Tạo dòng, biểu hiện và tinh sạch nhân tố tăng trưởng nguyên bào sợi 1 (FGF 1) người trong eschrichia coli

Tap chi Cong nghe Sinh hoc 12(4): 615-621, 2014

T30 DOING, BIEU HI) N VA TINH S3CH NHAN TO TANG TRUONG NGUYEN BAO
S01 1 (FGF-1) NG1Td1 TRONG ESCHERICHL4 COLI
Ding Tit Truirng, Trinh Minh Thwyng, Trin Vin Hien
Truang DO hoc Khoa hoc At nhien, DO hoc Quoc gia thanh pha Ha Chi Minh
Ngiy nhin bii: 02.4.2014
Ngiy ratan ding: 30.9.2014
TOM TAT
FGF-1 (fibroblast growth factor 1) la mit protein da chic nang. Protein nay ci khit fling kich thich phin
bao, hoot his si,r sang cia to bao, tham gia vao nhieu qua trinh sinh hoc nhtr su phat trien phoi thai, sire chila
cac mo, tai tao to bao, thic day su hinh thanh mach mau va su phat trien cue nguyen bao sqi fron; qua trinh
chea lanh vet thuang. Vii flitting tiem nang nhu vtty, hien nay FGF-I da duqc ap din vao rat nhieu linh vtrc
nhu san xuat m9 phalli, chum tri cac benh el) lien quan den tim mach, chum limit vet thucrng. Nhim to ra
protein FGF-I tai to hqp vii gilt thanh hqp 19 cho nhimg Ong dung i Vi4t Nam, chain tai tien hint' tao ding E.
coli ci kha nang bieu hien protein tai to hqp FGF-1. Gen fgfl (423 bp) duqc khutch dal tO plasmid pIDT-fgf/
bAng phuong phap PCR va ding his vao plasmid pET-His. Plamid tai to hqp mang gen fgfIdAt duii su kiem
soat cia promoter T7 duqc dAt ten la pET-fg/7. Gen fg/1 trong plamid pET-fgf/dtrqc kiem tra bing PCR,
enzyme giii hon va giai trinh tu. Sau di, plasmid pET-fgf/dtrqc hien nap vao chimg E. coli BL21(DE3). 'Chi
nuoi cay lac chant tai to hcrp trong mei truing LB, be sung IPTG de cam Ong T7 promoter, FGF-1 duqc bieu
hien dui% dang the vii trong to bao chit cia E. coli vii ham lucrnt chiem lchoang 22% tang protein sau 16 gib
cam Ong khi phan tich bang SDS-PAGE, lai Western va phan mem Quantity-One. The vii china FGF-1 duqc
hoa tan bang dung dich Guanidin 4M va tai gap cuen bang bang phuong phap pha loing nhanh, thu nhan duqc
FGF-1 6 clang holt tan. Phuong phap sic ky ai lac vii heparin duqc sir dung de tinh soch FGF-1. Ket qua cho
they FGF-1 duqc thu b phan down dung ly vii nang cla NaCI I,2M va vii nang di NaCI 0,4M c6 c6 de tinh
soch Wong Ong la 93,4% va 50,1%. Chi c6 protein i Rhin doon ming de mutii cao c6 hoot tinh tang sinh in
vitro ding nguyen bao sqi chuit NIH 3T3 xap xi sun phim FGF-1 among mai, vii E1350 = 657 pg/ml.
khea: FGF-1, th'j vui, tinh ch" r i Igc voi heparin. thu hoot tinh

MO DAU
Nhan to tang truing nguyen bac. sqi 1 (fibroblast

growth factor (FGF)-1) hay cern goi la nhan to tang
truing nguyen bio sqi c6 tinh acid (acidic fibroblast
growth factor — aFGF) la met protein da chic nang.
FGF-1 c6 kha nang kich thich phan bio, hoot h6a su
song sot to bio, tham gia vao nhieu qui trinh sinh
hoc nhu su phat trien phoi thai, sira china cic mo vi
tit too to bio, thic day su phat trien to No.* mo vi
to chic vot 19 dm to bao nei mo, thic day su hinh
thinh mach miu vi su phit trien ctia nguyen bao sqi
trong qui trinh china linh vet thucmg... (Langer et
al., 1993, Zakrzewska et al., 2008). Vii nhOng chic
nang nhu viy, hien nay FGF-1 da duqc irng citing
vao rat rthieu linh vac nhu sin xuttt m9 pham, chtra
trj cic benh c6 lien quan den tim mach, china linh vet
thucmg.
Trong y hoc, nhting nghien ciru ye FGF-1 da
duqc chimg minh la ci khi nang kich thich su tang

sinh mach vi sira china cic hu hong, ca tim. Sau khi
duqc dua vao ca tim, FGF-1 thic day su hinh thinh
mach 6 cic mo thieu miu cic be (ho(ic thieu oxy)
nheng benh nhan dang 6 giai dog' cu6i. Do viy,
FGF-1 Bang duqc nghien cia thir nghiem lam sang
pha I vi II de chira tri cic benh ye tim mach (Cuevas
et al., 1997, Htun et al., 1998, Schumacher et al.,
1998). Ngoii ra, FDA can dua ra khio sit mei di
dinh gia FGF-1 trong dieu trj viem loit mo do tieu
FGF-1 c6 the duqc sir dung dieu trj cic vet
thuang man tinh vi chan thwmg bao gem loit do Cr
miu tinh mach, vet sera paw thuit va b6ng.

Trong m9 pham, FGF-1 li met tic nhan duqc sir
dung phi bien giap duy tri met lin da khoe, cheng
leo hOa vi tre hia lin da do c6 tic dung kich thich su
gia tang dm cac nguyen bio sqi da; tang truing to
bio sung, sin xuAt chAt nen ngoai bio, kich thich sin
xuAt cic yeti to tang truing an thiet khic di phuc
hei da; vi kich thich tic tang truing. Vii riffling uu
615


DOng Tat Twang et al.
diem nhu vay, FGF-1 dirge sir dung ngay tang nhieu
trong cac serum mg pham chi:mg lao h6a.
Truck nherng tiem nang ung dung dui FGF-1
trong y hoc va mg phim, chimg toi tien hanh to clang
to bao E. coil c6 kha nang bieu hien vucrt mac FGF-1
lam nguen nguyen lieu
va tinh such protein nay
cung cap cho viec img dung FGF-1 tai Viet Nam.

a

VAT LIEU VA PHIYONG PHAP
Chung chu va plasmid
Chung vi khuin E. coli DH5a [F- endA 1 hsdR17
recAl gyrA96 NacU169
(rk-/mk) supE44 thi
(080 lacZ 4M15)] (Takara) duqc sir dung de nhan
ban plasmid. E. coli BL21(DE3) [F-, ompT, hsdS
(re- ma). gal (DE3)] duqc sir dung lam to bao chu

bieu hien FGF-1 nguai tai to hqp.
Plasmid pET-His (Novagen), kich thy& 4636
by china promoter T7 duce cam img bing IPTG
(isopropyl fl-D thiogalactoside), gen khang
ampicillin (Amp) dirge dung de too plasmid pETfgfl bieu hien FGF-1.
Ck phuang phap sinh hoc ph'in ter nhu tach chiet
plasmid, ghep n6i gen, cat kiem tra gen bang enzyme
ban che, to to bao E. colt kha bien va bien nap
plasmid vao to bit° E. coli duqc thvc hien theo
Sambrook va cong stir (Sambrook et al., 2001).
Thiet ke plasmid tai to hop pET-fgf/ mang gen
fgfl ma hoa cho protein FGF-1
Gen fgfl ma h6a cho protein FGF-1 (ma se
tren ngfin hang gen?, c6 kich thuerc bao nhieu,
lay ca gen hay met down gen de bieu hien) duqc
khuech dui tar plasmid pIDT-fgf/ (cung cap beri
Be mon COng nghe Sinh hoc Phan tir va Mei
tnrerng, Twang DH Khoa hoc Ttir nhien TPHCM)
bing PCR vai Op mei bit Op dfic hien vol hai
dill gen FGF1F/FGF1R San phim PCR duqc xir
Ijr vai enzyme cat han che NdeI va BamHI va
tinh che bing EZ-10 Spin Column DNA Gel
Extraction kit (BioBasic, Canada) theo Imams
dAn cua nha san xuat. Plasmid pET-His dirge cat
mer yang bang cep enzyme NdeI Ara BamHI, bit
hogt enzyme bang phenol/chloroform va tinh che
bing EZ-10 Spin Column PCR Products
Purification kit (BioBasic, Canada). Sau do san
pham PCR va plasmid da duqc xir ljr bang NdeI
va BamHI dirge nei vOi nhau de tao thanh

plasmid pET-fgfl.
616

.h

Sing Iric the Wen flop va giai trinh tkr DNA
Gen fgfl va plasmid pET-His sau khi xir IS/ bang
enzyme NdeUBainHI duqc nei v6i nhau bang enzyme
T4 ligase. Sau qua trinh nei, sari pham Wei duce bien
nap vao to bao E. coil DH5a va tien hanh sang lac
dung vi khan mang plasmid tai to hqp tren mei
truerng thach LB china ampicilin 100 tg/ml. Tiep theo,
khuan lac mac duqc ten mei Mrerng china khang sinh
dirge nu8i cay va tach chi& plasmid tai to hqp.
Plasmid tai to hqp duqc kiem tra bing cach cat
gi&i han yeti hai enzyme NdeUBamHI va PCR vai
cep moi FGF1F/FGFIR theo chucmg trinh: 95°C/5
phut; 30 chu .kg: 94°C/45 phut, 55°C145 &ay,
72°C/45 phut; ket chic, 72°C/10 phut. Deng tiled, gen
fgfl duqc giai trinh tv 6 c8ng ty Macrogen (Han
Quec). Viec phan tich trinh tkr gen duqc tien hAnh
tren phin mem Jellyfish.
Dieu hitn protein FGF-1
Ching E. coil BL21(DE3)/pET-fgf/ duqc nuoi
trong mei truang LB-Amp100, lac 250 v/p, 37°C den
khi OD600 dot 0,6 - 0,8 thi tien hanh be suns IPTG
den ming de) 0,5 mM de cam Ong stir bieu hien
FGF-I, tiep tic nueoi lic them 16 gia. Sinh khei to
bao dirge thu loi va huyen phi trong dem lanh
(10mM Tris-HCI, pH7, ImM EDTA), d#t trong di

15 phut. Te bao dirge pha von bing Ultrasonic Cell
Disruptor (Mg) sau de) ly tam 13000 v/p, 4°C, 10
phut de thu phin doon protein tan (dich noi) va
protein khang tan (can). Protein tong so dung cho
SDS_PAGE duce chum bi bang each them dung
dich xir 1Y man vao huyen phi to bao, gia nhiet 5
phut 6 95°C, ly tam 13000 v/p, 4°C, 15 phut de loci
be con.
Phin tich SDS-PAGE va lai Western
Sr bleu hien FGF-1 duqc phan tich bing SDSPAGE 15%. Protein duce phat hien bing Coomassie
Brilliant Blue, khei luring phan tir FGF-1 duqc so
veri thang phan tir luring thap (Amersham). Sr hien
dien cua FGF-1 duqc kiem chimg bing phuang phip
lai Western vai khang the khang dac hien FGF-1
(R&D). Ham luring cua FGF-1 trong to bio E. coil
duqc xic dinh tuang dei bang phin mem Quantity
One (Bio-rad).
Tai gip cu#n FGF-1 hr the vii
The vii (pha protein khOng tan) thu nhan t6 E.
coli BL21(DE3)/pET-fg/7 duqc rira 3 Ian bang dung
dich rira the vii (Tris-HCI 20mM, pH 8,0) c6 bo sung
Triton-X100 ding de 1% de loci met s6 protein tap,
sau de rira 3 lin bing dung dich rira the vii khang be


Top chi COng nghe Sinh hoc 12(4): 615-621, 2014
sung Triton X-100. The vai sau khi dra duqc h6a tan
trong dung djch Gu-HCI 4M de neng da protein dot 4
sau do duqc pha bang nhanh bang dung djch
tai gap cuOn (Tris-HC120mM, urea 2M, sucrose 10%,

tween80 0.1% pH 7.5) de nOng d'6 protein dot 0,2
mg/ml, khuay deu 6 4°C trong yang 14 gib.

giai hart deu dinh tuang ung vai gen fgfl, san phim
co kich thuac 4601 bp.

20000 by

Tinh chi FGF-1 bing sic kj, ai Isrc vIri heparin
C6t HiTrap Heparin HP (Ge Healthcare, They
SI) duqc can bang bang dung djch A (Tris-HC1 20
mM, pH 7,5). Dung djch the vai tai gap cuan duqc
nap vac. cat HiTrap Heparin HP vai tic d6 2,5
sau d6 cat duct tai can bang bang dung
djch A. Tien Wirth dung ly cac protein barn cat bang
bon hqp dung djch A va dung djch B (Tris-HCI 20
mM, NaCI 2M, pH 7,5) vai ti la 20%, 40%, 60%,
80%, 100% dung djch B, tot da 2,5 ml/phut. Cac
phan down protein duqc thu nhan, dian di SDSPAGE, xic djnh da tinh sach bang phan mem
Quantity One (Bio-rad) va djnh luqng FGF-1 bang
phuang phi!) Bradford.
Dinh gii host tinh tang shill in vitro ctia FGF-1
sau doh chi
Hoot tinh sinh hqc cua FGF-1 sau khi tinh the
duqc danh gia thong qua tiler nghiam tang sinh in
vitro tren d6ng nguyen bao sqi chuat. Te bao NIH
3T3 duqc nu8i trong moi tnemg DMEM/F12 vai
10% huyet thanh (fetal bovin serum) den khi dot
da bao phu khoang 80% be mat binh nuoi thi den
hinh tech va thu nhan to bito don bang enzyme

trypsin 0,25%. Sau 46, to bio NIH 3T3 duqc
huyen phu trong moi tru6ng DMEM/F12 vai 10%
huyet thanh va bo sung vac. dia 96 gieng a mat d0
5x103 to bio/gieng, ti a 37°C, CO2 5%. Sau 24 gia
u, mei tru6ng cu duqc but b6 va thay bang moi
truong DMEM/F12 duqc b.& sung FGF-1 tinh the
vii n6ng 80 tang bac 10 tar 10 pg/ml den 10
Sau 48 gib, moi gieng nu8i city duqc 1:46
sung 10 al MTT 5 mg/ml va do d6 hap the quang
a btrac song 550 nm.
KET QUA VA BI$N LUAN
Thlit ki plasmid tai to hqp pET-fg/7
Gen fgfl (423 bp) ma h6a .FGF-1 da duqc
khuech dal plasmid pIDT-fgf/ bang phuang phap
PCR (Hinh 1) va xir IS, too deu dinh bang cap
enzyme Ndel va BamHI. Plasmid pET-His cans
duqc cat m6 Ong bang cimg mat cap engzyme cat

500 b
400 by

423 bp

75bp
Hinh 1. PCR khuech dai gen fgll. 1: Thang chuan DNA 1
kb plus; 2: San pharn PCR gen fgfl.

San pham not gen fgfl va plasmid pET-his duqc
bien nap vac* vi khuan E. coli DH5a, sau d6 tien
14nh sing lqc (long E. coli mans plasmid tai to hqp

bang khang sinh ampicilin va kiem tra plasmid tai to
va giai trinh ar. Ket qua
hqp bang PCR, cat giai
PCR plasmid tai to hqp yeti moi FGF1F/FGFIR va
hai enzyme Ndel, BamHI deu cho sin phan co kich
thy& 423 by tucmg img vai kich thuac dm gen fgfl
duqc then vac) (Hinh 2). Eking thei, genie/ duqc
giai trinh ttr va so sixth vai trinh ttr thiet ke bang
phan mem Jellyfish, ket qua so sanh cho they gen
fgfl trong plasmid tai to hop khong co sai sot ten
trinh tu nucleotide, va ding khung djch ma tren
plasmid pET-His (ket qui kh6ng trinh bay a day).
Bien hiin gen fgfl ma hos FGF-1 trong E. coli
BL21(DE3)/pET-fgf/
Nhiun danh gia sir bieu hian va tim hieu sir hien
dian cua FGF-1 a clang the vai hay clang h6a tan
trong to bio chit, chimg E. coli BL2I(DE3)/pETfel duqc nu8i city lac trong LB-Atup100 va cam
Ung bang IPTG. Sinh Ishoi to bao duqc thu nhan sau
16 gib cam img va tien hanh phi ming to bao de
phan tich ink protein tong so, phan down tan (chill
FGF-1 tan) va phan down killing tan (chira FGF-1
dung the
Ket qua phan tich SDS-PAGE cho they, khi
duqc cam mg bai IPTG, thing tai to hqp di tong
hqp mat luqng len protein co kich thtroc khoang 16
617


Ding Tit Tnrong et al.
kDa (Hinh 3A, gieng 3), trong khi do protein nay

khong co trong dich tong khi kitting cam img IPTG
(Hinh 3A, gieng 2). So sanh veri khoi lucmg phan tir
ly thuyet cua FGF-1 khoang 15,8 kDa, cho 111'4 vach
protein dam nay hien dien trong dich tong dtrqc cam
Crn4 co the la aFGF tai to hop bleu hien tUpET-fg1/.
Nham khang dinh vach protein duce bieu hien la
FGF-1, chung ten phan tich them bang lai Western
vii khang the dac hieu khang FGF-1, tren ban phim
xuat hien vach tin hieu tucmg frng vari vi tri cita vach
protein xuat hien trong dich tong protein khi duqc
cam img IPTG (Hinh 3B, gieng 3), kat qua nay

20000 by

kiting dinh vach protein dam xuat hien khi duqc cam
ung chinh la FGF-I.
Ben canh do, khi phan tich phan doan khong tan
va tan dich to bao da duce phi ming cho
protein FGF-1 chi duqc bieu hien 6 dang the
the viti
trong trong to bao E. coli (Hinh 3A, B, gieng 4).
Dinh lung bang phan mem Quantity-One, ch6thay
FGF-I bieu hien chiem 22% tong protein dm to bao
E. coli BL2 I (DE3)/pET-fgf/ (Hinh 3C, gieng 4).
Nhang ket qua ten chirng to ch6ng t6i da thanh
cong trong viec tao chimg E. toll BL21(DE3)/pET7/ie7
bieu hien FGF-1 ngtr&i tai to hqp.

20000 by

500 by
400 by

423 by

5000 by
4000 by

4601 by

500 by
400 by

423 by

75bp
75 bp

A

B

Hinh 2. Keit qua kiern tra gen fgfl trong plasmid tai to hqp pET-fgf1. PCR vdri cap mdi FGF1F/FGF1R (Hinh 2A); Cat bang
hai enzyme Ndel, BamHI (Hinh 2B). 1A: Thang DNA 1 kb plus, 2A: chirng am, 3A: gen fgfl, 4A: san pham PCR, 18: Thang
DNA 1 kb plus, 2B: plasmid tai to hqp tuft khi cat, 3B: plasmid tai t6 hop sau khi cat bang Ndel/BamHl

kDi

1

97,0 -- 66,11 -45,0 —

3(1,0 —

20.1

2

3

4

1I

4IP •

14,4 —

Hlnh 3. Str bieu hian FGF-1 trong E. coli BL.21(DE3)/pET-fgf1. Dit)n di SDS-PAGE (A); lai Western yeti khang 014 dOC hiOu
khang FGF-1 ngtrtri (B); dinh krqng bang Quantity-One (C); 1: thang protein; 2: protein tong so K. mAu kh6ng cam UN
IPTG; 3: protein tong s6 to mhu cam Ong IPTG: 4: phan doan protein kh8ng tan khi cam Ong IPTG; 5: phan doan tan khi
cam Ong IPTG.

618


Tgp chi Cong nghe Sinh hoc 12(4): 615-621, 2014
TM gip cuOn FGF-1 tu. the vfii
Guanidin IA chit hien
manh lam cho cac

protein bj ket cum trong the vAi du& ra va tan trong
dung djch. Sau khi pha loang bang dung djch tai gap
cuOn, ming dO Guanidin giam xuting tao,dieu kien
cho cac protein tai ciu trAc tar lei. Met so chit nhu
urea, sucrose, tween80 duqc bo sung de qua trinh tai
gip cuOn dien ra tot hen, ngan chAn stir ket coin tra
lei cua cac protein.

4, gieng 2) khang swat hien nheng vach protein nay;
lieu nay cluing to dung dich guanidin 4M da 116a tan
hill het protein trong the viii. Sau khi tai gip cuen,
trong pha protein tan xuat hien vita protein Wang
img vai FGF-1 (Hinh 4, gieng 4), (Icing th6i khang
co protein bj ket cum trong qua trinh tai gip cuOn
(Hinh 4, gieng 5). Nhu vAy, FGF-1 da duce tai gap
cuOn va ton tai b clang tan sau khi tai gap men va
sin sang cho nhAng 'mac tiep theo.
Tinh chi FGF-1 bing sic Ic* iii lrc voi heparin

kDa T 1 2 3 4 5
97,066,045,0-

L.
7
4 :
1

30,020,114,4 -

I I

•

Illnh 4. Phan tich protein trong qua trinh UM gap cuOn FGF1 tren SDS-PAGE. T: Thang protein chuan; 1: Th6 viti
trtr6c khi h6a tan; 2: COn sau khi hoa tan the viii; 3: Dung
dich the viii h6a tan bAng Guanidin 4M; 4: DO protein sau
khi tai gap cu6n; 5: C6n sau khi tai gap cuOn.

The viii truac khi hoa tan (Hinh 4, gieng 1) va dung
djch the viii boa tan bang guanidin 4M (HInh 4,
•
gleng 3) co cfic. vach protein tuong img. vai nhau;
trong khi do, phan cAn sau khi hoa tan the yin (Hinh

FGF-1 co hi luc manh vai heparin, twang tic
gicra FGF-1 Ira heparin là lien ket ion gala polyanion
tren heparin (nhorn sulfate) v6i Lys113, Arg119,
Lys128, Asn18, Asn114 va GIn127 tren be mAt cua
phan tir FGF-1 (Fan et al., 2012, Zakrzewska et al.,
2009). Khi trong dung djch mutii co ntmg de cao,
lien ket gift FGF-1 NIA heparin bj pha va.
Phan door, 20% (Hinh 5A, gieng 4) va 60% dung
djch B (Hinh 5A, gieng 5) deu co protein co khoi
luqng phan tin I9 thuyet cua FGF-1 Ichoang 15,8 kDa.
Cac protein FGF-1 tai to.hqp trong cac phan doan
nay da duqc khang djnh bang lai Western (Hinh 5B,
gieng 4 va 5). Ben canh d6, khi phan tich phan doan
dung ly 20% va 60% dung djch B bang phAn mem
Quantity-One, ket qua cho thiy FGF-1 thu nhAn
duqc chiem tuong Ang IA 50,1% va 93,40% luqng

protein tinh sach duqc (Hinh 5C, gieng 4 Ira 5). Hieu
suit tinh sach va hieu suet thu hei cua toin qua trinh
&rot trinh bay trong bang 1. Ket qua nay phi hqp
vai ding b8 cua Moringlane va dOng tic gia (1990),
FGF-1 co boat tinh duqc thu nhan khi dung ly voi
dung djch china NaCI 1,2 M trong tinh the sac k' ai
lvc heparin (Moringlane et al., 1990).

Hinh 5. Phan tich protein trong cac phan doan tinh the ai Ivc heparin. Dien di SDS-PAGE (A): lai Western vii khang the
dac hieu khang FGF-1 ngu*i (B): Phan tich do tinh sach bang phan mem Quantity One (C). T: Protein chuen: 1: Dung dich
tru.eic khi qua cot: 2: Dung djch ra khoi cot sau khi nap meu: 3: Dung dich riya cot: 4: Phan doan 20% dung dich B: 5: Phan
doan 60% dung djch B.

619


Dang TAt Trtrimg et al.
Bang 1. Phan tich Wang FGF-1 va hiau suet thu hei FGF-1 caa town qua trinh tinh che.
Luang protein
t6ng (mg)

Ty la
FGF-1 (%)

Ltmng
FGF-1 (mg)

Meu the vui ttrai ban au

12,00

49,30

5,92

Phan doan 20% dung djch B

0,65

50,10

0,32

5,41

Phan doan 60% dung dich B

2,26

93,40

2,11

35.64

Dinh gia host tinh tang sinh in vitro ctia FGF-1
sau tinh the
Do thj danh gia hog tinh tang sinh cua FGF-I c6
dong dac trung cho mgt thin nghiem tang sinh. Ket
qua cho thay c6 su tuang quan giira ruing dO FGF-1

va su tang sinh clang to bao NIH 3T3, ,khi gia tang
nOng dO FGF-I trong moi twang nuoi cay thi mat dO
to bat) NIH 3T3 c6 su gia tang tuang img. Tir do thj
suy ra duot gia tri ED50 cua cac phan doan.
Ket qua cho they mAti FGF-1 thu (kw tir phan
doan 60% dung dich B c6 EDso = 657 pg/ml cao ham
mau FGF-1 thucrng mai voi ED50 = 535 pg/ml.
Nguyen nhan khien gia tri ED50 cua Tau FGF-1 thu

Mau suet thu
hal (%)

dtrqc tir phan (loan 60% dung djch B cao hen meu
thucrng mai co the do de tinh sach ctia mau FGF-I
phan doan 60% dung djch B dot 93,4% thap hen so
voi met' FGF-1 thuang mai c6 de tinh sach > 97%.
Ket qua ding cho they !lieu FGF-1 thu duqc tir phan
doan 20% dung dich B cho gia tri ED50 cao hen 7 Ian
matt thucrng mai. Ket qua nay chUng minh rang mall
FGF-I thu dugc tir phan clop 60% dung dich B
(twang img voi 1,2M NaCI) c6 hog tinh tang sinh to
bao va FGF-1 thu duac tir phan doan 20% dung dich
B (twang img yeti 0,4M NaC1) kitting co hog tinh
tang sinh to beo. twang tu vai nghien cfru cua
Moringlane va thing tic gia (Moringlane et al.,
1990).
FGF-1 thucrng mai
_e_ FGF-1 pltht doan 60%B
FGF- I phiut doan 20%B

0
C

-2

0

2

6

I

8

Log([FGF- I]) (pg/ml)

KET LUAN
Chung toi de too throe chOng E. coli
BL21(DE3)/pET-fe bieu hien FGF-1 ngtrai tai to
hqp khi, cam ang bang IPTG. FGF-1 ton tai dtrai
dong the yid kitting tan trong to bao chat dutyc tai
gip cuqn bang phuang phap pha bang nhanh giitp
thu nhan dtrgc FGF-1 a dung tan. Till qua qua
620

trinh tinh sach bing sac kj, ai luc voi heparin.
FGF-1 co hoot tinh tang sinh to bao NIH 3T3 dtrac
thu nhan a phan doan 1,2M NaCI co dO• tinh sach
93,4%. Day la nghien ciru dau tien ye bieu hien ye

thu nhan FG.F-1 c6 hoot tinh sinh hoc 6 Viet Nam
ye IA tien de cho cac nghien cuu quy trinh cong
nghe sin xuat FGF-1 tai to hop sau nay cho nhiing
ang dung trong y hoc ye my pham tai Viet Nam.


Top chi Ging nghe Sinh hQc 12(4): 615-621, 2014
Lirl cim
Cong trinh nay dux thitc hien tit kink
phi hang nam caa truang Doi hQc Khoa hoc Ty.
nhien, Doi hQc Quoc gia HCM cap ltng nam cho Be
mon Cong nghe Sinh hoc Pita h - Mai truang.
TAI

THAM KHAO

Cuevas P, Reimers D, Carceller F, Martinez-Coso V,
Redondo-Horcajo M, Saenz de Tejada 1, Gimenez-Gallego
G (1997) Fibroblast growth factor-1 prevents myocardial
apoptosis triggered by ischemia reperfusion injury. Eur J
Med Res 2(11): 465-468
Fan W, Yan W, Xu Z, Ni H (2012) Formation mechanism
of monodisperse, low molecular weight chitosan
nanoparticles by ionic gelation technique. Colloids Surf B
Biointerfaces 90: 21-27.
Htun P, Ito WD, Hoefer IE, Schaper J, Schaper W (1998)
Intramyocardial infusion of FGF-1 mimics ischemic
preconditioning in pig myocardium. J Mo! Cell Cardiol
30(4): 867-877

Langer R, Vacanti JP (1993) Tissue engineering. Science
260(5110): 920-926
Moringlane J, Spinas R, Bohlen P (1990) Acidic fibroblast
growth factor (aFGF) is present in the fluid of brain
tumour pseudocysts. Acta Neurochirurgica 107(3-4):88-92
Sambrook J, Russell DW (2001) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Labo-atoty
Press.
Schumacher B, Pecher P, von Specht BU, Stegmann T
(1998) Induction of neoangiogenesis in ischemic
myocardium by human growth factors: first clinical results
of a new treatment of coronary heart disease. Circulation
97(7): 645-650
Zakrzewska M, Marcinkowska E, Wiedlocha A (2008)
FGF- I : from biology through engineering to potential
medical applications. Crit Rev Clin Lab Sci 45(1): 91-135
Zakrzewska M, Wiedlocha A, Szlachcic A, Krowarsch D,
Otlewski J, Olsnes S (2009) Increased protein stability of
FGF1 can compensate for its reduced affinity for heparin.
J Biol Chem 284(37): 25388-25403

CLONING, EXPRESSION AND PURIFICATION OF HUMAN FIBROBLAST GROWTH
FACTOR 1 (FGF-1) IN ESCHERICHIA COLI
Dang Tat Truong, Trinh Minh Thuong, Tran Van Hien
University of Science, Vietnam National University HoChiMinh City
SUMMARY
FGF-1 (Fibroblast growth factor-I) is a pluripotent growth factor which can possess broad mitogenic and
cell survival activities, and is involved in a variety of biological processes, including embryonic development,
cell growth, tissue repair, cellular regeneration, angiogenesis, and so on. With such potentials, FGF-1 has now
been applied to many fields such as cosmetics, treatment to cardiovascular diseases, wound healing. In this

study, we proceeded to overexpress recombinant protein FGF- I in E. coli for low-price FGF-1 production
towards applications in Vietnam. Thefgfl gene of 423 by encoding for human FGF-1 was synthesized by PCR
using plasmid pIDT-fgf/ as template and cloned into plasmid pET-His under the control of strong T7 promoter.
The gene in correction of recombinant plasmid, termed pET-fgfl, was confirmed by PCR, restriction enzyme
digestion and DNA sequencing. After that, plasmid pET-fgf/ was transformed into E. coli BL21(DE3) to
establish the recombinant E. coli BL21(DE3)/pET-fg/7. When the recombinant E. coli BL21(DE3)/pET-fgf/
was shakily cultured in LB medium containing ampicillin, subsequently added with IPTG to induce the T7
promoter, FGF- I was over-expressed and accumulated as inclusion body in cytoplasm with content accounted
for 22 percent of total protein after 16 h induction using SDS-PAGE, Western blot, and Quantity-One softwave
analyses. Inclusion bodies harboring FGF-1 was solubilized by Guanidin 4M and was refolded using diluted
method to isolate solubilized FGF-1. Heparin-affinity chromatography was conducted to purify FGF-1. Results
indicated that the recombinant protein was eluted at NaCI 1.2M and NaCI 0.4M with the purity of 93.4% and
50.1%, respectively. The former purity had in vitro NIH 3T3 stimulating capacity equal to that of commercial
product, having EDso = 657 pg/ml.
Keywords: FGF-I. inclusion bodies, heparin-affinity chromatography, bio-assay

Author for correspondence: E-mail: tvhieuQhcmus.edu.vn
621