hiến lược mới trong việc biểu hiện erythropoietin (EPO) trên dòng tế bào CHO DG44 bằng vector tricistronic

• Nghien cu'u - Ky. thuot

Chi&I luvc
trong viec bleu hien
erythropoietin (EPO) tren dung to bao
CHO-DG44 bang vector tricistronic
Nguy& Anh Xuan', Lao Dire Thuan3, Doan Ngoc HAP, Do Minh Si u*
Khoo Sinh hoc, Truang Dai hoc Khoa hoc Ter nhien — Dai hoc Quoc gia Tp. Ho Chi Minh
COng iv TNHH CNSH Dirac NANOGEN
' Khoa Ging ngh "(3 sinh hoc, Mtrang Dai hoc. Mir Tp. 114 Chi Minh
'Vin Stir khoe nghe nghiep vd Moi trtrang
E-mail:

Summary
Erythropoietin (EPO) gene was cloned into the expression vector pDIGF-EPO, which carries other two
expression cassettes (GFP and DHFR). The dihydrofolate reductase deficiency (DHFR-) CHO (DG44)
cell line was transfected by this vector using lipofectin method. The GFP-based system was used to
shorten the cloning duration and enhance the opportunity to select super clones. The DHFR-based
system was used for gene amplification. The transfected cells were selected and processed through 2
cycles of FACS followed by gene amplification and single cloning by limited dilution method. GFP gene
from the single cell clone was removed by recombination. An optimized fed-batch was developed to
culture the EPO expressing cells. The cells could survive for 14 days; the protein titer was 902.6 ± 19.96
mg/mL (comparable to the previously reported values for EPO production in pilot scale). The purified
recombinant erythropoietin from the fed-batch had the molecular weight within the range of 30-40 kDa
as checked by SDS-PAGE. Its isoform pattern by isoelectric focusing (IEF) electrophoresis satisfied the
requirement of isoforms issued by the European pharmacopoeia 7.0 (EP 7.0) with ratios in appropriate
ranges. Peptide mapping and sialic acid content analysis strongly passed the requirement stated in
EP 7.0. Biological activity of the obtained EPO-alfa versus the 3rd WHO International Standard for
Erythropoietin for bioassay was 112.545 ILI/mg protein.
Keywords: Recombinant human erythropoietin (rHuEpo), CHO-dh&-e cell, GFP, single clone, gene
amplification, recombination.

Dat van de

EPO to nhien hoan toan giong nhau ye trinh tu'
acid amin va CO chu6i oligosaccharid giong nhau
trong c5u talc hydrat carbon. EPO tai to hop CO
hoat tinh sinh hoc twang tu' nhu' EPO nOi sinh

Erythropoietin (EPO), mot nhan to tang troang
kith thich tao hong ceu, giup cai thien clang 1(6
ch5t luvng cuOc song cue benh nh5n mac chOng
suy th5n ho5c chOng thieu mau tem tong. EPO
Bong vai trO quan trong trong so tang sinh va biet
h6a cue cac to bao tier, than hong chu trong tOy
xuang 111 . Ph5n Ian EPO duvc tao ra chu yeu bai
th5n de dap Ong vai tinh tang thieu oxy trong
mo, mot phen nha (10%-14%) do gan tong hop
(gan la ca quan chinh san xuet ra EPO a bao
thai) 141 . Trong nhOng nam gen day, viec Ong dung
cue EPO tai to hop trong dieu tri benh ngay tang
ph6 Nen ten toan the gi&i. EPO tai to hap va

NhOng to bao dOng vat nhu, CHO duvc Ira
chon de san xuet cac protein sinh duvc nhu'
EPO bai cac bien doi sau dich ma dm to bao phu
hop vai ceu truc protein cua ngudi ye mat chac
nal-1g cOng nhu' duvc dOng hoc 16 . ViOC sir dung
to bao CHO cho nuoi cay san xuet protein vol
quy me Ian ngay tang ph6 bien bai cac (long to
bao nay c6 the thich Ong vai viec phat trier) trong

mai twang huyen ph0 khOng chive huyet thanh.
Ngoai ra, cac clang to bao nay duvc tong nh5n la
cac to bao an toan cho viec san xuAt protein tai to

TAP CHI DUCK HQC - 11/2015 (SO 475 NAM 55)

25

'91


• Nghien civu - Ky thuOt
hap m. Tuy nhien, de Iva chgn disc d6ng to bao
dOng vat the hien cac protein muc tieu (bien dOi
sau dich ma) veri hieu suet cao, met s6 yen de 41
dircyc giai quyet nhir hO the -1g bieu hien kern hieu
dO
qua, thol gian chuyen gen va tao d6ng,
bieu hien protein the') & quy m6 Ian. Day le nhCrng
khO khan gap phai trong hau het cac dong to bao
hien nay 18.91. Trong nghien ctru tnyac day, Meng
va cong sv da phat then he thong dong bieu hien
gen muc tieu vcil gen chon loc DHFR (ma hoe cho
enzym dihydrofolat reductase) va gen GFP (ma
hoe cho protein phat hujmh quang xanh) de Iva
ch9n d6ng to bao bieu hien protein tai to hcyp muc
tieu vai bleu suet cao nhat [121. Deng to bao tot nhat
dvgc chgn 19c dva ten he thong chon I9c DHFR
va he thong phan tech to bao dva ten tin hieu
hujmh quang cue GFP (FACS).

Trong nghien cull nay, chimg t6i diva ra mot
chitin lucyc mai cho viec tao dong to bao bieu
hien On dinh protein EPO tai to hap. He thong
vector tricistronic gem gen muc tieu, gen GFP
(tao thuan Igi cho viec rut ngan th6i gian tao
d6ng va tang kha nang chgn 19c d6ng to bao tOt
nhet) va gen DHFR (dung ch9n loc ten dong to
bao khuyet cluVng DHFR CHO-DG44). Biyac
tiep theo cue nghien CCPU le lc* be gen GFP bang
phvang phap tai to hgp sau khi chon duvc to
bao don dong bieu hien protein muc tieu tOt nhat.
Qua trinh nay se giiip giam hay loai be su' bieu
hien die GFP.
VOt lieu, phvang phap nghien ciru
Thief ke vector
Trong nghien ciru nay, plasmid mang gen ma
hoa EPO tai to hgp ngueri (EPO), gen GFP (ma
hoe protein GFP), gen khang Zeocin va gen chon
loc DHFR (ma hea dihydrofolat reductase). Trinh
tcy Hu-EPO duvc thiet ke va tong hcyp bai hang
Genscript (Piscataway, M9). Gen eGFP duvc
phan tech tip plasmid pEGFP-N1 (Clontech, M9).
Gen DHFR duvc phan tech tiv plasmid pSV2-dhfr
(ATCC, M9). Promoter CMV duvc sir dyng de
Trinh
dieu hoa bieu hien EPO tai to hgp
tcy intron dm gen beta-globin tren the (GenBank:
V00882.1) va \rung CpG cica gen HNRPA2B1
ngt.rol ma hea cho 2 loai protein hnRNP A2
va B1 (GenBank: D28877.1) duvc be sung de

26

tang cv6ng kha clang bieu hien gen muc tieu.
Ket thuc qua trinh bieu hien GFP bang phvang
phap tai to hgp. Hai vi tri tai to hgp tuvng dOng
FRT duvc them vao truac promoter va sau
trinh tty gen GFP. De dam bao cho GFP bieu
hien thap km gen ma hoe EPO, trinh to IRES
duvc chen vao truac promoter dm GFP. VOng
promoter (SV40) ducyc sir dyng de dieu h6a
bieu hien DHFR. Plasmid ducyc d6ng hoa vao
to bao vi khuen E. co/i Top1OF theo hvang den
cica nha san xuet (Invitrogen, Mg). Plasmid bleu
hien duvc tinh sach bang b0 kit tech chiet DNA
(Qiagen, M9).
Quy trinh chuyen gen
Te bao CHO-DG44 (ngan hang cGMP,
Invitrogen, M9), dong to bao mang dOt Nen gen
reductase dihyrofolat (DHFR) nem troll nhiern sac
the se 2, duvc Sir dung rOng rai trong san xuet
protein tai to hgp th6ng qua phvang phap chon
loc khuyet dveyng DHFR. Te bao CHO-DG44
duvc nu6i cey trong m6i truteng PowerCHO2
(Lonza, M9) co be sung hypoxanthin va thymidin
(HT, Sigma-Aldrich, Mg) 6 37°C, 5% CO2.
De chuyen plasmid vao to bao CHODG44, nghien cOu sir dung WO kit chuyen
gen Lipofectamine LTX with Plus Reagent kit
(Invitrogen, Mg). Quy trinh thvc hien duvc tien
hanh theo Wang den cue nha san xuet. Nghien
cipu ben hanh chuyen vector tricistronic vac) to

bao CHO-DG44. Sau 48 gi6 chuyen gen, to
bao chuyen gen Neu hien GFP duvc quan sat
bang kinh hien vi hujmh quang va sv bieu hien
protein EPO trong dich nuoi cey to bao duvc dinh
luvng bang bi) kit human EPO ELISA (Stem Cell
technology, Canada).
Chon loc va tao clang
bieu hien EPO tai to hop

to bac) CHO-DG44

Qua trinh chon 19c citycyc tien hanh bang cach
nu6i to bao de' chuyen gen trong m6i trirevng chon
19C be) sung 400 pg/ml zeocin, 50 nM methroxat
(MTX) ve khuyet cluOmg HT (Sigma-Aldrich, M9).
Sau ch9n loc, to bao mang plasmid 'Jinx nu6i
cey tang sinh trong moi tupteng duy tri c6 be sung
50 pg/mI zeocin. Te bao chuyen gen bleu hien
manh GFP dirgc chon 19c 2 len ten he thong
may FAGS Calibur (BD Biosciences, M9) va sir

TAP CHI DUOC HOC - 11/2015 (SO 475 NAM 55)


• Nghien civu - Ky thUOt
dung cho nhirng nghien ciyu tiep theo.
Te bao sau khi lkya chon bang GFP tiep tuc
duvc ben hanh chon IQC thong qua khuech dai
gen. Te bao duvc cam irng trong mei trtyang
nuoi cey bO sung MTX vai ruing dO tang dan tO,

50nM den 500 nM. Sau dO, to bao duvc phyc
hoi va tiep tuc nuoi tang sinh. DOng to bao dun
(single clone) bieu hien On dinh protein muc tieu
tiep tuc duvc tao ra bang phiyang phap pha
bang tai han. Sang loc nhiyng dong to bad dun
co bleu hien GFP (quan sat dkrai hien vi hu9nh
quang) va EPO (dinh luvng bang phiyang phap
ELISA) cao nhat. Cu6i cOng, su' bieu hien GFP
duvc loaf bó bang cach chuyen plasmid pOG44
mang gen ma hoa recombinase (Invitrogen, M9)
vac) d6ng to bao Ikra ch9n. Hieu suet tai to hap
cluvc clanh gia bang he thong may FACS Calibur.
Huai ch tang sinh to bac) san xuAt EPO tai
to hcrp tren quy mo this, nghi§rn
Doing dan to bao sau khi chon I9c theo quy
trinh neu ten (bleu hien manh nhat EPO tai to
hap) duvc sir dung de nuoi chy tang sinh bang
m6i tru,Crng PowerCHO2 (Lonza). Trkrac tier), to
bao duvc nuoi cay trong Ong nuoi cey lac 50 ml
(BD Biosicence, M9), sau dO du,cyc nuoi cey tang
sinh len the tich cao hon (600m1) trong he thong
nuoi cey Tube spin flask (TPP, Thyy ST) ket hcyp
vai tO nuoi cey lac (Kuhner, Thuy ST) . Cu6i ding,
qua trinh nuoi cey to bao san xuet EPO tai to
hap duvc thy,c hien trong binh nuoi cey lac 5 L
(Thompson, M9). Te bao duvc nuoi bey vai mat
dO ban clau 0,5 x 106 to bao/mL trong 2 L m6i
tnyOng san xuet O' 37°C, 5% CO2, tOc de lac 100
vong/phOt. Sau 3 ngay nuoi cey, 500 mL dung Bich
feed (bao Om Feed 3 tiy Excellgene, Galactose,

Manose, Insulin,Transferrin va h6a chat khac de
tang qua trinh gen dkveyng cua EPO tO, Sigma)
duvc b6 sung hang ngay. Qua trinh nuoi cey to
bao san xuet EPO ket th0c khi ti le phan tram to
bao sting dual 70% (khoang 12-14 ngay). Dich
nuoi cey to bao sau dO duvc thu nhan va tinh
the bang he thong sac ki Akta - Mabselect Sure
(GE Healthcare, Anh) theo hu,deng dAn cua nha
san xuet. Protein sau tinh sach duvc kiem tra,
danh gia cac chi tieu nhu' ceu truc h6a hoc, c10
tinh sach, nOng dO protein, hopt tinh sinh hoc dkya
ten cac quy trinh de" duvc cong b6 [Jelkmann,
TAP CHI DUOC HQC - 11/2015 (SO 475 NAM 55)

Lee, Breidbach].

Ket qua va ban luan
Vector bieu hiOn EPO tai to hap
1111M111111

Hinh 1: Ca'u truc vector pDIGF-EPO
Chien Vac bleu hien protein EPO ten dong
to bao CHO-DG44 trong nghien ciyu nay mang
lei hieu qua cao khi xem xet troll khia canh thOi
gian tao d6ng va lu,cyng protein duvc tao ra.
Wing bleu hien rHu-EPO bao Om gen muc tie('
(Hu-EPO) duvc dieu hoa biyi promoter/enhancer
CMV. Diya ten ket qua cong b6 cua patent WO
2004067701A2, viing CpG dm gen HNRPA2B1
ma h6a cho protein hnRNPA2 va B1 duvc sir

dyng nhu' le yeu to tang twang bleu hien gen
(enhancer) [3] . Viing CpG duvc chipng minh nhu,
la yet, CO tang cu,ang bieu hien gen ngopi lei va
co nhieu thuan Igi khi thiet ke vector cho viec tao
dong bieu hien [11) . Nghien ciyu cua Buchman va
cong
(la chOng minh sky hien dien cua vOng
intron thuOc gen beta-globin b tho cO kha nang
tang ckyo'ng su' bieu hien gen cao Op 2 den
500 len 12] . Ket hap cac yeu to tang cuteng tren,
su' bieu hien cua GOI tang len dang ke (dir lieu
khong trinh bay).
Trinh ty, IRES (internal ribosome entry site) le
trinh tu' cluvc tim they trong virus co kha nang huy
dOng ribosome de bieu hien protein mot each clOc
lap va dOng vai tr6 quan tong trong qua trinh phat
then cac vector polycistronic. VOng IRES thu nhan
tO, virus Encephalomyocarditis (EMCV) hien nay
diyac sir dung chu yeu trong heu het cac vector.
Tuy nhien, luvng protein bieu hien diyai su' dieu
flea cua EMCV IRES thuteng kh6ng du de dat duvc
cac yeu cau thkyc nghiem hay chiya tri. Nghien
27


• Nghien civu - Ky. thulat
Tao &mg to bao CHO-DG44 bieu hiOn EPO
tai to hqp

cue Licursi va ding sy, cho they viec sir dung vOng

IRES cue nhan to tang biking nOi mach (vascular
endothelial growth factor) va protein cam irng
collagen loai 1 (type 1 collagen-inducible protein)
tang cutrng kha nang bleu hien gen cao han so vai
cac vector china EMCV IRES 11°1. Trong nghien ciru
nay, chimg tai sir dung trinh ty, nay de dieu h6a sy'
bieu hien cue GFP (marker chon 19c). Cau triic cua
vector pDIGF-EPO du'o'c m6 to trong hinh 1.

Sau 48 gib' chuyen gen, su' bieu hien EPO
va GFP tam tho'i duvc kiem tra Ian lout bang
phutrng phap ELISA va phuvng phap quan sat
mOu dutri kinh hien vi huknh quang. DO, lieu tit,
su' bieu hien GFP cho they hieu suet chuyen gen
dat 50 - 70% va to bao bleu hien EPO & mirc 2,3
pg protein/te bao/ngay (bang 1).

Bang 1: Sy, bieu hien EPO va GFP cua to bao CHO-DG44

Ham luvng EPO (pg/te bao/ngay)
Ti le bleu hien GFP (%)

Sau 1 ihn sorting Sau 2 ihn
sorting

0

2,08 ± 0,46

6,82 ± 0,83

9,01± 0,62

12,30 ± 1,07

0,02

14,78 ± 3,27

32,57 ± 6,33

78,22 ± 8,96

94,66 ± 9,13

Sau 2 ngay chuyen gen, to bao duvc chip
19c bang m6i truirrig nuoi cey khuyet dutrng HT-,
b6 sung khang sinh va MTX nham ch9n ra cac
to bao chuyen gen On dinh. Protein GFP duvc
chirng minh la huru ich trong he thong chon 19c
beri protein GFP cho phap tao ra mot phuvng
thirc ch9n I9c nhanh, dun gian va hieu qua
ten cac to bao da duvc chuyen gen th6ng qua
phkrang phap thing chuyen. Dkra ten k9 thuet
phan tech va thu nhen to bao co chon loc thong
qua tin hieu huknh quang (FAGS), nghien ciru
sir dung chien luvc nay de phat hien cac to bao

bieu hien manh protein myc tieu. Cac dong to
bao c6 tin hieu GFP cao nhat trong moi trutmg

duvc cho la co kha nang bieu hien protein myc
tieu EPO vai hieu suet tirang tu'. Trong nghien
ciru nay, sau hai Ian phan tech va thu nhen to
bao bang k9 thuet FAGS, khoang 94% to bao
hieu hien GFP cho tin hieu vai cutrng (10 khac
nhau (hinh 2). Ket qua nay cho phap nghien ciru
loai 136 cac to bao bieu hien thap protein myc tieu
EPO. Ngobi ra, cac to bao chuyen gen On dinh
cho they sy, tang len dang ke luvng EPO duvc
tao ra.

Bien hiOn On dinh

1)61 cluing

00 pm

Dieu hi#n
on dinh

Bleu hiOn
tam thol

DOi

Thong sei

100 pm

100 ma

FACS Ian 1

FACS lOn 2

100 pm

Hinh 2: Su' bleu hien GFP cua to bao CHO-DG44 truoc va sau khi chuyen gen.
Ti le to bac, phat huknh quang truoc va sau khi chuyen gen, phan tech to bao ducr xac dinh bang kSi
thuet FACS. Tin hieu phat huknh quang cua t6 bao duvr kiem tra duial kinh hien vi huknh quang.
Cac to bao sau khi Itra chon theo quy trinh
neu ten tiep tuc nuoi cey tang sinh va tao dOng
to bao don bang phuvng phap pha loang tai
han nharn chon i9C dong to bao bieu hien On
dinh nhet. Dich huyen phu tip dia 96 gieng duvc
thu 12 ngay sau khi chuyen to bao vao dia nuoi
ch. 48 dong to bac tot nhat duvc nuoi ten dia

24 gieng bang cach lye ch9n cac to bao tir die 96
gieng chuyen qua dia 24 gieng. Sau 5 ngay nuoi,
dich huyen phO tip cac gieng cua dia nuoi cey 24
gieng duvc phan tich. Sau dO, 12 dong to bao tot
nhet duvc chon ra va chuyen vao die nuoi cey
6 gieng de tiep tuc nuoi cey tang sinh. Dina tren
kat qua phan tich tip dich nuoi cey to bao, 6 &mg

28

TAP CHI INICIC HOC - 11/2015 (SO 475 NAM 55)

• Nghien an' - Ky thUOt
to bao bieu hien tot nhat (EPO 1 - 6, bang 2)
duvc Rya chon va nuoi cay tang sinh trong 6 ngay
sir dyng he thong lac On nhiet (Kuhner). Dora Von
ket qua thu nhan duvc tir nuoi cay 16c (rn't do to
bao ban dau 5 x 105 to bao/mL, the Lich 100 mL
mai tru,o'ng nuoi cay), d6ng to bao bleu hien tot

nhat (EPO 5) duvc Iu'u tiff, trong ngan hang giOng
va kY hieu EP00414. Kat qua danh gia hiOu su6t
san xuOt protein muc tieu va duirng cong tang
tru,O'ng cho cac clang to bao tot nhat duvc trinh
bay trong bang 2.

Bang 2: Hiau suat caa cac dOng to bao don bieu hiOn EPO
Ngay 3
Deng M bao

NO), 6

Mat d6
Ham lurrng protein Luang protein/
(108 M bao/m/)
(pg/m1)
to bao/ngay

Thai gian nhan dOi
(gia)

Mat c16 M bao
(106 to bao/m1)

Ham luring protein
019/m0

EPO 1

2,23 ± 0,25

135,2 ± 6,33

20,2 ± 0,97

33,38 ± 0,13

4,24 ± 0,31

365,8 ± 10,25

EPO 2

2,21 ± 0,39

122,1 ± 7,49

18,4 ± 1,23

33,58 ± 0,25

4,37 ±0,27

371,2 ± 11,36

EPO 3

2,37 ± 0,45

136,3 ± 5,01

19,2 ± 0,85

32,07 ± 0,36

4,62 ± 0,39

312,9 ± 15,26

EPO 4

3,09 ± 0,51

141,7 ± 8,23

15,3 ± 0,98

27,40 ± 0,24

4,91 ± 0,52

344,8 ± 14,33

EPO 5*

3,13 ± 0,62

198,6 ± 7,22

21,2 ± 1,51

27,21 ± 0,19

5,23 ± 0,68

405,6 ± 17,66

EPO 6

2,61 ± 0,47

153,7 ± 9,16

19,6 ± 0,98

30,20 ± 0,27

4,31 ± 0,29

311,2 ± 12,38

(*) D6ng to bao clan &Frio Iva chon de tgo ng5n hang M bac): EP00514

Hien nay, cac enzym tai to hap tai cac vi tri
chuyen Net (site specific recombinases) duvc
phat trien thanh nhirng cong cu sir dung trong
k9 thuat thao tac ten gen vi chi:mg co the xec tac
chinh xac cho cac phan irng cat, cher), dao doan
hoac chuyen vj tri dm DNA thong qua cac trinh
ty, nhan biet dac hieu [5]. Nghien ciru sir dung
he thong Flp-In (Invitrogen) chira vi trf tai to hgp
Flp (Flp Recombination Target site - FRT) de

Doi clukng

loai 136 gen GFP. Su' bleu hien GFP da giam
Bang ke khi du'a he thong nay (vector pOG44)
vao to bao CHO-DG44 bieu hien GFP. Theo hinh
3, kat qua cho thay to bao EP00414 sau khi loai
be GFP van bieu hien EPO vai ham luvng tuvng
ty,. Trong khi d6, miyc de bleu hien GFP giam
dang ke (8,98%) so vai truirc khi chuyen vector
pOG44 (93,64%). Te bao sau khi loai be GFP
duvc lu,u trir va ky hieu EP00514.

Tnr■fre khi (Ai to hop Sat) kit( tai to ivy')

100 mm

Hinh 3: Sy bgti hiOn GFP ctla t6 bao CHO-DG44 tnferc va sau khi tai to h9p.
Tile to bao phat huknh quang trubv va sau khi tai to hop duryc xac Binh bang ksi thuOt FACS.

Tin hiOu phat huknh quang cCia to bao dupy ki4rn tra dtpai kinh hidn vi huknh quang
Nu8i cay to bao va san xuAt EPO tai to hoop

Hieu su'At cua EP00514 duvc danh gia
khi nuOi cay theo phirang phap nuOi cay fedbatch (quy mo 5 L) vol mai tru,teng duvc toi
u'u (mei truing du'o'c be sung theo theri gian,

the tich cuOi 2500 mL) sir dung binh nuoi cay
5 L (Thomson) va he thong to cay lac (Kuhner).
Luvng protein du,gc xac dinh bang phuvng
phap ELISA sir dung be kit human Erythropoietin
(EPO) ELISA (Stemcell Technologies). Kat qua

TAP CHI DU6C HOC - 11/2015 (SO 475 NAM 55)

29


• Nghien CCPU - K9 thu.at
duvc trinh bay trong bang 3. Voi phiyang phap
nue' cey fed-batch duvc tai tyu [lea, EP00514 cO
the duy tri trong vong 14 ngay. Ham luvng protein

dat 902,6 ±19,96 µg/mL, twang &rung vai cac
Gong be truVc dO ve san xuet EPO.

Bang 3: Ket qua nuOi cey d6ng to bao EP00514 theo phuung phap nu6i cey Fed-Batch
Ngay

Mat d6

(106 to bao /m1)

Ti le 16 bao
song (%)

Ngay 0

0,55

99,21 ± 0,25

2000 mL MOi throng Power CH02

Ngay 1

0,66 ± 0,09

98,51 ± 0,16

40 mL Feed 3 dupc136 sung mai ngay

Ngay 2

1,43 ± 0,11

98,62 ± 0,22

Ngay 3

2,32 ± 0,25

95,87 ± 0,31

Ngay 5

2,84 ± 0,23

97,26 ± 0,25

Ngay 6

3,65 ± 0,37

94,19 ± 0,47

Ngay 7

3,53 ± 0,26

93,07 ± 0,51

+ Insulin + Transferrin N-glucosamin

Ngay 8

3,52 ± 0,38

95,65 ± 1,33

+ Galactose + Mannose

Ngay 9

4,36 ± 0,52

92,37 ± 3,51

Ngay 12

2,76 ± 0,63

90,64 ± 4,73

Ngay 13*

2,62 ± 0,61

73,44 ± 4,33

Ham lucmg EPO
(lig/m1)

221,3 ± 12,11

Mei trutmg + Feed

+ Galactose + Mannose + Non essential amino acid + Uridin
+ L-Glutamin + Insulin + Transferrin

425,1 ± 13,24

668,7 ±21,12

+ NEM + Uridin

+ Uridin + L-Cystin, + NEM + N-glucosamin + Pluronic + Insulin
+ Transferrin
+ NEM + N-glucosamin + Uridin

902,6 ±19,96
(*) thu hoach vao ngay 14

Phan tich EPO tai to tiger)
Protein tai to Nip EPO tinh sach tip d6ng to
bao EP00514 nu6i cey fed-batch c6 trong luvng
phan tir khoang tip 30 - 40 kDa khi duvc kiem tra

bang phiyang phap SDS-PAGE (hinh 4). KM qua
nay phi) hap vat tieu chuan duvc &Fa ra trong
DLyo'c dien Chau Au (European pharmacopoeia
7.0 - EP 7.0).

---------.--.^----------

3

Hinh 4: Ket qua phan tich mAu protein EPO.
(A) Ket qua phan tich peptid cua meu EPO bang phuung phap peptid mapping. Mau EPO (tren) va md'u chain qu6c to EPO
(EDQM, duet). (8) Ket qua phan tich isoform cua EPO bang phuung phap than di diem clang dien (IEF).
Gieng 1: AA& EPO, gidng 2: Thang chuan pl, gieng 3: Mau EPO chuan qu6c te. (C) Ket qua phan tich trong lucmg phan ter EPO

bang phurtng phap SDS-PAGE. Gieng 1: Mau EPO; gidng 2: Mau EPO chuan quOc td, gieng 3: Thang chuan protein.

Ket qua khao sat ti le isoform bang phiyang
phap dien di diem deng dien (IEF) cho they EPO
tao ra dam bao cac yeu cau ve isoform vai cac t9
le nem trong khoang giO1 Flan. Cu the, isoform 1
khong duvc phat hien they, trong khi dO isoform
6 chiern phan Ian lirong isoform (bang 4). KM
qua phe sec 1(9 peptide mapping cho they EPO
ce phe peptid twang veyi EPO chuan guoc te.

Ham luvng acid sialic cua protein sau tinh such
la 14,90 ± 0,30 mol sialic acid/mol EPO (du•a ten
phtycvng phap dm EP 7.0). Kat qua nay ming
th6a man cac you cau dm EP 7.0 10 mol sialic
acid/mol EPO).

30

TAP CHI DUVC HOC - 11/2015 (SO 475 NAM 55)

Hoat tinh sinh hoc cua EPO tao ra duvc
darth gia ten m6 hinh chuOt cai khoe manh


• Nghien cU'u - K9 thuOt
(20 — 22 g) va so sanh vai EPO chug!' quOc to
theo tieu chuen c6a WHO danh cho EPO (3rd
WHO International Standard for Erythropoietin—
ma NIBSC: 11/170). Haat tinh sinh h9c cua EPO

duvc tinh Wan dkra tren chi so hOng cau
cOa
chuOt khi tiem EPO. Ket qua cho they EPO tao ra
co hopt tinh 112.545,00 ± 6,32 Ill/mg protein. Ket
qua nay them man yeu cau dm EP 7.0 100.000
111/mg protein).
Bang 4: Ti le isoforms caa EPO
Insoform

Ti4 phhn tram chun (%)

Wet qua (%)

Insoform 1

0-15

0

Insoform 2

0-15

0

Insoform 3

1 -20

4.9

Insoform 4

10 - 35

12.7

Insoform 5

15 -40

23

Insoform 6

10 - 35

29.5

Insoform 7

5-25

23.9

Insoform 8

0-15

4.9

Nghien aru sir dung he thong chon loc mai
giup giam clang ke thai gian tao dong (so Iu'Q'ng
the he cua d6ng duvc chon 19c la 30) va tang
twang kha nang thu nhan cac dong to bao bieu
hien manh protein EPO. Ter d6, cac d6ng to bao
co the duct thiet lap va san xuet protein tri lieu vai
min do, twang dirang so vai cac he thOng ch9n
loc hien nay. Dec biet vai nganh tong nghiep can
tOc do tao ra cac nguyen lieu cho cac nghien ciru
lam sang giai dopn I là het sin quan tong, he
thong chon loc duvc de xuet trong nghien ciru
nay hi vong tao ra dirgc scr khac biet nhet dinh.

Ket kfan
Da nghien
va phat trien thanh tong dong
to bao CHO-DG44 bieu hien On dinh protein EPO
tai to hgp vai hieu suet dat 902,6 ±19,96 mg/mL.
Cac ket qua dm nghien dm nay cho they EPO
tai to hgp di 'c tao ra co trong luvng phan ter, ti
le isoform, cau tr6c peptid krang
EPO chuen
quOc to theo Duvc dien Chau Au (EP 7.0). Ngoai

ra, hopt tinh sinh hoc cua EPO dat 112.545 IU/mg,
twang &rang vai EPO chuen quOc to WHO (3rd
WHO International Standard for Erythropoietin).

Tai liOu tham khao

1. Ananth G. S. (2008), "The role of recombinant
erythropoietin in childhood cancer", The Oncologist,
13, 157-166
2. Buchman A. R., Berg P. (1988), "Comparison
of intron-dependent and intron-independent gene
expression", Mol. Cell Biol., 8, 4395-4405
3. Crombie R. L., Irvine A. S., Lipinski K. S., Williams
S. G. (2004), Improved genetic elements providing
high levels of expression, Patent WO 2004067701 A2.
4. Diane M. R., Fitzroy D., Elliott P. (1994),
"Erythropoietin: A review", J. Nati. Med. Assoc., 86,
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5.Dymecki S. M. (1996), "Flprecombinase promotes
site-specific DNA recombination in embryonic stem
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line developments in speed and efficiency", Adv.
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genome", Nature biotechnology, 9, 718-720.
8. Jelkmann W. (2007), "Control of erythropoietin
gene expression and its use in medicine". Methods
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9. Kim J. Y., Kim Y. G., Lee G. M. (2012), "CHO
cells in biotechnology for production of recombinant
proteins: current state and further potential", Appl.
Microbiol. Biotechnol., 93, 917-930
10. Licursi M., Christian S. L., Pongnopparat
T., Hirasawa K. (2011), "In vitro and in vivo comparison

of viral and cellular internal ribosome entry sites for
bicistronic vector expression", Gene Ther., 18, 631636.
11. Mariati, Yeo J. H., Koh E. Y., Ho S. C., Yang
Y. (2014), "Insertion of core CpG island element into
human CMV promoter for enhancing recombinant
protein expression stability in CHO cells", Biotechnol.
Prog., 30, 523-530.
12. Meng Y. G., Liang J., Wong W. L., Chisholm
V. (2000), "Green fluorescent protein as a second
selectable marker for selection of high producing
clones from transfected CHO cells", Gene, 242, 201207.

(Ngay nhOn bai: 13/06/2015 - Ngay duyOt (fang: 02/11/2015)

TAP CHt DUQC HQC - 11/2015 (SO 475 NAM 55)

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