Tạo dòng biểu hiện tinh chế và xác định hoạt tính của tác nhân kích thích tạo quần lạc đại thực bào từ chuột trong hệ thống escherichia coli

Tgp chi Cong nghe Sinh hoc 13(1): 47-53, 2015

TAO DONG, BIEU HL$N, TINH Cid VA XAC DINH HOAT TINH CiJA TAC NHAN
KICH THICH TAO QUAN LAC DAI THIX BAO Tif CHUQT TRONG H$ THONG
ESCHERICHIA COLI
Nguyen Thanh Thio, Nguyen Bich Thio, Tren Van Hieu
Trtremg Dgi hoc Khoa hoc tit nhien, Ali hoc Quac gia Thanh phd Ho Chi Minh
Ngay nhan bai: 19.3.2014
Ngay nhan ding: 30.9.2014
TOM TAT
Granulocyte macrophage-colony stimulating factor (GM-CSF) la Man to kich thich tao quin the 4 bao
tham gia vao qui trinh dip Ong mien dich nhu bach au, dal thin bao, duOng bao (4 bao mast), to bao TK,
nguyen bao sqi . DO la met cytokin c6 kha ning tic deng len nhieu lopi to bio mau va ci nhieu tiem ning ring
Truic khi c6 the duqc Ong
dung nhu trong dieu tri neutropenia, ay ghip tay xuong hoc lam ti duqc
dung tren ngtrai, GM-CSF an duqc thu nghiem tucmg dump tren m8 hinh deng vet. Trong nghien dm nay,
mGM-CSF ducrc bleu hien trong Escherichia coil (E. coli) Whim tao nguon nguyen lieu cho nhang thir nghiem
tren mo hinh chuet. Gene mgm-csf mi h6a protein mGM-CSF (ITN khuech dpi to plasmid pIDT-GM-CSF
bang phucmg phip PCR, xir Ii bing enzyme at giii han vi duqc gin chin vao plasmid pET28a. Plasmid tii to
hqp pET-mGM sau d6 duqc bier' nap vao chung bleu hien E. coil BL21(DE3). Sau qua trinh cam irng bieu
hien yeti IPTG, ket qua phin tich bing SDS-PAGE cho they mGM-CSF bieu hien duei clang the vii trong to
bao chit ctia E. coil BL21(DE3). Ket qua lai Western voi khing the dic hieu khing mGM-CSF khing dinh
protein duqc bleu hien vugrt mirc chinh la protein mGM-CSF. Protein mGM-CSF thu nhan duqc tit ding the
vii kh6ng ci host tinh trong to bao E. coli dime tii gip .cuen vi tinh sach bang sic Id trio deli cation. Cac ket
qua phan tich bang lai Western va dinh luqng bang phan mem xic dinh dim de cho they di thu nhan duqc
mGM-CSF v6i de tinh sach dot 94,9%. Cuei cing, mGM-CSF duqc dinh gia hoot tinh sinh hoc daa tren kha
ang kich thich tang sinh ding to bao FDC-P1 va cho ket qua ED50= 3022 pg/ml.
Tit khoa: mGM-CSF, tai gap cuen, FDC-P1, thfr hoot tinh

M6 DAU
Granulocyte macrophage-colony stimulating

factor (GM-CSF) là met cytokin c6 khi Wing tic
Ong len nhieu loci to bito miu. N6 ci khi ning kich
thich tang sinh in vitro va biet hoi cic to bio miu
tien than tra thinh 4 bio bach cau ho va dai thin
bio, tic Ong len chic nang ctia 4 bao mien dich, tir
di tang phin ling mien dich bam sinh cling nhu mien
dich dac bieu (Enzler, Dranoff, 2003). Do di, GMCSF c6 nhieu tiem ning ins dung, dac Wet trong ho
trq dieu tri cac khiem khuyet ye miu, ning cao hoot
deng di too to bio miu sau hia tri lieu hoc ciy
ghip ttiy xucmg (Armitage, 1998), he trq mt.& va
trong qui trinh dieu tri berth bach cAu ac tinh ding
tuy (Richard et al., 1995), nu8i ciy to bio tua img
dung trong dieu hi ung thu (Shurin, Salter, 2009) va
lim ti duqc vaccine (Jones et al., 1994; Hamilton
and Anderson, 2004).
Vii tiem ning ling dung reng rii cilia GM-CSF,

ching Vol mong mu& xay dung met qui trinh sin

xuAt GM-CSF gia thinh re trong nuic. Tnrac khi c6
the duqc ling dung tren ngtroi, nhing nghien cuu ve
GM-CSF can phil triti qua nhimg thin nghiem tua ng
duang tren mo hinh dOng vat. Mat khic, GM-CSF la
kh8ng c6 phan ling cite° gift cic loii, GM-CSF cita
nguai kh8ng c6 tic dung len to bio chuOt vi ngtrqc
lei (Enzler, Dranoff, 2003). Deng thud, su duing his
khong &lb huang ten host tinh dui mGM-CSF vii
bang chimg la mGM-CSF tai to hqp tir E. coli du
kh8ng duqc glycosyl hoi van c6 hoot tinh sinh hoc
cao ham protein hr nhien khi thin nghiem in vitro

(Schrimsher et al., 1987; Wingfield et aL, 1988)
Nhiun cung cap nguyen lieu cho nhitng thin nghiem
GM-CSF ten mo hinh chuet sau nay nhu thin nghiem
lam ti duqc vaccine, gitip hoi phuc he thong to bio miu
mu h6a hay xa tit chung to i nen hinh ding h6a gen
mgm-csf ma hos mGM-CSF vao plasmid bieu hien
pET28a di tao thinh plasmid tii to hqp pET-mGM.
Plasmid tii to hqp tiep tuc duqc bien nop vat) E.coli
BL21(DE3) nham too ra chung ce kha ning bleu hien
47


Nguyen Thanh Thao et aL
mGM-CSF. Protein mGM-CSF thu nhan dual dang the
duqc tai gip cuen, tinh sach va thu nghiem hoat
tinh sinh h9c in vitro tren dang to bito FDC-Pl.
VAT LIEU VA PHUONG PHAP
Ching chi vi plasmid
Chung vi lchuin E. coli DH5a [F- endA 1 hsdR17
(rk-/mk) supE44 thi A," recA 1 gyrA96 dlacU169
080 lacZ 2u1115)] duqc sir dung de nhan ban
plasmid. E. coli BL21(DE3) [F-, ompT, hsdS (rema), gal (DE3)] duqc sir dung lam to bao chi bieu
hien mGM-CSF tii to hqp.
Plasmid pET-28a (Novagen, M9), kich think
5369 by mang promoter T7, gen khang kanamycin
(Kan) dirge ding de lam vector bieu hien. Plasmid
pIDT-GM-CSF (cung dip bei Be mon Ong nghe
Sinh h9c Phan tir Ara Mei truing, Truing DH Khoa
hcc Ttr thien TP. HCM) duqc sir dung lam vector
cung cap nguon gen mgm-csj

Cic phtrang phip sinh hqc phin trlr
Nhu tach chiet plasmid, too to bao kha hien, bien
nap plasmid vao to bio E. coli dirge nen hanh theo
Sambrook & Russell (2001).
Thiet ke plasmid tai hqp pET-mGM mang gen
mgm-csf
Gen mgm-csj c6 kfch thtroc 389 by
(GI:109734154) mi h6a cho protein mGM-CSF
duac khuech dai tir plasmid pIDT-GM-CSF bang
phan img PCR yeti cep mOi doe hieu cho gen co
mang trinh tv cat cia hai enzyme cat giei han Xhol
vi Ncol. Sin pliant PCR duqc tinh the bang be kit
EZ-10 Spin Column DNA Gel Extraction (BioBasic,
Canada) theo huang dAn cint nha sin xuat va duqc
xir H vai hai enzyme Xhol vi Ncol. Sau khi cat bang
enzyme han che, gen mgm-csf ductc not trio pET-28a
di duce cat bang hai enzyme nay de too thanh vector
tai to hqp pET-mGM mang gen mgm-csf.
Sing 19c the hien nap vi kiem tra plarnid tai to
Ito
Vector di to hqp pET-mGM duqc bien nap vac,
to bao E. coli DH5a. The hien nap mqc thinh khuan
lac tren mei truing LB ca bo sung kanamycin 30
vg/m1 duqc sang I9c de chqn ding mnc tieu bang
PCR khuan lac yeti cap moi d#c bleu cho gen mgmcsf theo chuang trinh: 95°C/5 phut; 30 chu kSr:
94°C/45 giiy, 55°C/45 giay, 72°C/ 45 giiy; ket thfic,
72°C/10 pita Sin phiun PCR duqc kiem tra bing
48

dien di tren gel agarose 1%. Plasmid tir the bien nap

cho ket qua sang 19c duang tinh tit') tuc duqc kiem
tra bang PCR vei c#p mOi bit cOp tren vector nim
ngoai vung MSC la T7 promoter va T7 terminator.
Plasmid tai to hqp cho ket qua ducmg tinh
phan img PCR duqc giai trinh 6 ding ty Macrogen
(Han Quoc). Sr Wang ding giea trinh ar gen trong
plasmid vi trinh to thiet ke duqc kiem tra bang phan
mem Jellyfish.
Cam ling bieu hiOn mGM-CSF
E. coil BL21(DE3)/pET-mGM duqc nuoi trong
mei truing LB c6 be sung kanamycin yeti nOng de
30 µg/ml, lac 250 v/p, 37°C den khi ODA Qat 0,6 0,8 thi tien hanh be sung IPTG 0,5 mM de cam Ong
str bieu hien gen. Sinh khei to bao duqc thu sau 4 gib
cam Ong va duce pha va bang sang sieu am sir dung
may Ultrasonic Cell Disruptor (M9) de thu protein
tong so. Cir 1 ml djch huyen phi to bao se duqc sieu
am 6 4°C vOi 6 xung 15W, mei xung dai 30 giay.
Protein pha tan va khong tan duqc phlin tach bang ly
tam vOi tot d6 13000 Ong/phut trong 20 phut, 4°C.
Protein di to hqp duqc kiem tra bang SDS-PAGE vi
Western Blot sir dung khang the dac hieu khang
mGM-CSF (Santacruz, Mj,) vi &Km phat hien bang
ECL kit (GE Healthcare, Anh).
Tii gap cuOn mGM-CSF
The vii sau khi thu nh#n duqc nia sach va hoa
tan trong dung djch Urea 2M, pH 12,5 vii ti l'e 0,5 g
the yid : 20 ml dich has tan. Khuiy i nhiet de phOng
10 phut, sau d6 pha ling tn,rc tiep vao dung djch tii
gap cuen (25mM Tris base, 2M Urea, 0,1% Tween
80, 0,5 mM Cystine, 2,5 mM Cystein, pH 9,5) de

n6ng di) protein cu6i cling dat khoing 100 — 200
sau d6 khuiy nhe 16-18 tiOng i 10°C. Protein
sau khi tai gAp cuen dtrqc 19c hoc ly tam loci be
bao quan 6 4°C de phut vu cho cac thi nghiem
ve sau.
Tinh sach mGM-CSF
Protein mGM-CSF c6 diem ding dien pI=6
(Schrimsher et al., 1987) vi v#y thing OH sir dung
phtrcmg phip tinh sach bang sic kjI trao di cation
tren hg dicing sic
AKTA Explorer (GE
Healthcare, Anh) vOi cet sic ky Hitrap SP FF 1 ml.
Qua trinh tinh sach duqc nen hanh nhu sau: (1) can
bang OA bang 10 ml dung djch NaOAc 20 mM, pH
5; (2) nap 10 ml mAu vao cet; (3) rea cac thanh phan
gin kh6ng at hieu bang 10 ml dung djch NaOAc
25mM, pH 5; (4) dung ly bang 60% dung djch
NaOAc 20 mM, pH 5, 40% dung djch NaOAc 20


Tgp chi Cong nghe Sinh hpc 13(1): 47-53, 2015
mM, NaC1 1M pH 5. Cac phin down protein thu
'thin duqc kiem tra bing dien di SDS-PAGE a cac
dieu kien c6 vi khong c6 chit kit* ceu not disulfide
nhu 2-mercaptoethanol hay DTT.

37°C va doc ket qua do mit (10 quang a btrac sang
450nm.
KET QUA VA THAO LUAN

Thir nghiem host tinh mGM-CSF in vitro
Thiet ke plasmid pET-mGM

Hoot tinh sinh hoc cita mGM-CSF duqc xfic
djnh thong qua Idle nang dap 1mg tang sinh cia clang
4 bao FDC-P1(ATCC CRL-12103). Te bac) duqc
nuoi cey tong mei truang DMEM 10% FBS, mit dO
4 bao 2.105 4 bao/ml, trong ti ii 37°C, 5% CO2, (10
Am 85%. Mk protein an tit* nghiem duqc pha
loing bic 10 tir neng dp 2x106 pg/ml xueng 2x10'
pg/ml. Ching toi tien hanh thin nghiem vai 50p1 mau
protein/gieng va 50111 djch 4 bao/gieng. Sau 48 Oa,
kha nang tang sinh cue to bio duqc Vic djnh bing
cfich bo sung 100 CCK-8/gieng, u them 3 gia

1

by

Gen mgm-csf (389 bp) ma hem cho protein mGMCSF di duqc khuech d3i thanh cong tir plasmid pIDTGM-CSF bang phial img PCR vai moi dic hieu (Hinh
1A). Sin phem not cua gen v.11 plasmid (Urge bien nap
vao to bao E. coli DH5a. The hien nop duqc sang loc
bang khang sinh kanamycin va phuang phip PCR. Ket
plasmid tech chiet tir khuin 10c dugt
quit.giai trinh tir
tuyen chon cho they trinh ttr gen mgm-csfIrong plasmid
tai to hqp tuang (long 100% yeti trinh tu thiet ke (Hinh
1B). Nhu voy plasmid pET-mGM di duqc thiet ke
thanh cong.

11

21

41

31

51

61

71

=1111-91T .-.

5TOCCACCCACCC3C9CACCCATUCTITTMCCGOCCTFOGNIOCATCTLGAGGCCAMJAZALGCCCTOJIACCMT

011097331 36

ATOCCACCUCCC6C19.ACCCATCACTOICACCCOGCCT9013■039.41011GA0GCCAT911604•00CCTS3ACCICCT

20X0

C40443300. supicecceacCeperssecc4c440141eaccc0/941100449.414114041109051444.060reeu4.acetcc t

5000

.01-CST 19-6 603TGALATGCCTUTCACAT941.5103•0300TAGAAGIVOTVITTAALTAGITCITCRUACAALCTIACATOTOTGC

01
011011734156

3000

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Ill

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151

GGATGACATGCMTC/ICA1TOAATGAA(1/009,146.40TCOTCTCTAACGICTTCTCCTITIJAAALCTWATISTOTGC

C33346994 1103144.4144C140.0494 ccomtga03449c449540140141eueeflagmcceccum03•034 tow 41419199
161

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O01-CST 0-0 AOACCCOCCTGUZATATTCOACCA04101=ACO0000.41T9CACCAAACTCA4GGGCGCCTTOAACATGACAOCCAOC

1 500

011007 36134 AOACCCGCCTGA•GATERCOAOCA0009CTAC3000C541.919,ACCAAACTCA5GGGCGCCTIWACATGAMGCCAOC

Consmaus 440690099941440•1411.404994030101490930944cmaceasscr.46409;e9eoccg sat 4 la scapec ago
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5011-CA v-4 TACTACCAGACATAGTOCCCCCCABC7tC003114C00511430,UACKAAGITACCAGCTATOCOGATTITATACACAG
500 — 1111110
600 —

await

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TALTALCAGACJIMACTOCCCCCGIACTCCOOJAACOGICTOTILYAC1.9

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10.4099340aucige.eeesaet.0040seqp.w.4664440443tcaecoccr....0e9eattcc•u0.49
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O101-9339 .-13 0C7110405CT9TC-9301-114917CCCC1119.0.470CAA1AWC4611T.A.4104
CCITAAAACCTTITMACTLITATCCCCTITOUTOCAAWACCIATCWJAL

Canne6499 e411marece.90900902a50ceecettc40331419403644096010044444

A

B

Hinh 1. Kat qua thu nhan gen mgm-csf.. (A) San phArn khuyech dal gen mgm-csf. 1: thang DNA 1kb plus, 2: gen mgm-csf,
(B) Kat qua sap gieng cat trinh tu, gen mgm-csf thu nhan v61 trinh to cong b6 tren ngan hang gen (GI: 109734154).

Bien hiOn protein mGM-CSF trong to bho E. coli
BL21(DE3)/pET-mGM
Ket qua cam (mg bieu hien protein tren ban dien
di SDS-PAGE cho they, so vai khi khong duqc cam
ang (Hinh 2A, gieng 2), chimg E. coli
BL21(DE3)/pET-mGM sau khi duqc cam 1mg bai
IPTG di sinh tong hqp mGM-CSF vai luqng kha
Ian, c6 kich thy& khoing 14,4 kDa (Hinh 2A, gieng
4) va phen Idm protein duqc bieu hien a clang the vii
(Hinh 2A, gieng 5). Ket qua nay phi hqp vai cong
bt) cue DeLamarter et aL, (1985). Tuy nhien

nghien ciru nay, Itrqng protein mGM-CSF chi chiem
10% tong luqng protein to bao. Trong khi Q6, protein

mGM-CSF cue ching toi duqc bleu hien 0 intim dO
cao, chiem 26,9% tong Wong protein to bao (kat qua
khong trinh bay). Protein mGM-CSF tai to hqp da
duqc khing djnh bang lai Western vai khang the
chuyen biet khang mGM-CSF. Cfic protein tai to hqp
mGM-CSF trong dung djch protein tong so, protein
pha khOng tan (the vii) va pha tan deu c6 kha nang
nhin biet vi lien ket tot vai khang the khang mGMCSF (gieng 4, 5, 6 Hinh 2B). Nhtr v4y, c6 the khing
djnh protein mGM-CSF duqc cam (mg bieu hien
trong chting E. coli BL21(DE3)/pET-mGM.
Tinh stich mGM-CSF
mGM-CSF a clang the vii sau khi tai gip cuOn de
49


Nguyen Thanh Thao et al.
thu nhAn dang tan duct tien hinh cho qua cOt sic ki trao
doi cation. Dung ly de thu nhAn mGM-CSF bAng each
dung dung dich NaOAc 20mM, NaCI 1M, pH 5 de thay
doi luc ion. Khi dung dich dung ly di qua cot, cation Na+
se barn vao chat nen tich dien am va day cac protein ra
ngoai. Ket qua dien di cho thay kohan doan khong gan cOt
(Hinh 3A, gieng 3) chira mOt phan protein mGM-CSF va
cac protein khic c6 trong phan dap truerc khi qua cOt
(Hinh 3A, gieng 2). Nguyen nhan ducrc du doan la do
prong mlu nap vuo qua kha nang bat cua cOt hoc do
mOt . phan tir mGM-CSF chua hinh thanh clang cau

hinh clan den tich dien khac di nen khong the On len cOt.
MAt khac, a buac nia eot (Hinh 3A, gieng 4) kh6ng xuat
hien vach protein nao. Trong khi d6, a phan clop 40%
dung dich ly giai, tuang ling 400mM NaCI (Hinh 3A,
gieng 5), chang toi da thu duqc vach tuang ling protein
mGM-CSF. Sir dung phan mem Quantity One cho thay

protein thu nhOn &roc c6 dO tinh sach dat 94,9% (Hinh
3B, gieng Nhu viy, chi vii mOt bulk tinh such bang
sac IcY trao dal cation, chang tot da thu nhar&roc protein
mGM-CSF yen dO tinh such cao, tuang ducmg vii ding
bo cua Schrimsher (1987). Tuy nhien, trong nghien ciru
cua cac tic gia nay, protein mGM-CSF phai trai qua hai
burc tinh such bang sac i(51 IQC gel va trao doi anion.
Dieu nay lam gia tang ding ke chi phi cling nhu lam
giant hieu suit thu nhAn mGM-CSF tinh sach.
Protein rnGM-CSF co hai cau not disulfide nOi phan
tir. DE kiem tra stir hinh thanh cau not disulfide dm
mGM-CSF tai to hop trong cac phan (loan thu nhan ducrc
trong qua trinh tinh che, chung Col tion hinh dien di SDSPAGE cac phan doan protein nay vii dung dich bien tinh
mau khong be, sung tic nhan khir cau not disulfide nhu
DTT hay 2-mercaptoethanol.

kDa 1

45

20 —
19A —t
9.5

A
Hinh 2. Ket qua bleu hien mGM-CSF. Dien di SDS-PAGE (A); lai Western veri khong the OC hieu khong mGM-CSF (B). 1:
thang protein phan tir Itygng thep, 2: protein pha tong E. coli BL21(DE3ypET-mGM; 3: protein pha tong E. colt
BL21(DE3)/IPTG; 4: protein pha tong E. coli BL21(DE3)/pET-mGMAPTG, 5: protein pha khong tan E. coli BL21(DE3)/pETmGM4PTG, 6: protein pha tan E. coli BL21(DE3YpET-mGMAPTG.

I'D.
97,0
011,0
45,0

30,0

20,1

14,4

A

B

Hinh 3. Kiem tra protein trong cac phan doen tinh such mGM-CSF. Dien di SDS-PAGE (A); Dinh luvng bang Quantity One
(B). 1: Thang protein phan tir luvng thep, 2: phan doan trtierc qua cet, 3: phan doan khong
cOt, 4: phan doan rira got,
5: phan doan dung ly.

Trong phan doan truoc qua cOt (Hinh 4A, gieng 4), kDa va 20,1 kDa cua thang phin tti luong thAp) co hai
vi tri cua protein muc tieu mGM-CSF (giira vi to 14,4 vach, du doan tuang Ung vii hai clang kh6ng co va co
50

•

Top chi Cong nghe Sinh hoc 13(1): 47-53, 2015

caunal disulfide caa mGM-CSF tai to hqp. Phan doan
khong barn cpt (Hinh 4A, giang 3) chaa vach phia tat,
trong khi de phan doan dung ly (Hinh 4A, gieng 2)
chua vach phia dual caa protein vatic tie'u. Chang toi du
doin, ck protein hinh thanh duqc cau n6i disulfide da
gin len cet va duqc thu nhfin trong qua trinh dung ly,
trong khi de cac protein khong hinh thanh eau not
disulfide di bj iota khi nap mau.

Protein mGM-CSF tinh such tiep tic duce kiem
tra bang phucmg phip SDS-PAGE va lai Western v6i
khang the chuyen Wet khang mGM-CSF (Hinh 4B).
Kit qua cho thay, tren ban phim c6 vach tin hieu
hien re Wang irng v6i each c6 kich thuOc nim giaa
hai vach 14,4 kDa vi 20,1 kDa tren bin dien di SDSPAGE. Nhu v#y mau protein thu nh#n duqc sau qua
trinh tinh such chinh la mGM-CSF.
2 3 4

1 2 3 4

kDa
07,0
56,0
45,0

10,0

30,1

14,4

A

Hinh 4. Kiem tra protein trong cac phan doan tinh the mGM-CSF trong dieu kien then di SDS-PAGE khong CO tac nhan
khir c5u nOi disulfide (A) 1: Thang protein phan tir luvng th5p; 2: ph5n doan dung ly: 3: ph5n doan khOng grin Get: 4: phan
doan turac qua cot. Dien di SDS-PAGE va lai Western blot (B) 1,3: mGM-CSF tai t6 hap: 2,4: thang protein phan tir lung
thAp.

Dinh gii host tinh mGM-CSF

Hoat tinh sinh hoc in vitro caa protein mGMCSF duqc dinh gia dua ten kha nang kich thich tang
sinh d6ng 4 brio FDC-P1, bei day la clang 4 bac phi
thuec mGM-CSF. Chung tei tien faith &nth gil kha
rang tang true:rig caa to brio 6 day neng de mGMCSF to 10'2 den 106 pg/ml.

0.4

0.3

•

0.2

Ket qua phan tich qua phAn mem Graphpad
Prism (Hinh 5) cho d6 thj c6 hinh clang dac thing

sigma cart met thu nghiem tang sinh, dieu nay cho
dray protein mGM-CSF di kich thich duqc su tang
sinh va duy tri stir sang ctia doll 4 bio FDC-Pl. Clc
thong se phan tich cho thay to bio dap img vi bat
din phis trien manh 6 Ming de 103pg/ml, tang On
djnh den neng de 106 pg/ml. Thir nghiem cho gia trj
ED50 = 3022 pg/ml. Gil trj ED50 nay la cao ham so
v6i cac nghien ciru di cong be try& ye hoat tinh
sinh hoc dm protein mGM-CSF thu nhOn to E. coli
hay 4 brio deng 141 (Burgess et al., 1977;
Schrimsher et al., 1987). Tuy nhien,. viec djnh luqng
hoot tinh sinh hoc met cytokin c6 the thay doi b tong
he thong thir nghiem va timg dieu kien nuoi cay. Vi
vily, can c6 nhang thu nghiem so sinh vai matt
c6 the &nit gia chinh Vic hoot tinh sinh
chart
hoc cart mGM-CSF tai to hqp (Meager, 2006).

a

0.1
0.0

l'o
Log[mGM-C SF)

Hinh 5. 06 thi hiring quan giCra tang tarang t6 bao FDCP1 va nOng dO mGM-CSF.

Day la c8ng be clau tien 6 Viet Nam ve vi'ec to
clang vi thu nhim mGM-CSF c6 hoot tinh sinh hoc tir

he thong vi khan E. coli. Tren the gioi, mGM-CSF
tai to hqp tir E. coli di duqc bieu hien IA dlnh gil
hoot tinh sinh hoc trong cong be caa DeLamarter et
al., (1985). Trong nghien dm nay dm chang till, gen
51


Nguyen Thanh Thao et al.
ma hem protein mGM-CSF duqc bieu hien trong E.
coli BL21(DE3). Protein mGM-CSF tai to hqp duqc
bieu hien 6 =iv dO cao va phin 16n protein duqc bieu
hien dm% dung the viii. Sau bu6c tai gip cuOn va tinh
such, ch6ng toi da thu nhen duqc mGM-CSF v6i dO
tinh such cao doting thin dA loui be duqc nhiing phan ter
mGM-CSF hinh thanh khong &mg ciu n61 disulfide.
Protein mGM-CSF sau khi duqc tai Op cuOn va tinh
such di' the hien hog tinh sinh hoc, duqc khan djnh
qua kha nAng kich thich tang sinh dong 4 bao FDCPI . Ket qua cita nghien ciao nay la ca sof cho cac this
nghiem tren m8 hInh chuOt sau nay.
KET LUAN
Gen ma h6a mGM-CSF di duqc bieu hien thanh
ding trong chin% E. coli BL21(DE3). mGM-CSF duqc
bieu hien chit yeu trong to bao chit cita E. coli
BL21(DE3) duai dung the yid. The vOi duqc sit dung de
tai gip cuOn va tinh the nhAm thu nhin mGM-CSF co
hoot tinh yeti dO tinh such cao. Sau qua trinh tinh such
chung toi thu nhin duqc mGM-CSF yeti dO tinh such
94,9%. Ket qua dui nghiem hoot tinh cho thAy mGMCSF co kha rang ldch thich tang sinh dong 4 bao FDCPI voi gia tri ED50 3022 pg/ml.
Lei cam an: Cling trinh nay duac tlurc hien tit kink
phi hang num cda Throng Dai hQc Khoa hoc to

nhien, Dai hpc Qudc gia HCM clip hang num cho BO
mon Cong nghe sinh h9c phan to viz mai truang.
Cam on GS. Nicos Nicola Wien nghien cult Y khoa
Walter and Eliza, Uc)
cung cap dong to bao
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TAI LIEU THAM KHAO

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Top chi Cong nghe Sinh hoc 13(1): 47-53, 2015

CLONING, EXPRESSION, PURIFICATION AND BIOACTIVITY IDENTIFICATION OF
murine GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR (mGMCSF) IN ESCHERICHIA COLI
Nguyen Thanh Thao, Nguyen Bich Thao, Tran Van Hieu2
University of Science, Vietnam National University HoChiMinh City
SUMMARY

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that stimulates a variety of
blood cells and therefore has many potential applications, such as chemotherapy side-effect reduction, bonemarrow cell immobilization, vaccine adjuvant and so on. Before GM-CSF can be used in human, it is required
an initial trial on animal model. In this study, mGM-CSF was expressed in Escherichia coli (E. coil) to provide
material for testing in mouse model. A gene mgm-csf coding for mGM-CSF protein was amplified from pIDTGM-CSF plasmid by PCR method, digested with restriction enzymes and inserted into pET28a plasmid to
create a recombinant plasmid pET-mGM. The gene in the obtained plasmid was checked by PCR and
sequencing. The plasmid was then transformed into the expressing strain E. coli BL21(DE3). After induction
using IPTG, GM-CSF was over-expressed in the form of inclusion body in the cytoplasm of E. coli
BL21(DE3) and was confirmed by Western blot with anti-GM-CSF antibodies. The inclusion bodies was then
refolded and subjected for cation chromatography purification. The purified recombinant protein was mGMCSF with the purity up to 94.9 percent of total purified fraction. Finally, mGM-CSF bioactivity was assessed
via the proliferation of FDC-P1. Result showed that the EDse of mGM-CSF was 3022 pg/ml.
Keywords: Expression, mGM-CSF, refolding, FDC-PI, bio-assay

Author for correspondence: Tel: +84-8-38307079; E-mail:
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