theo CO s6 10 d^n khi dp dye cua mau nam trong
p chi Cong ngh? Sinh hpc 10(2): 319-325,2012
ngudng do cua may quang pho. ODMO ciia mSu len men dugc tinh bing gia trj OD do dugc nh3n vdi dg pha loang.
Phan lich protein
Ham lugng ciia protein tai td hgp dugc xac dinh theo phucmg phap da dugc mo ta truac day (Do Th) H u y k CM/,. 2008).
KET QUA VA THAO L U A N
Tang sinh khoi te bao vii san l u g n g TrxFIiC bAng cong ngh^ len men timg nie bo sung giAn doa \ :i bo sung lien tuc nguon dinh du'ii'ng
Te bao dugc nuoi cay Irons; niiu trudng MYTGly (la moi trudng da dugc ldi uu Uong ckc nghien cim trudc) d 37°C, tdc do khuay 500-1000 v/p va lupng khi luu diong tir 0,4 den 0.8 \ \ in de giiJ cho ndng do oxy hoa tan trong binh len men luon dgt khoang 20" o Den cudi pha len men batch, nhiet dp trong cac binh len men dugc ha vuong 28°C, tien hanh bd sung tliem ngudn dinh dudng va chat cam img smh long hgp TrxFiiC. Cac binh duac thiet ke nhu (Hinh 1).
Trong qua trinh len men bieu hien protein, chiing E. coli tai td hpp mang gen fliC sinh trudng hieu khi. Vi vay, neu chimg sinh ttuong manh, chiing se sii dyng o \ \ hda tan cd trong binh de thuc hien
cac phan ung trao ddi chit Tir do, lugng oxy hoa tan trong binh (DOT) sg c6 xu hudng giim, Cd thi ndi, thdng so DOT la mgt trong nhfmg dau hi?u cho biet tdc dg sinh irudng ctia te bao va gian tiep cung cap thdng tin ve ngudn carbon, dinh dudng frong mdi trudng, len men batch I, ngudn oxy trong binh giam den tgn ciing va lang cao dpi ngpt (Hinh 2A) chung td toe dp sinh trudng cua vi khuan da bit dau giam mgnh den dirng sinh trudng, Khi dupe bd sung them YT (Binh 2), chiing sinh trudng them mgt thdi gian ngin rdi dimg lai vi ngudn carbon da bi can kiet (Hinh 2A. 2B)
Khi dugc bo sung them glycerol (dii dugc bd sung dan dgc ho^c cd them YT, te bao trong cac binh len men edn Igi deu sinh trudng manh den mat dp te bao dgt khoang 13 g te bao kho/lil mdi trudng tuang duong vdi OD(,M dat khoang 35-40, Tuy nhien, trong binh len men bd sung gian doan ngudn dinh dudng YTG, toe dg sinh trudng le bao cd nhanh hon mgt chiit so vdi len men bd sung lien tuc nhung
\oi lugng nho nguon dinh dudng. Trong thi nghiem nay. len men limg me cd bd sung gian doan nguon dinh dudng, TrxFIiC dugc tong hgp tot nhat va sinh khoi t£ bao ciing tang nhanh (Hinh 2B. 2C), San lugng TrxFliC dat dugc khoang tren 1,8 g/I, tuong duong vol 0,14 g/g 16 bao kho. Quy trinh len men nay vira thu dupe lupng Idn protein tai td hpp, vira rill ngin thdi gian len men, tii do giam duac chi phi nhien lieu
Hinh 1. So do bo tri thi nghiem cac binh len men kilm tra sy tang sinh kh6i, s^n lifgng TrxFliC b§ng cong nghe len n fed-batch b6 sung giSn doan (GD) va b6 sung lidn tyc (LT) nguin dinh duong
DoThj Miiyen elal
1:1;. :i:
I:!:*:!:
Hinh 2. Oi'ru] lli.ii Irong qua Irinh I6n men (A) '.in men liii h,iti h hn sung gi.in doan vd b6 siiiiij lir (v6ng/phut), Q l6c dO sgckhi, T iihlfeldO, HR tmi
m
kiKM iS b.io (B) v.1 Sc^n lugng TrxFliC Ihu duoc (C) bdng cflng ngh§ lgn 111' ngu6n <lii)h ilir&ng 02 oxy c6n Igi Irong binh, RPM l4c dO khujy
I ail',: sinh klioi le l>.i<> li.itii; cong NI;II(' len men limg llic bo sung giim (loan kel lii^*|) hii MUII: lii'ii tuc nguon dinh dudng
I'rong ihi nghivni ii,i>. cac binh li-n men dcii dugc du\ tri loc dp kliu.i> liu Ja 150(1 \/p \ii Uic dd khi luu thdng qu.i binh l,i I w i n sau khi cam img. Mgl sd nghien ciru chi ra r^ng. ihdi dicin cam img sinh long hpp protein nil quan Irnng, Tiiy theo limg loai protein ma cd prolein dugc long hgp lot nga\ kill te bao duac cam irng d ODMHI thii p. iioiig
kill iiiiii sii prolem l^i cim dugc long hpp khi sinh khdi le b.io da du Ion Vi the, song song vdi Met khai) silt (|u> trinh len men. te bao dupe khao sal kli.i ndng ,siiili long hgp ' l r \ F l i C khi dupe cam img ir die thin dii'in kh.ic nha.i: ki'l thiic liin men batch V.I [;nr,i lan len men t i n g me bd .sung gian dogn iiijiiDii dmh dudng. ( .ic binh tir 2 den 6 dugc duy In DOI 2()"ii bang nhivt dd. Sau khi bd sung lien lye dmh duijng, DOT dugc duy tri nhd tdc dp bo sung dinh dudng, C.ic binh len men duoc thiel ke nhu (Minh 3).
Hg thdng li'ii men da binh CireUi
Cim ung IPTG khi OD = 24, bA sung li^n luc glycerol
Cam ung IPTG khi OD = 39, b6 sung Ii6n Igc glycerol
C ^ wng IPTG khi OD
= 24, b6 sung I tdn Igc
YTG
Hinh 5 Kh6ng cdmung
IPTG.
b6 sung Ii6n tgc YTG
:[^Binh6 C ^ img IPTG khi 00 = 44, b6 sung lien Igc YTG
Ket thuc len men tirng mc giAn do^n V T ( ; , c6 bo sung lien tuc nguon dinh duong Hinh 3. So d6 b6 tri Ihi nghiem c ^ binh I6n men kiim tra sy lang sinh khii vS s^n iu'gng TrxFliC b^ng c5ng ngh| l&i men fed-balch b6 sung giin dogn k6l hgp b6 sung lifin tyc ngufin dinh du&ng.
Tgp chi Cong nghe .Smh hiic 10(2) 319-325,2012 Len men batch va U'rng me bd sung guin doan dmh dudng va s.iii dd dugc lid sung hen luc ijKcciol hoac YTG, neu lc ban dugc ciim img o Ol) c m (Binh '. Binh (i) thi h;iiii liuuii; TrxFliC ihu dugc i;il Ihap, Ngirgc lai. Irong en hai Iriri-nig hgp, lc biio dmrc cam img khi OI),,,,,,-24 (liinh 2. liinh 4) (hi dc-ii dio ham lugng TrxFliC cao. d^ic biel klu bd sung lien (tic YTG (Hinh 4). Len men limg me bo sung gi.iii doan ngudn dinh dudng kel hop vdi bd MIII!; lien lye
ngudn dinh dirirng da Liiii lang sinh khiii le bao len khoang 30 g/lii \;i san luong Ti,\FliC loi da thu dugc lu binh len iiieii la ' , * .li'lil mdi truinig, Klu id biio khdng dirge cam img 11'IG, Idcdiismh (riiirng cua Ic biio tiKTiig duang vivi loc tig smh trudng ciia ti biio dupe cam img boi IPTG o c;ic binh len men .sd 1-4
\a (i Dieu dd ehinig lo ndng dp IP'Ki cam img smh loii^ ligp l i s l l i C khdng anh luiang den sg smh Huong cua lc bao.
70 ,
V
* w ' K.-' i . - * ft.-" ^ - ^ n.-* ,.*•' * ^
Hinh <•. Slnhi khoi 16 bao (A) vA sSn lugng TrxFliC Ihu du'oc (B) bang cong nghe len men fed-balch bo sung gian doan kel hjp bo sun J lien tgc ngu^n dmh duo'ng BR Binh len men
Aok hu'dng cua thanh phiin dinh duong len miic do on dinh cua I r\ITi(
De tang smh khdi te b.io \a san lugng TrxFliC, chiing tdi da nudi ca> chung tai td hty|i trong 6 binh len men vdi tdc do khua> ldi da 151)11 \ p. lumig khi di vao binh 1 w i n \a lien hanh len men limg me bd sung gian doan ket hgp bd .sung lien luc ngudn dinh
dmrng. Ddng ihdi, de giam chi phi nguyen lieu dimg cho len men \a tim ra cdng thirc mdi trudng thich hpp cho len men lirng me, ngay sau khi ket thuc len men lirng me bd sung gian doan ngudn dinh dudng, eliuiig tiep luc duac len men timg me bd sung hen lue vdi cae ngudn dinh dudng khac nhau, thdi diem cam img khiic nhau (Hinh 5)
Hinh 5. Sa do bo iri thi nghiem cSc binh len men kilm tra su' ^nh huong cOa thanh ph&n dmh dudng bo sung len mirc d$
on djnh cOa TrxFliC.
D o T h j Huyen era/
Kel qua mgt lan nflra dil chi ra i.ui^'. liil cii c;ic man dugc cam img aOD«H, tbiipdeu ilm s,in liioni: piolein tai to hgp cao va ngupc lyi (Hinh 6), Dga SM< kel (|iia nay cimg vdi kei qua iren (Hiiili 4). chimg tdi nh(in thay. thdi difini cam img ihicli hty^) de thu lugng Idn protein uii td hgp ki siiu khi kel iliiic hnlcli. hill dau Icn men timg me bt> sung guin do^in ngudn dinh diriVng cQng la llic ODwm dyl klio,iiij; 24
Kill dugc bd sung lien lyc ngudn dinh duflng.
sinh khoi le bao King giin luong dinnig nhau 6 cac binh len men. Iii> iihicii, MU moi ngudn dinh dudng
bo sun(j khiic nhau, lupng TrxFliC thu dupe s2 khic nhau. O binh \&n men 1, ham lupng TrxFliC giam theo dun gian len men khi bd sung lien tuc glycerol (Hinh 6). Trong khi dd, neu dugc bd sung them cao niim men, hiim lugng TrxFliC dugc long hpp ty 1^
tliiiiiii vdi sinh khoi te b i o (Binh 3), Con neu them ca tryptone ciing vdi glycerol va cao nam men (Binh 5), hiim lugng TrxFliC cung dn djnh nhung do sinh khdi tii bdo c6 xu hudng giim sau mgt thdi gian len men nen siin lugng TrxFliC thu dugc kim hon so vdi binh Icn men 3 chi bo sung lien tyc cao nam men.
^
Hinh 6. Smh kh6i t l bdo (A) vS sdn lugng TrxFliC Ihu dirge (B) b^ng cflng ngh$ I6n men batch iSn hai kit hgp mi b i sung li§n tgc c^c ngudn dinh dudng khdc nhau, thdi dilm cdm i>ng khdc nhau, G: gifr sau khi cdm irng BR. Binh I6n men.
Len men thu TrxFliC a cic dieu ki^n nuoi cay loi ini Chung mang gen fliC dugc nudi dga theo quy trinh len men timg me bd sung gian doan ket hgp bd sung lien tuc glycerol, cao nam men sau khi ket thiic timg me gian doan va cam img IPTG dung thdi diem, khi OD500 cita te bao d^it gia tri khoang 24.
Thai gian len men ciia chimg tai to hgp keo dai 15 h vdi nhivi dg nudi cay sau cam img ta 30°C, pH 7. tdc dd khuiy tdi da 1500 v/p va lupng khi di vao binh I vvm. San lugng TrxFliC trong binh Ien men d^t 4.6 g/lit mdi trudng, tang khoang 9 lan so vdi Icn men d binh tam giac (Hinh 7).
g> 5 -5 45 i "
. 5 3 5
•a 25 e 3
e 0.5
1 L6n men Irong binh 2 Lin men Irong binh tam g,6c
1 2 TrxFliC
Hinh 7. So siinh sdn lugng TrxFliC thu dugc ir qui mfl lgn men binh tam giSc vd trong binh ISn men
KET LUAN
Len men timg me chimg E. coli lai to hgp mang gen fliC trong binh len men I lit bo sung gian d o ^ kel hgp bd sung lien tyc cao nam men, glycerol, cam Omg sinh idng hgp TrxFliC khi ODAOO ciia dich len men dgt khoang 24, lugng TrxFliC thu dugc dat khoang 4,6 g/lit mdi trudng.
Ldi cam on: Bdi btio ttinrc thirc hien bang kinh phi d\r dn htyp lac giua I 'let Sam-Thu\ Dien "Sdn xual cac protein tdi Id hpp sir dgng trong Ndng nghiep va Y dtr^c " do SIDA/SAREC tdi tra.
TAI LI$U THAM KHAO
Cuadros C, Lopez-Hernandez FJ, Dommguez AL, IWcClelland M. Lustgarlen J (2004) Flagellin fiision proteins as adjuvants or vaccines induce specific immune responses, Infect Immun 72(5): 2810-6.
Do Thi Huyen, Le Quynh Giang, VSn Thj Nhu Nggc, To Long Thanh, Truong Nam Hai (2008) Flagellin FliC ciia Salmonella Enterica serovar Typhimurium dugc bieu hi^
tot trong Escherichia coli BL21 dudi d?ng dung hgp wiri thioredoxin, Tgp chi Khoa hpc va Cong ngh$ 46(2): 49:5?^^
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F E D - B A T C H F E R M I M A T I O N F O R I N C R I : A S I > < ; T H E \ l E L D O F F L A G E L L I N F L I C F R O M R E C O M B I N A N T ESCHERICHIA COLI IN M U L T I - P A R A L L E L F E R M E N T A T I O N S Y S T E M G R E T A
Oo Thi Huyen', Le Qu> nh Giang'. Svcn-Olof Enfors^ Truong Nam Hai' ' Institute of Biolechiiolog\: Vidnani .Academy of Science and Technology 'Snedish Royal Inshtiite of Technology'
SLTV1MARY
Gene thC coding for llagellin FliC of Salmonella enterica serovar Typhimurium was expressed in Escherichia coh BL21 in the fusion form with chickens' immunopolentiator -thioredoxin (Trx), thus the recombinant protein was abbreviated to TrxFliC In order to have a large amount of the recombinant antigen for Salmonella vaccine or vaccine adjuvant developments, in this study, some fermentation parameters and procedure WCTC imestigaled in mulu-parallel fermentation system Greta (Sweden). In the bioreaclors, nutrient in medium and fermentaiion process played an important role in E eoli BL21 cells mass and TrxFliC yield.
Fed-balch fermentation by adding one dose of YTG (yeast extract, tryptone and glycerol) following the first batch fermentation, the cell mass increased quickly, volumetric productivity of TrxFliC reached over 1,8 g/l, correlate to 0.14 g/g dry cell mass However, fed-batch fermentaiion by combining addition of one dose of nutrient then by nutrient exponential feed increased significandy TrxFliC productivity. Point of time for adding IPTG to induce recombinant protein production was an imponani paramcicr For obtaining a huge of TrxFliC.
IPTG should be added during fed-balch fermentation when ODMI« reached approximately 24 During fed-batch with exponential feed, nutrient impacted in TrxFliC stability. Productivity of TrxFliC was lower when the recombinant stram was exponentially fed with YTG or glycerol, however the TrxFliC yield was gained more stable when the producer strain was exponential fed with YG (glycerol va yeast extract). In the optimal fermentation conditions and parameters, the obtained yield of TrxFliC was 4.6 g/l culture, increased 9 nmes if comparing to TrxFliC productivity in the flask fermentation
Keywords: E coh BL2I.fliC. fed-balch S. Typhimurium. TrxFliC, vaccine
' Author for correspondence: Tel/Fax: +84-4-37562790; E-mail: tnhai(d},ibt.ac.vn
